PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Acc&Pres/ClRCIDaIeDue.indd CHARACTERIZATION OF THE DIFFERENTIAL SUSCEPTIBILITY OF DOPAMINE NEURONAL POPULATIONS IN A MOUSE MODEL OF PARKINSON’S DISEASE BY Bahareh Behrouz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Neuroscience 2008 ABSTRACT CHARACTERIZATION OF THE DIFFERENTIAL SUSCEPTIBILITY OF DOPAMINE NEURONAL POPULATIONS IN A MOUSE MODEL OF PARKINSON’S DISEASE BY Bahareh Behrouz Parkinson's disease (PD) is a neurodegenerative disorder that causes severe motor impairments due to progressive loss of nigrostriatal (NS) dopamine (DA) neurons. Abnormal DA metabolism has been proposed to underlie the degeneration of these neurons, but not all DA neurons are affected to the same extent in PD. There is severe loss of NSDA neurons, adjacent mesolimbic (ML) DA neurons are less severely affected and tuberoinfundibular (Tl) DA neurons remain intact. The reason for this differential susceptibility amongst DA neurons is unknown. Elucidating mechanisms by which some neurons are preferentially lost while others are protected should lead to discovery of protective factors that can be translated into therapies for PD. The experiments in this dissertation characterize a model for PD using the toxin 1-methyl,4—phenyl-1,2,3,6-tetrahydropyridine (MPTP) that mimics the differential susceptibility of DA neuronal populations seen in PD. In the MPTP mouse model, NSDA neurons are highly susceptible, while MLDA neurons are less damaged and TlDA neurons remain fully protected. Furthermore, the immediate response of these neuronal populations suggests that MLDA neurons don’t lose as much DA as NSDA neurons immediately following treatment with MPTP, suggesting there might be heterogeneous populations of MLDA neurons, i.e. one that is susceptible and one that is resistant. On the other hand, TIDA neurons initially respond to MPTP treatment in a similar fashion to NSDA neurons, but rapidly recover. The recovery of these neurons from MPTP-induced DA loss is dependent on synthesis of new proteins following MPTP treatment. It is therefore likely that TIDA neurons upregulate the expression of proteins that allow for this recovery. Microarray analysis of mRNA expression in NSDA and TIDA neurons reveal several important pathways and factors that may protect TIDA neurons after exposure to MPTP. One of the genes upregulated in TIDA neurons following MPTP treatment is the protective protein parkin. Mutations in this gene cause autosomal recessive PD and increased expression of this protein infers protection to cells in a variety of models, leading to the hypothesis that parkin upregulation may contribute to the recovery of TIDA neurons following MPTP treatment. This hypothesis is tested by two separate approaches: exposing parkin knock out (KO) mice and wild type (WT) littermates to MPTP and stereotaxically injecting WT C57bl/6 mice with lentiviral particles expressing parkin shRNA in order to transiently knock down (K0) the expression of parkin. Animals receiving parkin shRNA lentivirus, but not parkin KO show stunted recovery of DA in response to MPTP treatment, suggesting parkin may be essential for the recovery of these neurons. Elucidating factors that are responsible for differential susceptibility to mitochondrial Complex l inhibition may be further translated into neuroprotective strategies that can prevent the ongoing degeneration of DA neurons in PD. This dissertation is dedicated to the love of my life, Andy. Thank you for inspiring, guiding and encouraging me to be the best I can be. And to my parents, All your sacrifices, love and support have made it possible for me to pursue my dreams. iv ACKNOWLEDGEMENTS I sincerely thank Andy, my parents, and Andy’s parents for all their love, encouragement, and support and my aunt Mariam for her continued guidance. I thank my sister, Rozina, for motivating me to pursue higher education and for raising such a fantastic son, Pouya, to be the pride and joy of my life. Drs. Lookingland and Goudreau, words alone cannot describe my gratitude for the exceptional training and guidance that I have received in your lab. I have been truly fortunate to have the opportunity to learn from you both. I would like to thank my committee members Drs. MohanKumar, Gallo and Amalfitano for their guidance and advice, Dr. Sisk for her continued support throughout the years and for being a phenomenal role model and Dr. Wade for sparking my interest in Neuroscience. I wish to thank the faculty, staff and students of the Neuroscience, Pharmacology/Toxicology and Neurology Programs. I also thank the members of the Goudreau/Lookingland laboratory who have made this experience fun and exciting. I will miss you. TABLE OF CONTENTS List of Tables .................................................................................................... xi List of Figures ................................................................................................... xiii List of Abbreviations ...................................................................................... Xviii Chapter 1. General Introduction .............................................................. 1 A. Statement of Purpose ..................................................................... 1 B. Parkinson’s Disease ...................................................................... 2 C. Degeneration of NSDA Neurons and Motor Abnormalities in PD .......................................................... 3 Normal NSDA Neuronal Function 3 NSDA Control of Basal Ganglia ................................................ 6 NSDA Neurons and Control of Basal Ganglia in PD ............ 8 D. Environmental and Genetic Factors and PD ................................. 8 E. Pathways Implicated in PD ............................................................... 11 Mitochondrial Function and Reactive Oxygen Species...........,. 11 Protein Degradation 15 Abnormal DA Metabolism ............................................................... 19 F. Differential susceptibility of DA neurons in PD ........................... 23 MLDA Neurons ............................................................................. 26 TIDA Neurons ................................................................................. 28 G. Animal models of PD ....................................................................... 32 Neurotoxin-Based Models ........................................................... 32 MPTP ....................................................................................... 33 Rotenone ................................................................................ 36 6-OHDA ................................................................................... 38 Paraquat .................................................................................. 39 Genetic and Gene Transfer Models ............................................ 39 a-Synuclein .............................................................................. 40 a-Synuclein KO or KD... 41 Overexpression of WT or Mutant a- Synuclein ................... 41 LRRK2 ...................................................................................... 44 Parkin ....................................................................................... 45 Parkin KO ................................................................................. 45 Overexpression of Parkin 46 Pink1 ......................................................................................... 46 011 ............................................................................................ 47 H. Dissertation Objective ..................................................................... 48 Chapter 2. Materials and Methods ............................................................. 50 vi A. Animals ................................................................................................ 50 Generation of Parkin Knock-Out Mice .................................... 50 Genotyping ................................................................................. 50 B. Drugs .................................................................................................... 54 Chronic MPTP ............................................................................. 54 Single Injection MPTP ................................................................ 54 Cycloheximide ............................................................................ 55 GER-12909 .................................................................................. 55 Lentiviral shRNA Against Mouse Parkin ................................. 55 C. Tissue Preparation ............................................................................. 57 D. Neurochemical Analyses 60 E. Western Blot Analyses ....................................................................... 61 Protein Extraction ..................................................................... 61 Electrophoresis/Transfer Conditions ...................................... 62 Protein Detection and Quantification ..................................... 62 F. Immunohistochemistry ..................................................................... 66 G. Unbiased Stereological Cell Counting ............................................. 66 H. Rapid Immunofluorescent Staining ................................................ 68 l. Laser Capture Microscopy (LCM) .................................................... 70 J. RNA Isolation from LCM Samples .............................................. 72 K. RNA Amplification ............................................................................. 73 L. Microarray Hybridization and Initial Analysis ............................... 74 M. Microarray Data Analysis and Statistics ....................................... 79 Normalization, Background Correction and Data Summarization ................................................................. 79 The Use of Updated Annotation Files .................................... 82 Statistical Analyses .................................................................. 83 Cluster Analysis ........................................................................ 83 Pathway Analysis with DAVID ................................................ 86 Analysis of Candidate KEGG Pathways .................................. 86 N. RNA Isolation from Microdissected Tissue Punches ............... 87 0. Reverse Transcription ..................................................................... 88 P. Real Time Quantitative PCR ............................................................ 88 Q. Stereotaxic Lentiviral Injections ................................................... 92 R. Statistical Analysis ........................................................................... 96 Chapter 3: Characterization of Differential Susceptibility of TIDA, NSDA and MLDA Neurons in the Chronic MPTP Mouse Model... 97 A. Introduction ....................................................................................... 97 B. Hypothesis ......................................................................................... 98 C. Experimental Design ........................................................................ 98 D. Results ................................................................................................ 103 E. Discussion ........................................................................................... 113 vii Chapter 4: Characterization of the Immediate Neurochemical Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP A. Introduction ...................................................................................... B. Hypothesis ......................................................................................... C. Experimental Design .......................................................................... Time Cause of Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP ................................... Effects of Blockade of DAT on the Response of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP... Effects of Protein Synthesis Inhibition on the Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP ................................................... Effects of Immediate vs. Delayed Protein Synthesis Inhibition on the Response of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP .................................. D. Results ................................................................................................ Time Course of Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP ................................... Effects of Blockade of DAT on the Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of MPTP ..... The Impact of Protein Synthesis Inhibition on the Neurochemical Responses of NSDA, MLDA and TIDA Neurons to a Single Injection of MPTP ................................... The Impact of Immediate vs. Delayed Protein Synthesis Inhibition on the Neurochemical Responses of NSDA, MLDA and TIDA Neurons to a Single Injection of MPTP ........................................................................................... E. Discussion ............................................................................................ Differential Response of TIDA, NSDA and MLDA Neurons After a Single Injection of MPTP .............................................. Mechanism of MPP+ Uptake is Different in TIDA Neurons vs. NSDA and MLDA Neurons ................................................... Recovery of TIDA Neurons is Dependent on Synthesis of New Proteins after MPTP Treatment .................................................................................. F. General Conclusions ........................................................................... Chapter 5: Characterization of the Genomic Responses of TIDA and NSDA Neurons to a Single Injection of MPTP A. Introduction ........................................................................................ B. Hypothesis .......................................................................................... C. Experimental Procedures .................................................................. viii 119 119 121 121 121 125 127 129 131 131 137 144 149 154 154 157 159 160 162 162 162 163 0. Results ................................................................................................. 164 Genes with Differential Expression in ARC and SNpc in Control and MPTP Treated Mice ............................................. 164 Unbiased Pathway Analysis of Microarray Data with DAVID .......................................................................................... 167 Cluster Analysis of Genes Significantly Higher in ARC than SNpc in MPTP- Treated Mice .................................................... 176 Analysis of Candidate Genes... 179 Real Time PCR Analysis of Candidate Genes .......................... 181 E. Discussion ............................................................................................ 185 Analysis of Pathways using DAVID Annotation Tools ......... 185 Identification of Candidate Clusters .................................... 187 Analysis of Candidate Genes ................................................ 189 Genes and Pathways that Potentially Protect TIDA Neurons from Toxicity ............................................................. 191 Chapter 6: The Involvement of Parkin in the Recovery of TIDA Neurons from MPTP-Induced DA Loss 191 A. Introduction ....................................................................................... 191 B. Hypothesis ......................................................................................... 198 C. Experimental Design ........................................................................ 198 Parkin Protein Levels Following a Single Injection of MPTP ......................................................... 198 Neurochemical Response of TIDA Neurons to MPTP in Parkin KO mice and WT Littermates .................................... 198 Neurochemical Response of TIDA Neurons to MPTP Following Lentiviral Knock Down of Parkin ......................... 199 D. Results ............................................................................................... 199 Parkin Protein Levels Following a Single Injection of MPTP ....................................................... 199 Neurochemical Responses of TIDA Neurons to MPTP in Parkin K0 and WT Mice ......................................... 201 Neurochemical Responses of TIDA Neurons to MPTP Following Lentiviral Knock Down of Parkin ......................... 204 E. Discussion ............................................................................................ 209 Chapter 7. Concluding Remarks 214 DA Theory for Selective Vulnerability of NSDA Neurons in PD ...... 214 Basal Differences Between NSDA and TIDA Neuronal Populations 215 Response of TIDA Neurons to MPTP .................................................. 217 Hypotheses Regarding the involvement of Parkin in Recovery of TIDA Neurons ........................................................................................ 218 Protection of NSDA Neurons in PD 224 Appendix 226 References 270 1-1 2-1 2-2 3-2 3-3 4-2 4-3 4-4 5-1 5-2 LIST OF TABLES Genes involved in Mendelian inheritable forms of Parkinson’s disease ........................................................................................................ 10 Sequence and catalog information for parkin and non-target shRNA lentiviral particles purchased from Sigma ............................................. 56 Description of primary antibodies used in the experiments in this dissertation ...................................................................................... 63 Forward and reverse primers used for real time PCR analysis ............ 89 The experimental endpoints used for indices of neuronal function in the chronic MPTP mouse model ............................ 102 Norepinephrine (NE) concentrations in median eminence (ME), striatum (ST) and nucleus accumbens (NA) of control and MPTP treated mice .............................................................................................. 107 Serotonin (S-HT; A) and 5-hydroxyindoleacetic acid (S-HIAA; 8) concentrations in median eminence (ME), striatum (ST) and nucleus accumbens (NA) of control and MPTP treated mice............. 108 The experimental endpoints used for neuronal function following treatment with a single injection of MPTP ........................... 124 Norepinephrine (NE) concentrations in median eminence (ME), striatum (ST) and nucleus accumbens (NA) of saline control and MPTP treated mice .................................................................................... 134 DOPAC concentrations (ng/mg protein) in ME, ST and NA after a single injection MPTP with protein synthesis inhibition using cycloheximide .................................................................................. 148 DOPAC concentrations (ng/mg protein) in ST, NA and ME after a single injection MPTP with protein synthesis inhibition using cycloheximide ................................................................ 153 DAVID Pathway analysis of genes expressed higher in ARC than SNpc after treatment with MPTP ................................................... 169 Relative expression levels of genes that regulate apoptosis in ARC vs. SNpc of saline and MPTP treated mice ................................. 171 xi 5-3 5-4 6-1 A-1 A-3 A-4 DAVID Pathway analysis of genes higher in SNpc than ARC only after treatment with MPTP ........................................................... 178 Normalized mRNA expression levels of LRRK2, synuclein, D11, and Pink1 in ARC and SNpc of mice injected with saline or MPTP.... 184 The effects of parkin shRNA administration and a single injection of MPTP on norepinephrine (NE), serotonin (5-HT) and (S-HIAA) concentrations in the median eminence (ME).............. 207 Genes with statistically higher expression in ARC than SNpc of saline treated controls ......................................................................... 227 Genes with statistically higher expression in SNpc than ARC of saline treated controls ......................................................................... 237 Genes with statistically higher expression in ARC than SNpc of animals treated with MPTP ................................................................ 246 Genes with statistically higher expression in SNpc than ARC of animals treated with MPTP .................................................................. 258 xii 1-2 1-3 1-4 1-5 1-6 2-1 2-3 2-4 2-5 2-6 LIST OF FIGURES Images in this dissertation are presented in color. Schematic diagram depicting the neurochemical events in a NSDA axon terminal ................................................................................ 5 Function of the basal ganglia via the direct and indirect pathways in normal (left) and PD patients (right) ..................................................... 7 The mitochondrial electron transport chain ........................................ 13 The Ubiquitin Proteasome System (UPS) 17 DA non-enzymatic (left) and enzymatic (right) metabolic pathways 22 Distribution of dopamine and norepinephrine neuronal populations in a sagittal view of the rat braIn 25 Differences in the DA transmission, uptake and feedback inhibition in NSDA or MLDA neurons (top) and TIDA neurons (bottom) .................................................................................................... 30 Diagram describing distribution, bioactivation and site of action of MPTP ............................................................................................. 34 Genotyping of wild-type (WT), homozygous parkin knock-out (KO), heterozygous (HT) mice ........................................................................... 53 Diagrams of coronal brain sections illustrating the location of micropunches used to dissect NA and ST ............................................. 58 Diagrams of coronal brain sections illustrating the sections used to dissect ARC (top), VTA and SNpc bottom) .............................. 59 GAPDH levels in ARC and SNpc of control and MPTP treated mice...65 Time course of RNA degradation during rapid immunofluorescent staining for TH ......................................................................................... 69 Laser capture of tyrosine hydroxylase-immunoreactive (TH-IR) cells of SNpc ................................................................................ 71 xiii 2-7 2-8 2-9 2-10 2-11 2-12 2-13 2-14 2-15 3-1 3-2 3-3 3-4 3-5 Visualization of individual arrays hybridized with amplified mRNA from ARC and SNpc of control animals ................................................ 76 Visualization of individual arrays hybridized with amplified mRNA from ARC and SNpc of MPTP treated animals .................................... 77 Assessment of RNA quality in individual arrays ................................. 78 Mean expression levels (Y-axis) of all genes in each array (X-axis) before (top) and after (bottom) normalization with GCRMA ........... 81 Cluster analysis of genes significantly higher in ARC than SNpc after treatment with a single injection of MPTP ................................. 85 GAPDH mRNA levels in ARC and SNpc of control and MPTP-treated mice ................................................................................. 90 Bilateral stereotaxic injection of lentiviral vectors into ARC of control and MPTP-treated mice ............................................................ 93 Stereotaxic injection of lentiviral vector encoding for green fluorescent protein (GFP) into ARC of mice ........................................ 94 IBA1 staining for brain sections from mice that received stereotaxic injection of lentiviral vector encoding for green fluorescent protein into ARC ................................................................. 95 Time course of chronic MPTP administration ...................................... 99 The effects of chronic MPTP administration on DA levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ............................................................................................................ 104 The effects of chronic MPTP administration on DOPAC levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ............................................................................................................ 105 The effects of chronic MPTP administration on the ratio of DOPAC/DA in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ........................................................................ 106 Tyrosine hydroxylase immunoreactive (TH-IR) cells in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and arcuate nucleus (ARC) of control and MPTP treated mice ......... 110 xiv 3-7 4-1 4-2 4-3 4-5 4—6 4-7 4-8 4-9 4-10 The effects of chronic MPTP administration on tyrosine hydroxylase immunoreactive (TH-IR) cell numbers in the arcuate nucleus (ARC), substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) ......................................................................................................... 111 The effects of chronic MPTP administration on Nissl positive cell numbers in the arcuate nucleus (ARC), substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) ........................... 112 Paradigm for the single injection MPTP time course experiment ...... 122 Experimental paradigm for the combination of GBR pretreatment with a single injection MPTP ................................................................. 124 Experimental paradigm for the combination of cycloheximide pretreatment with a single injection of MPTP .................................... 128 Experimental paradigm for the combination of cycloheximide treatment with a single injection MPTP ............................................... 130 The time course of effects of a single injection of MPTP on DA levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ......................................................................... 132 The time course of effects of a single injection of MPTP on DOPAC levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ................................................................ 133 The time course of effects of a single injection of MPTP on S-HT levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ....................................................................... 135 The time course of effects of a single injection of MPTP on 5HIAA levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA) ........................................................................ 136 The effects of GBR pre-treatment and single injection MPTP administration on DA levels in median eminence (ME) .................... 138 The effects of GBR pre-treatment and single injection MPTP administration on DA levels in striatum (ST) ...................................... 139 XV 4-11 4-12 4-13 4-14 4-15 4-16 4-17 4-18 4-19 4-20 5-2 5-3 5-4 The effects of GBR pre-treatment and single injection MPTP administration on DA levels in nucleus accumbens (NA) ................. 140 The effects of GBR pre-treatment and single injection MPTP administration on DOPAC levels in median eminence (ME)............. 141 The effects of GBR pre-treatment and single injection MPTP administration on DOPAC levels in striatum (ST) ............................... 142 The effects of GBR pre-treatment and single injection MPTP administration on DOPAC levels in nucleus accumbens (NA) ........... 143 The effects of cycloheximide and MPTP administration on DA levels in median eminence (ME) ....................................................... 145 The effects of cycloheximide and MPTP administration on DA levels in striatum (ST) ............................................................................... 146 The effects of cycloheximide and MPTP administration on DA levels in nucleus accumbens (NA) ........................................................ 147 The effects of immediate and delayed cycloheximide and single Injection MPTP on DA levels in ME ...................................................... 150 The effects of immediate and delayed cycloheximide and single Injection MPTP on DA levels in ST .......................................................... 151 The effects of immediate and delayed cycloheximide and single Injection MPTP on DA levels in NA ........................................................ 152 The pattern of expression of genes differentially expressed among regions in the control (top) and MPTP treated (bottom) animals... 165 The number of genes with statistically higher expression in ARC (top) and SNpc (bottom) under control and MPTP treated conditions ................................................................................................ 166 Relative expression levels of genes that regulate apoptosis in ARC vs. SNpc of saline and MPTP treated mice ........................................... 170 Expression levels of genes related to DA metabolism, transport and secretion in ARC vs. SNpc of saline and MPTP treated Mice ............. 175 xvi 5-5 5—6 5-8 6-1 6-2 6-4 6-5 6—6 6-7 6-8 7-1 Cluster Analysis of genes up-regulated in ARC but not SNpc after treatment with MPTP ............................................................................... 177 Expression levels of Parkinson’s disease related genes in ARC vs. SNpc of saline and MPTP treated Mice ................................................ 180 RT—PCR analysis of normalized parkin expression levels in ARC and SNpc in controls and MPTP treated mice ............................................ 182 RT—PCR analysis of normalized UCH-L1 expression levels in ARC and SNpc in controls and MPTP treated mice ............................................. 183 Structure and E3 ligase activity of parkin ............................................. 194 The proposed role of parkin in selective degeneration of DA neurons ................................................................................................. 197 The effects of a single injection of MPTP on parkin levels in median eminence (ME) and striatum (ST) ......................................................... 200 The effects of a single injection of MPTP on DA levels in the median eminence (ME) of parkin knock out (K0) and wild type (WT) littermates 202 The effects of a single injection of MPTP on DA levels in the median eminence (ME) of parkin knock out (K0) and wild type (WT) littermates ...................................................................... 203 The effects of parkin shRNA administration and a single injection of MPTP on DA levels in the median eminence (ME) .............................. 205 The relationship between DA concentration and parkin levels in ARC of mice that received both parkin shRNA (143, 145 or 147 sequences) and MPTP ................................................................................................... 206 The effects of parkin shRNA administration and a single injection of MPTP on DA levels in the striatum (ST) ................................................. 208 The proposed involvement of parkin in pathways that lead to cell death in Parkinson’s disease ........................................................... 223 xvii 5-HIAA 5-HT 6-OH DA AADC AD ANOVA ARC Complex I DA DAT DAVID D11 DOPA DOPAC DOPAL E1 E2 E3 FDR LIST OF ABBREVIATIONS S-hydroxyindoleacetic acid Serotonin 6-hydroxydopamine L-Aromatic amino acid decarboxylase Aldehyde dehydrogenase Analysis of variance Arcuate Nucleus Nicotinamide adenine dinucleotide (NADH)—ubiquinone reductase Dopamine Dopamine transporter Database for Annotation, Visualization and Integrated Discovery Oncogene DJ1 Dihydroxyphenylalanine Dihydroxyphenylacetic acid Dihydroxyphenylacetaldehyde Ubiquitin activating enzyme Ubiquitin conjugating enzyme Ubiquitin ligating enzyme False discovery rate xviii GAPDH GBR GO GPe GPi GSH H202 HPLC-EC IBR K0 K0 LCM LNAA LRRK2 MAO ME Mito MLDA MPDP MPP+ hAPTP Glyceraldehyde-3—phosphate dehydrogenase GBR-12909 Gene Ontology Globus pallidus external segment Globus pallidus internal segment Glutathione Hydrogen peroxide High performance liquid chromatography with electrochemical detection In between ring Immunoreactive Knock out Knock down Laser capture microscopy Large neutral amino-acid transporter Leucine-rich repeat kinase 2 Monoamine oxidase Median eminence Mitochondria Mesolimbic dopamine 1-Methyl-4-phenyl-2,3-dihydropyridinium 1-Methyl-4-phenyl-pyridinium 1-Methyl-4 phenyl-1,2,3,6-tetrahydropyridine xix NA NADH NE NorBNI NSDA 02- OH- OONO- PD Pinkl PKC PM Probenecid RDU RING ROS SEM SNpc ST SOD TH TI DA Nucleus accumbens Nicotinamide adenine dinucleotide Norepinephrine nor-binaltorphimine Nigrostriatal dopamine Superoxide Hydroxyl radical Peroxynitrate Parkinson's disease PTEN induced putative kinase 1 CaZ+—calmodulin-dependent protein kinase Perfect-match Dipropylsulfamoyl-benzoic acid Relative density unit Really interesting new gene Reactive oxygen species Standard error of the mean Substantia nigra pars compacta Striatum Superoxide dismutase Tyrosine hydroxylase Tuberoinfundibular dopamine XX UPS U50488 UCH-Ll VL VMAT-2 VTA WT Ubiquitin proteosome system Trans-3,4-dichloro-N-methyl-N-[2-(1- pyrrolindinyl)cyclohexyl]-benzene-acetanide methanesulfonate hydrate Ubiquitin carboxy-terminal hydrolase L1 Ventral lateral thalamic nucleus Vesicular monoamine transporter Ventral tegmental area Wild type xxi Chapter 1. General Introduction A. Statement of Purpose Parkinson's disease (PD) is a debilitating neurodegenerative disorder that causes severe motor impairment with underlying progressive degeneration of nigrostriatal (NS) dopamine (DA) neurons. While symptomatic therapies exist, there is currently no way to retard or halt the degenerative process. Abnormal DA metabolism has been proposed as a mechanism for the specific degeneration of NSDA neurons. However, all DA neurons are not affected to the same extent in P0. In fact, while there is severe loss of midbrain NSDA neurons, the adjacent mesolimbic (ML) DA neurons are affected to a much lesser degree than NSDA neurons and hypothalamic tuberoinfundibular (TI) DA neurons remain unaffected in PD. Other pathways implicated in the pathogenesis of the disease (e.g. mitochondrial dysfunction, protein degradation and oxidative stress) are central pathways that should be essential to all cells and do not explain this differential susceptibility. The overall aim of the research discussed in this dissertation is to characterize the differential susceptibility of DA neuronal populations in an animal model of PD using pharmacological, neurochemical, molecular and biochemical approaches. The underlying hypothesis is that mechanisms intrinsic to TIDA neurons, which are unaffected in the disease process, protect these neurons from neurodegeneration that occurs in susceptible NSDA neurons. If this hypothesis is correct, elucidation of these factors could lead to development of therapies to protect NSDA neurons in PD, and thereby prevent the progressive motor deficits associated with this devastating disease. 8. Parkinson's Disease PD is a complex, multifactorial disorder that affects more than a million people in North America alone (Cookson et al, 2007). The disorder is characterized in the clinic by its debilitating motor symptoms, which include resting tremor, rigidity, bradykinesia (slowness of movement) and postural instability (Fahn, 2003). A definite diagnosis of PD requires post-mortem observation of NSDA neuronal loss, which underlies the motor symptoms of PD, as well as the presence of proteinaceous intracellular inclusions called Lewy bodies. The reason for selective vulnerability of NSDA neurons and the formation of Lewy bodies is not known, and there is currently no therapy to slow or stop the progressive nature of these pathological abnormalities in PD. Current therapeutics for PD focus primarily on reducing the severity of motor symptoms using medications that replace DA activity at post-synaptic receptors. The most common treatment for P0 is the precursor L-DOPA, which is decarboxylated predominantly by glial cells in the brain to form DA and reinstate DA transmission lost due to degeneration of substantial numbers of NSDA neurons. Coadministration with carbidopa blocks decarboxylation of L-DOPA in the periphery, allowing for fewer side effects. Other treatments include DA agonists, monoamine oxidase 8 inhibitors, and catechol-O-methyltransferase inhibitors. While these medications provide some relief to patients, they only alleviate the symptoms and are unable to slow or stop the progressive degeneration of NSDA neurons in PD. C. Degeneration of NSDA Neurons and Motor Abnormalities in PD NSDA neurons, emanating from substantia nigra pars compacta (SNpc) and projecting to the striatum (ST), are key components of the extrapyramidal motor system and their degeneration underlies the motor deficits of PD. These motor deficits can be alleviated by mechanisms that restore striatal DA concentrations (Dauer and Przedborski, 2003). Understanding normal NSDA neuronal function is essential to discovering mechanisms that may underlie their degeneration in PD. Normgl NSDA Neuronal Fwtion: NSDA neurons have long projecting axons and form classic synapses with target medium spiny neurons in ST. The tightly controlled synthesis, storage, release and metabolism of DA in these neurons (Figure 1-1) allows for regulation of the activity of the basal ganglia that ultimately control voluntary movement. Synthesis of DA begins when dietary tyrosine is transported into DA axon terminals via the large neutral amino—acid transporter (Oxender and Christensen, 1963; Oldendorf and Szabo, 1976; Palacin et al., 1998), where it is hydroxylated to 3,4-dihydroxyphenylaIanine (DOPA) by the rate limiting enzyme tyrosine hydroxylase (TH) (Levitt et al., 1965; Kumer and Vrana, 1996). In the cytoplasm, the enzyme L—aromatic amino acid decarboxylase (AADC) quickly decarboxylates DOPA into DA (Kumer and Vrana, 1996). The vesicular monoamine transporter-2 (VMAT-2) rapidly and efficiently packages newly made DA into synaptic vesicles. Any newly synthesized cytosolic DA that is not packaged is degraded into 3,4-dihydroxyphenylacetic acid (DOPAC) through a two-step process catalyzed by mitochondrial monoamine oxidase (MAO) and aldehyde dehydrogenase (AD). Cytoplasmic DA regulates its own synthesis by inhibiting the activity of TH via end-product inhibition (Okuno and Fujisawa, 1985; Andersson et al., 1988; Haavik et aL,1990) Vesicular DA is released into the synaptic cleft upon propagation of an action potential where it can bind to post-synaptic 01 or 02 receptors, thereby enhancing or inhibiting the firing of post-synaptic neurons by CAMP-dependent mechanisms (Onali et al., 1985; Monsma et al., 1990). DA released into the synapse can also activate pre- synaptic DZ autoreceptors and thereby inhibit further synthesis or release of DA (Christiansen and Squires, 1974; Roth, 1975). Excess DA is removed from the synapse by the high-affinity reuptake DA transporter (DAT) and is either repackaged into vesicles or rapidly metabolized to DOPAC (Kilty et al., 1991). NSDA Axon Terminal DOPAC DOF’AC Mito N M y/ DOPAL VMA Tyrosine—> DOPA—v DA — a — TH --> AADC Tyrosine Figure 1-1. Schematic diagram depicting the neurochemical events in a NSDA axon terminal. Tyrosine is taken up into the axon terminal by the large neutral amino-acid (LNAA) transporter. Tyrosine is converted to DOPA by the rate limiting enzyme tyrosine hydroxylase (TH). DOPA is decarboxylated to DA by L-aromatic amino-acid decarboxylase (AADC) in the cytoplasm. Newly synthesized DA is transported by vesicular monoamine transporter-2 (VMAT) into synaptic vesicles for release. Newly synthesized DA can also be degraded by mitochondrial (Mito) MAO. DA released into the synapse can bind to post-synaptic 01 or D2 receptors (Di/02 DA-R) or 02 autoreceptors (DZ DA-R). DA released into the synapse is removed via the DA transporter (DAT). Recaptured DA can be either repackaged into synaptic vesicles or degraded by MAO and aldehyde dehydrogenase (AD) to DOPAC. DOPAC is removed from the axon terminal through diffusion. (Drolet, 2006) NSDA Control of Basal Ganqlia The release of DA from NSDA neurons in the ST regulates the activity of the basal ganglia which collectively refers to the SNpc, ST, globus pallidus and the subthalamic nucleus (Bergman, 1998). The interplay amongst these nuclei determines the control of voluntary motor behavior. As depicted in Figure 1-2, DA released by NSDA neurons in ST acts on excitatory 01 and inhibitory DZ receptors to activate the direct pathway and inhibit the indirect pathway, respectively. Activation of the direct pathway leads to inhibition of areas of the globus pallidus and substantia nigra reticulata, which in turn removes their tonic inhibition from the thalamus thereby promoting voluntary movement (Jackson et. al. 1994). The indirect pathway normally serves to inhibit the voluntary movement. Therefore, inhibition of the indirect pathway also promotes voluntary movement (Jackson and Westlind-Danielson 1994). As a simple analogy, Dl mediated activation of the direct pathway is akin to pressing the gas pedal in a car, whereas, 02 mediated inhibition of indirect pathway is similar to letting off the brake; both promote forward motion or in this case voluntary movement. Normal Brain pD Brain Frontal Spinal | Frontal I - Thalamus Cortex Thalamus DZ Striatum Q1 D2 Striatum 01] ‘ , ....... . ...... ,5 ............. Indirect Direct Indirect Direct G'Obus GIobus pallidus pallidus externa| external """"""""" ent m segment - . .5” PF Subthalamic Subtlhalamic ] nucleus nucleus _ fly Globus pallidus internal & Globus pailidus Internal 5‘ Substantia ni ra reticulata Substantia ni reticulata Figure 1-2. Function of the basal ganglia via the direct and indirect pathways in normal (left) and PD patients (right). In normal basal ganglia DA released from the SN activates the direct pathway, thereby inhibiting the globus pallidus internal segment and substantia nigra reticulata and disinhibiting the thalamus. DA released in the ST also inhibits the indirect pathway which leads to disinhibition of the globus pallidus external segment and inhibition of the subthalamic nucleus, which in turn causes less excitation of the globus pallidus internal segment and substantia nigra reticulata allowing for the thalamus to be disinhibited. Increased activity of the thalamus leads to excitation of the frontal cortex, which leads to activation of the spinal cord and increased voluntary movement. In PD patients (left) significantly less DA is released into the ST thereby less activation of the direct pathway and inhibition of the direct pathway can occur. This causes increased inhibition of the thalamus and complicates voluntary motor movement. Blue lines represent inhibitory and red lines represent excitatory synapses. NSDA Neurons and Control of Basal Ganqlia in PD The loss of NSDA neurons leads to alterations in the activity of the neural circuits within the basal ganglia that regulate movement (Figure 1-2). The loss of DA in ST leads to an inhibition of the direct pathway and excitation of the indirect pathway, which results in lower firing rates of neurons in the motor cortex. The loss of motor cortex activity leads to lower activation of spinal cord motor neurons that activate peripheral muscle movement and a hypokinetic movement disorder. Since the loss of NSDA neurons underlies the cardinal motor symptoms of PD, halting the ongoing degeneration of these neurons will prevent the continued worsening of motor deficits in PD. D. Environmental and Genetic Factors and PD Most cases of PD are considered sporadic and of unknown source. While environmental factors have been implicated in the pathogenesis of the disease, no Specific factor has been identified. Epidemiological studies have revealed that rural living, well-water consumption, pesticide use and occupations such as mining and Welding are all associated with an increase risk for PD (Priyadarshi et al., 2000; Priyadarshi et al., 2001; Firestone et al., 2005; Jankovic, 2005). Other environmental factors such as coffee and alcohol consumption and cigarette smoking have been associated with reduced risk for the disease (Honig, 2000; Allam et al., 2002; Checkoway et al., 2002; Allam et al., 2004). However, the mechanisms by which these factors may influence disease risk remain unclear. While most cases of P0 are not inherited, several genetic mutations in families with a Mendelian pattern of inheritance have been identified and are listed in Table 1- 1. These genetic mutations have shed some light on disease pathogenesis by implicating a number of interconnected molecular pathways (Hofer and Gasser, 2004). These studies also demonstrate that multiple mechanisms can lead to the degeneration of NSDA neurons and result in PD symptoms. Understanding the overlap in molecular mechanisms between these heritable and sporadic cases of PD may elucidate the complex pathways that underlie this disease. These pathways are described in detail below. Chromosomal Position Mutations Pathology SNCA 4q21 Dominant Lewy bodies, NSDA and hippocampal neuronal loss LRRK2 12p12 Dominant Lewy bodies and NSDA loss PARK2 6q25.2—q27 Recessive Primarily NSDA loss PINK1 1p35—p36 Recessive Undetermined DJ1 1p36 Recessive Undetermined Table 1-1. Genes involved in Mendelian inheritable forms of Parkinson’s Disease. SNCA = a- synuclein, LRRK2 = Leucine rich repeat kinase 2, park2 = parkin, pinkl = PTEN induced putative kinase 1, and DJ1 = oncogene DJ-1. 1O E. Pathways Implicated in PD Several molecular pathways have been proposed to play a role in the etiology of PD. There is consistent evidence for a role of mitochondrial dysfunction, malfunctions in the proteasome and other protein degradation pathways, as well as DA synthesis and metabolism. These pathways are explained below and evidence for the involvement of each, as well as interplay amongst these pathways is presented. Mitochondrial Finction and Reactive Oxygen Species The mitochondrial electron transport chain has five complexes located in its inner membrane (Figure 1-3). Complexes I, III and IV use energy from the transfer of electrons down the transport chain to move protons from the mitochondrial matrix to the inner-membrane space and create a proton gradient across the inner membrane. Complex V can use the energy from this proton gradient to convert ADP to ATP. Disruptions in the electron transport chain can reduce ATP levels, and thereby decrease the supply of energy in the cell. Furthermore, under normal conditions oxygen can interact with electrons in the transport chain to form reactive oxygen species (ROS). All aerobic organisms are constantly exposed to ROS (Halliwell, 1992) but ROS can be scavenged primarily by glutathione under basal conditions. ROS that are not scavenged can cause damage to a variety of critical biological constituents including DNA, proteins and lipids. Mitochondria are thought to be the most important source of cellular free radicals, producing superoxide radicals at ubiquinone and NADH dehydrogenase (Turrens et al., 1985). Superoxide dismutase (SOD) normally converts superoxide to 11 H202, which reacts with transition metals to generate the prime mediator of cellular damage, the hydroxyl radical. Alternatively, superoxide can also react with nitric oxide to produce peroxynitrile (Beckman and Crow, 1993). Under physiological circumstances, approximately 2% of the oxygen consumed by mitochondria is converted to superoxide and this percentage is much higher in dysfunctional mitochondria. 12 FADHZ-t FADComplex Ill Complex II Complex IV Complex l ADP ATP Complex V Figure 1-3. The mitochondrial electron transport chain. A hydrogen gradient is created in the inner membrane space utilizing enzymes on the inner mitochondrial membrane to oxidize NADH2+ to NAD+ (Complex I), and FADHZ to FAD (Complex II). Electrons are passed from Complex to Complex in the inner membrane of the mitochondrion, losing some of their energy at each step. These electrons provide energy to "pump" hydrogen protons (Complex V) across the inner mitochondrial membrane. The hydrogen protons tend to move down their concentration gradient providing energy to phosphorylate ADP into ATP. 13 The first clue that pointed to mitochondria as an important pathway in PD came when a group of individuals who injected themselves with synthetic heroin contaminated with 1-methyl,4-phenyl,1,2,3,6-tetrahyropiridine (MPTP) developed motor symptoms that closely matched symptoms of PD (Langston et al., 1983; Langston and Ballard, 1983). MPTP causes mitochondrial Complex I inhibition which leads to decreased ATP synthesis and formation of ROS (Chiueh et al., 1994). Like PD patients, MPTP users experienced loss of NSDA neurons and axon terminals, and progressive loss of motor function over time (Vingerhoets et al., 1994). A more direct link between PD and mitochondrial impairment came from description of Complex I deficiency in post-mortem substantia nigra of PD patients (Schapira et al., 1989). Since this discovery, several reports have demonstrated Complex I deficiency in substantia nigra, platelets and skeletal muscles of PD patients (Bindoff et al., 1989; Krige et al., 1992; Mann et al., 1994). Overall, the severity of Complex I deficits have been reported to be about 35% in PD patients when compared to a control population. However, within PD patients there seems to be a heterogeneous population, with some patients showing severe Complex I deficiency and others having normal Complex l activity (Schapira, 2008). From this data, one can hypothesize that Complex I deficits in PD patients may lower the threshold of mitochondria or ROS-mediated apoptosis. Some genetic cases of PD confirm the involvement of mitochondria and ROS in PD. PTEN induced kinase 1 (PINK1) and oncogene DJ1 (DJ1) are genes associated with familial PD and their products are directly linked to mitochondrial function (Beal, 2005). 14 PINK1 is a mitochondrial kinase which is present in 5-10% of Lewy bodies for familial and sporadic PD patients (Gandhi et al., 2006). The L166B DJ1 mutation is associated with increased localization of DJ1 to the mitochondrial membrane (Bonifati et al., 2003a), suggesting mitochondrial dysfunction may be involved in familial forms of PD. With vast evidence for the involvement of mitochondrial dysfunction in PD, its role in the pathogenesis of the disease still remains largely controversial (Coppede et al., 2006). It is unclear, for example, whether mitochondrial deficits are the cause or a downstream effect of the disease. Protein Degradation The ubiquitin proteasome system (UPS) is the major intracellular protein degradation system (Hershko 1998). As demonstrated in Figure 1-4, in the UPS proteins destined for degradation are covalently tagged with ubiquitin, a 76 amino acid residue protein, through a process that requires the sequential actions of ubiquitin activating (E1), conjugating (E2) and ligating (E3) enzymes (Hershko et al., 1983). Usually, the ubiquitylation process is repeated many times to allow the formation of a polyubiquitin chain on the substrate which is then targeted for degradation by the 265 proteasome. The 265 proteasome is a large protease complex composed of a barrel- shaped 205 proteolytic core and two 195 regulatory caps, one on each side of the barrel's openings. Individual ubiquitin monomers are regenerated in the process by the actions of de-ubiquitylating enzymes. It is noteworthy that a large number of cellular proteins (>1000), comprising various E15, E25, E35 and other related members, 15 are involved in the UPS (Hicke et al., 2005) and therefore the dysfunction of this system could lead to major cellular consequences. 16 ubi uitin poly-ubiquitin \ q monomer 2 6 T . . b' 't’ . _@ —® U IQUI In 0+0 peptides O Figure 1-4. The Ubiquitin Proteasome System (UPS). Depiction of the UPS, demonstrating ubiquitin release from pro-ubiquitin (Step 1) followed by ubiquitin conjugation to the substrate by adenylation of ubiquitin by ubiquitin-activating enzyme E1 (Step 2), transfer of activated- ubiquitin to the cysteine of a specific E2 ubiquitin-conjugating enzyme (Step 3), transfer of activated ubiquitin from E2 to a substrate-specific E3 ubiquitin-ligase (Step 4) and formation of a substrate-E3 complex and biosynthesis of the polyubiquitin chain (Step 5). The substrate protein is recognized by the 19S regulatory particle in an ATP-dependent step (Step 6) and must be unfolded and deubiquitinated before it can enter the interior of the 205 particle to come in contact with the proteolytic active sites (Step 7). The passage of the unfolded substrate into the core is called translocation and results in degraded proteins (MacDonald, 1999). 17 Several discoveries point to UPS dysfunction in PD. There is decreased activity of the 205 proteasome in SNpc of PD patients (McNaught and Jenner, 2001; McNaught et al., 2003) and proteasome activators are suppressed in the PD SNpc (McNaught and Olanow, 2003). Other factors such as increased levels of proteins with oxidative damage, increased protein aggregation and formation of Lewy bodies also implicate the UPS in PD. It is noteworthy to mention that one of the major components of sporadic PD Lewy bodies is aggregated a-synuclein (Spillantini et al., 1997). Duplication, triplications or mutations in the gene for a-synuclein that cause increased expression of WT a-synuclein lead to familial PD (Chartier-Harlin et al., 2004). This suggests that too much of this protein can cause neurotoxicity, potentially by aggregating and blocking proteasome function. This idea is consistent with other neurodegenerative disorders such as Huntington’s disease, where aggregating proteins are associated with neurotoxicity. Other genetic cases have implicated UPS dysfunction in the etiology of PD. The first gene demonstrated to cause autosomal recessive PD, parkin, is an E3 ligase (Kitada et al., 1998). Interestingly, disease causing mutations of parkin were found to compromise its normal role as an E3 enzyme (Sriram et al., 2005). PD patients with mutations in parkin show accumulation of insoluble Pael receptors, a substrate for parkin possibly implicating the UPS pathway (Yamamoto et al., 2005). The discovery of a missense mutation in UCH-L1, a de—ubiquitylating enzyme, in a pair of German siblings with inherited PD (Leroy et al., 1998) also lends support to the role of UPS in PD pathogenesis. The mutated version of UCH-L1 markedly reduces ubiquitin hydrolase 18 activity in vitro (Gong and Leznik, 2007). This evidence, however, remains controversial since UCH-Ll mutations have only been seen in one pair of siblings and the possibility remains that other genes or environmental factors were responsible for PD in these individuals. As is the case with the mitochondrial pathway, the involvement of the UPS in the pathogenesis of PD should be interpreted with caution. Many cellular processes can lead to proteasome dysfunction and, in turn, proteasome dysfunction can cause impairment of other cellular processes that are implicated in PD pathogenesis. For example, proteasome inhibitors can cause mitochondrial damage (Sullivan et al., 2004) and, vice versa, mitochondrial Complex l inhibitors can decrease proteasome activity (Hoglinger et al., 2003a). The interconnectivity of these pathways makes it difficult to assess the exact role of UPS in PD pathogenesis and discern whether proteasome dysfunction is a result or an active contributor to the disease. A_bnormgl DA Metabolism A major question that has been posed is why there seems to be selective vulnerability of NSDA neurons in PD. Although several brain regions are affected in PD, as demonstrated by presence of pathological abnormalities such as Lewy bodies, the DA neurons of SNpc and noradrenergic neurons of locus ceruelleus exhibit the most extensive cell loss. One element common to these severely affected neuronal populations is that they produce catecholamines. It is therefore not surprising that metabolism of catecholamines, particularly DA, which is also synthesized in 19 noradrenergic neurons, has been proposed to play a role in the neuronal loss in the PD brain. It is crucial for DA neurons to closely regulate synthesis, storage, release and reuptake of DA because certain metabolites of DA can be highly toxic to neurons (Graham, 1978; Graham et al., 1978; LaVoie and Hastings, 1999b, 1999a). The enzymatic and non-enzymatic metabolism of DA is depicted in Figure 1-5. DA oxidation by MAO results in the formation of dihydroxyphenylacetaldehyde (DOPAL) which is highly toxic (Burke et al., 2003; Legros et al., 2004; Ebadi et al., 2005). Under normal conditions, AD can efficiently and quickly convert DOPAL to DOPAC, a less toxic species that diffuses out of axon terminals. Accumulation of DOPAL in nerve terminals by inhibition of AD can exacerbate neurotoxicity caused by Complex I inhibition (Lamensdorf et al., 2000). The further enzymatic conversion of DOPAL to DOPAC can also damage the cells by producing ROS. CytOplasmic DA can also auto-oxidize non-enzymatically to produce a highly reactive DA-quinone molecule, superoxide (02') and H202 radicals (Figure 1-5). The DA- quinone molecules bind to cysteine residues causing covalent protein modification, conformational changes and loss of function of intracellular proteins (LaVoie and Hastings, 1999b). Neurons are primarily protected from ROS-induced oxidative damage by glutathione (Cooper and Kristal, 1997), which donates an electron to free radicals (02-, OH-, OONO- and H202) and reduces them to less toxic molecules. If there is an increase in ROS formation or decrease in antioxidant proteins, the cell can undergo oxidative damage. The added stress from abnormal DA metabolism, 20 combined with other abnormalities such as mitochondrial or proteasomal dysfunction may make catecholamine neurons susceptible to cell death. 21 Auto-oxidation Enzymatic Breakdown Cytosolic DA l MAO DAqulnone + H202 + 02- DOPAL l 1Fe+2 1N0 1A0 :"dmg OH' OONO' H202 + DOPAC 2 cysteinyl F°+ residues OH- / Damage to cell Figure 1-5. DA non-enzymatic (left) and enzymatic (right) metabolic pathways. The enzymatic metabolism of DA results in the formation of DOPAC and a hydrogen peroxide (H202) molecule which can react with iron to form hydroxyl radical (OH') through the Fenton reaction (Dringen et al., 2000). Non-enzymatic DA auto-oxidation results in the formation of DA- quinone molecules as well as H202 and superoxide (02‘). H202 can produce OH‘whiIe 02' can react with nitric oxide (NO) to form peroxynitrite (OONO'). 22 Genetic cases of PD are consistent with the hypothesis that abnormal metabolism of DA may be involved. For example, a—synuclein and parkin are proposed to be directly involved in regulation of DA metabolism and transmission ((Jiang et al., 2004a; Jiang et al., 2006)), and mutations in the genes for these proteins may cause an imbalance in this tightly regulated system. Furthermore, mitochondrial impairment can affect DA metabolism and therefore the genes involved in mitochondrial function may disrupt normal DA homeostasis and cause further toxicity to the cells. Overall, much evidence suggests that damage from abnormal metabolism of DA may contribute to the selective loss of NSDA neurons in PD. On the other hand, if DA were the main culprit for extensive cell loss in PD, all DA containing neurons in the brain should be affected to a similar extent. However, as evidence presented in the next section will demonstrate, this is clearly not the case. F. Differential susceptibility of DA neurons in PD There are several groups of DA neurons in the brain, located throughout the hypothalamus and midbrain (Figure 1-6). While NSDA neurons undergo the most extensive lesion in PD, other DA neurons in the brain are not affected to the same extent (Ahlskog, 2005). NSDA neurons undergo more extensive degeneration compared to the adjacent MLDA neurons in PD. Based on post-mortem analyses of patients with PD, approximately 80-90% of melanin-pigmented neurons are lost in the SNpc compared to 30-40% in the ventral tegmental area (VTA) (German et al., 1989; Fearnley and Lees, 1991) while melanin-pigmented TIDA neurons do not degenerate in 23 PD (Braak and Braak, 2000). This differential susceptibility is inconsistent with the hypothesis that abnormal DA metabolism underlies the extensive demise of NSDA neurons. Understanding the differences between these distinct neuronal populations and the mechanisms by which they respond to cellular stresses or metabolize cytosolic DA may shed light on protective or deleterious properties that cause the differential susceptibility of these neurons in PD. 24 22 °?-{.‘ 0038. 000 ‘3 a... " "0' .0 1.20 00 00302.2: m... .....v.........—.==V=§_? v03” >§w\\\\§ §§§ ‘1. > 00003.3 , T rue-.0 H m 93:05.0: 0* 0000350 man 30303003230 30:33. 00050203 5 m 3930. 505 0* 30 B» 635. .5 >0 03 3030503230 :33?! >m >5 03 0000350qu 30:30.". no 8.80.4. 8:025: 1.20 n 20008305“ m4 u 2:083 2080. 2.003 m" m... Hmmd. 3030500313 6023.03 man mcxm Home 25 MLDA neurons MLDA neurons are located in the VTA and send axons through the medial forebrain bundle that terminate in the nucleus accumbens (NA) and frontal cortex (Dahlstrom and Fuxe, 1964; Ungerstedt, 1971c, Cooper et al., 2003). MLDA neurons are involved in the control of motivation, emotions and reward and other higher cognitive functions. These neurons have been extensively studied in regards to addiction and reward-based behaviors (Di chiara et al., 2002) as well as schizophrenia and other affective disorders. MLDA neurons are very similar to NSDA neurons in that they have long projecting axons and form classic synapses with target medium spiny neurons of NA or pyramidal cells of the frontal cortex. MLDA neurons utilize the same enzymatic machinery to synthesize, package, and release DA (Figure 1—1). Furthermore, these neurons use the high affinity DAT for reuptake of DA and utilize DA autoreceptors to regulate their activity. Some differences between MLDA and NSDA neurons have been noted and may help explain the differential susceptibility of these neuronal populations in PD. One major difference is that the majority of MLDA neurons express the calcium chelator calbindin, whereas only 30% of NSDA neurons express this protein (Lavoie and Parent, 1991; Parent and Lavoie, 1993; Haber et al., 1995). Neurons that express calbindin, both in SNpc and VTA are less susceptible in PD and animal models of PD (Yamada et al., 1990; Lavoie and Parent, 1991), however a direct cause and effect relationship has not been established between calbindin and cell survival. Furthermore, if indeed 26 protective, the mechanisms underlying the protective properties of calbindin have yet to be elucidated. MLDA neurons also have lower levels of DAT and D2 receptor mRNA when compared to NSDA neurons (Hurd et al., 1994; Haber et al., 1995). On the other hand, MLDA neurons have a higher turnover rate of DA than NSDA neurons as measured by the ratio of DA metabolites to DA (Behrouz et al., 2007), suggesting DA breakdown may be more efficient in these neurons. The resistance of MLDA neurons is unlikely to be due to lower toxin entry via DAT since these neurons also display resistance to lipophillic toxins that freely enter the cells (Betarbet et al., 2000; Behrouz et al., 2007). On the other hand, the differences in the way these neurons handle DA synthesis, uptake and metabolism may contribute to their partial resistance in PD models and PD and require further examination. 27 TIDA neurons TIDA cell bodies are located in the ARC of the hypothalamus and their axons project to ME, where they release DA into the hypophyseal portal blood. DA released into the portal circulation is transported to the anterior pituitary where it tonically inhibits prolactin secretion by activating 02 receptors on pituitary lactotrophs. A portion of the TH positive neurons in the ventrolateral ARC lack L-aromatic amino acid decarboxylase and are not capable of synthesizing DA (Meister et al., 1988). The exact function of these ”DOPAergic" neurons is not known and the remainder of this dissertation will focus on the dorsomedial region of ARC, which projects to ME and releases DA. TIDA neurons possess and utilize the same enzymatic machinery as NSDA and MLDA neurons in order to produce DA from dietary tyrosine (Figure 1-1). These neurons also use the highly efficient VMAT-2 to package DA inside of synaptic vesicles so that it may be exocytosed upon propagation of an action potential. Cytosolic DA that is not packaged is rapidly metabolized by MAO and AD to DOPAC in a similar fashion to NSDA and MLDA neurons. There are two major differences between hypothalamic TIDA neurons and mesotelencephalic NSDA and MLDA neurons. Since TIDA neurons do not form classic synapses with their targets (Figure 1-7), they lack the high affinity DAT but instead utilize a low affinity reuptake system (Demarest and Moore, 1979a; Annunziato et al., 1980). Second, TIDA neurons have much lower expression levels of 02 autoreceptors 28 (Lookingland and Moore, 1984; Gunnet et al., 1987; Weiner et al., 1991) and therefore lack 02 mediated feedback inhibition. 29 COM / DOPAC L MAO TYROSINE ‘—=—; DOPA -—~o u THf ooc“’ TYROSINE poem I \ HQMEQ, TYROSINE DOPA —+ 0 TH DDC A‘\M A0 COMT D BLOOD ——-—> POSTSYNAPTIC CELL BODY OR DENDRITE D] ANTERIOR PIT UITARY Figure 1-7. Differences in the DA transmission, uptake and feedback inhibition in NSDA or MLDA neurons (top) and TIDA neurons (bottom). NSDA and MLDA neurons (top) which form classical synapses with post-synaptic targets, have high affinity dopamine transporter for reuptake of DA, and utilize DZ receptor mediated feedback inhibition. TIDA neurons (bottom) release DA into the hypophyseal portal blood, utilize a low affinity high volume transporter for uptake of DA, and lack DZ receptor feedback inhibition Figure from Lookingland and Moore (2005). 30 Due to similarities between TIDA, NSDA and MLDA neurons, it would be plausible to hypothesize that disruptions due to abnormal DA function would affect the TIDA system in PD. However, unlike other central DA neurons there is no loss of TIDA neurons in the post-mortem PD brain (Langston and Forno, 1978; Braak and Braak, 2000) and the mechanisms underlying this selective resistance to neurodegeneration are not known. As such, characterization of the properties of TIDA neurons compared to other DA neurons that degenerate in PD may elucidate mechanisms that impart protection. The major function of TIDA neurons is to inhibit the release of prolactin from the anterior pituitary. Disrupting TIDA DA synthesis and release, preventing DA access to pituitary lactotrophs, or blocking pituitary 02 receptors, all increase prolactin secretion. TIDA neurons, in turn, are activated by prolactin (Hokfelt and Fuxe, 1972; Lookingland et al., 1987a; Arbogast and Voogt, 1997; Lerant and Freeman, 1998). This serves as a long loop feedback system where prolactin can suppress its own secretion by increasing the activity of TIDA neurons. TIDA neuronal activation by prolactin has two components, a rapid (less than 4 h) activation of TH that does not require protein synthesis and may be associated with the catylitic properties of the enzyme (Pasqualini et al., 1994; Lookingland and Moore, 2005), and a delayed (12-16 h) stimulation of TH which requires ongoing protein synthesis (Arbogast and Voogt, 1991; Lookingland and Moore, 2005). Several studies have used circulating prolactin concentration as an indirect index of TIDA neuronal integrity since loss of TIDA neuronal function stimulates 31 prolactin secretion. A majority of studies indicate that basal levels of prolactin are not altered in PD patients, suggesting that TIDA neuronal function is preserved (Lawton and MacDermot, 1980; Agnoli et al., 1981; Eisler et al., 1981; Thorner and Login, 1981; Vogel and Ketsche, 1986; Kirkpatrick and Tamminga, 1988). G. Animal models of PD Many animal models of PD have been developed to date and yet no single model, either based on neurotoxins, genetic mutations or a combination of the two, recreates all the key features of disease. Each model has its advantages and disadvantages in regards to studying different aspects of this complicated neurodegenerative disorder. Some of the most commonly used models are discussed here. Neurotoxin-based Models Several neurotoxins have been used to model PD, based on the ability of these compounds to induce degeneration of NSDA neurons. The most common toxin models employ MPTP, 6-hydroxydopamine (6-OHDA), rotenone or paraquat. MPTP and rotenone inhibit mitochondrial Complex I and all four toxins cause the formation of ROS. MPTP is the only toxin linked with a human form of parkinsonism, is the most widely studied and will be therefore be most extensively discussed here. 32 AAPTP MPTP is a lipophillic compound and upon systemic administration penetrates the blood brain barrier and enters the brain. Once inside the brain, the majority of MPTP is taken up by glial cells due to the high number of these cells compared with neurons (Markey et al, 1984). Inside glial cells, MPTP is converted by MAO to 1-methyl, 4—phenyl, 2,3, dihydropyrinium (MPDP) and oxidized to its active and toxic metabolite 1-methyl-4-phenylpyridinium (MPP+). As depicted in Figure 1—8, at modest doses MPP+ causes toxicity to DA neurons by gaining access to the cell via DAT and depleting ATP stores via binding to mitochondrial Complex I as well as producing ROS (Chan et al., 1991; Zang and Misra, 1993). MPP+ can also serve as a substrate for MAO inside these DA neurons and result in inhibition of the enzymatic conversion of DA into DOPAC (Takamidoh et al., 1987). In addition, MPP+ can be taken up into vesicles by VMAT-2, thereby displacing vesicular DA into the cytoplasm (Reinhard et al., 1987; Liu et al., 1992). 33 @- Blood-brain barrier A /i \/ \ fisporter l \ ® (\ A / Ymfim MAO-B @7‘ \0‘ Figure 1-8. Diagram describing distribution, bioactivation and site of action of MPTP. 1- Methyl, 4-phenyl, 1,2,3,6—tetrahydropyridine (MPTP), a lipophillic molecule rapidly crosses the blood brain barrier and is metabolized, primarily by glial cells, to its toxic and active metabolite MPP+. 1-methyl-4-phenylpyridinium (MPP+) can then enter dopaminergic neurons through the dopamine (DA) transporter. Once inside the cell, MPP+ can inhibit Complex I of the mitochondrial electron transport chain, disabling ATP production and causing the formation of reactive oxygen species. MPP+ can also be sequestered inside of synaptic vesicles, thereby displacing DA into the cytoplasm. Figure from Villa and Przedborski (2003). 34 Acute, sub-chronic and chronic MPTP administration regimens have been used to model different stages of PD, ranging from a single dose to chronic administration via osmotic minipumps over an extended period of time. All these dosing regimens cause loss of DA from the axon terminal region of NSDA neurons followed by retrograde cell death. The extent and mechanism of cell loss, however, depends on the dosing regimen. Paradigms using high daily doses (e.g. acute model with a total of 80 mg/kg MPTP over 4 injections in one day) cause severe, rapid and necrotic cell death. On the other hand, regimens employing repeated exposure to low daily doses (25 mg/kg per day or less) cause less rapid and primarily non-necrotic cell death (Bezard et al., 1997; Tatton and Kish, 1997; Petroske et al., 2001; Smeyne and Jackson-Lewis, 2005). The lower repeated dosing regimen better resembles PD where progressive non-necrotic cell death occurs (Mochizuki et al., 1996; Anglade et al., 1997). Chronic or sub-chronic treatment with MPTP also produces motor dysfunction, and in some cases of prolonged treatment, Lewy body—like inclusions (Petroske et al., 2001). Decreased UPS function has also been reported with MPTP treatment (Fornai et al., 2005), further implicating the, mitochondrial and UPS pathways in the etiology of PD. While NSDA neurons are severely damaged, TIDA neurons are resistant to modest acute doses of MPTP or MPP+ when administered either systemically or centrally (Melamed et al., 1985; Sundstrom et al., 1987; Willis and Donnan, 1987; Mogi et al., 1988). The complete resistance of TIDA neurons to MPTP toxicity is postulated to be due exclusively to extrinsic factors. It is generally believed that decreased MPP+ uptake into these neurons due to lower expression of DAT may underlie the resistance 35 of TIDA neurons. Others have suggested that differential distribution, reduced bio- activation by astrocytes, or sequestration of MPP+ into vesicles may be responsible for the resistance of TIDA neurons (Hirsch, 1992; Hirsch et al., 1997; Uhl, 1998). Although the MPTP mouse model mimics several aspects of PD, other aspects of the disease are not replicated in this model. These characteristics include the lack of progressive neurodegeneration after termination of MPTP administration and the lack of Lewy body formation in most regiment dosages of MPTP. Interestingly, people who injected themselves with MPTP have progressive NSDA loss of function (Vingerhoets et al., 1994) suggesting the lack of progressiveness of this model may be due to species differences. The absence of progressive degeneration remains a concern in most other toxin models as well. With its high level of specificity to DA neurons, ease of use, and reproducibility from rodents to primates, MPTP remains the most widely used toxin model of PD. Rotenone Rotenone, commonly used as an insecticide and fish poison, can bind to and inhibit the activity of Complex I of the electron transport chain. Rotenone’s lipophillic property allows it to easily penetrate the blood-brain barrier and enter all cells in the brain, where it accumulates in mitochondria and binds to nicotinamide adenine dinucleotide (NADH)—ubiquinone reductase (Complex I) and inhibits the transfer of electrons, decreases ATP and increases ROS production from mitochondria (Betarbet et aL,2002) 36 Chronic treatment of rats with rotenone causes relatively selective loss of NSDA neurons and formation of Lewy body-like inclusions (Betarbet et al., 2000; Thiffault et al., 2000). A loss of UPS function has also been reported in rotenone treated rats, connecting the mitochondrial and UPS pathways in this model of PD. These pathological changes are accompanied by motor dysfunction such as abnormal animal posture and decreased spontaneous movement (Betarbet et al., 2000; Thiffault et al., 2000) Differential susceptibility amongst DA neuronal populations has also been demonstrated in the chronic rotenone model, with NSDA neurons as the most susceptible, MLDA neurons less susceptible, and no loss of TIDA neurons or axon terminals (Betarbet et al., 2000; Behrouz et al., 2007). These features make rotenone an attractive model; however several major limitations have hindered the widespread use of this toxin for the study of PD. Although this model has been reproduced by several groups (Betarbet et al., 2000;. Behrouz et al., 2007), the NSDA lesions have been consistently reported to be present in only 50% of the animals that received rotenone, the severity of the NSDA lesion was highly variable, and there was non-selective degeneration in other neuronal types not usually affected in PD (Hoglinger et al., 2003b). In addition, the use of rotenone in vivo is technically challenging requiring survival surgery and implantation of an osmotic minipump, and while this model has been characterized in rats, there have been no successful reports of its use in mice. 37 6-0HDA 6-OHDA was used to model PD 40 years ago and was the first neurotoxin to produce selective degeneration of NSDA neurons (Ungerstedt, 1968). 6-OHDA is a hydroxylated analogue of DA (Blum et al., 2001) and selectively damages DA neurons after its entry into cells via DAT and norepinephrine transporter. Once inside the cell, 6- OHDA can accumulate in the cytosol and damage the cell by formation of quinone molecules and ROS (Przedborski and lschiropoulos, 2005). 6-OHDA can also inhibit mitochondrial Complex l (Betarbet et al., 2002; Schober, 2004). However, it is unclear whether Complex I inhibition is due to a direct binding of 6-OHDA to the enzyme, or indirect effect of ROS damage to mitochondria. Intracranial 6-OHDA injection produces a selective, potent, rapid and reproducible degeneration of NSDA neurons (Jeon et al., 1995). One advantage of using 6-OHDA is that it can be unilaterally injected in order to kill NSDA neurons on only one side of the brain, allowing the contralateral side of the same brain to be used as a control, as well as creating an asymmetric turning behavior to the contralateral side in response to DA agonist administration that correspond to the extent of the lesion (Ungerstedt, 1968). As such, quantification of the circling provides an in vivo index of the NSDA lesion severity. Disadvantages of this model include the lack of specificity for dopaminergic neurons and the rapid necrotic cell death, which does not mimic the slow, progressive, apoptocic degeneration observed in PD (Luthman et al., 1989; Jeon et al., 1995). 38 Furthermore, due to its inability to cross the blood brain barrier, 6-OHDA must be stereotaxically injected into the SNpc, medial forebrain bundle or ST to damage NSDA neurons. Paraquat The herbicide paraquat has also been used to model PD. Paraquat is structurally similar to MPP+ and enters DA neurons via DAT (Shimizu et al., 2001; Shimizu et al., 2003). Once inside the cell, paraquat can cause ROS formation and damage cells. Systemic paraquat administration leads to the destruction of NSDA neurons (Manning-Bog et al., 2002; McCormack et al., 2005). This loss of neurons is reported to be accompanied by the formation of Lewy body-like inclusions. This model, however, has not been well characterized and it is unclear whether the selectivity, time course and mechanism of cell death mirror that of PD (Thiruchelvam et al., 2000). Genetic and gene transfer models In addition to the toxin based models, several genetic models have been developed with null mutations (knock outs; KO), one extra copy of the gene, or point mutations of the genes implicated in PD pathogenesis. These models have provided information about the function of these genes as well as how these functions may relate to PD. Furthermore, combination of genetic and toxin models can mimic gene/environment integrations in PD. Gene transfer models have also been used to 39 knock down (KD), over express, or express WT or mutated versions of genes in adult animals, which have proven useful in bypassing developmental genetic compensation that occur in KO models (Periquet et al., 2005). These genetic models and their implications in elucidating the etiology of PD are discussed below. a-Synuclein a—Synuclein is primarily found in neural tissue and is named for its cellular localization in the pre-synaptic axon terminal regions as well as in nuclei. Several point mutations (A53T, A30P, E46K) as well as duplication and triplication of the gene result in autosomal dominant PD (Polymeropoulos et al., 1997; Kruger et al., 1998; Zarranz et al., 2004). While the functions of a-synuclein are not well understood, it’s involvement in several pathways including chaperone function, synaptic plasticity (Abeliovich et al., 2000; Murphy et al., 2000; Cabin et al., 2002), and regulation of reserve vs. readily releasable pool of vesicles (George et al., 1995) have been demonstrated. a—Synuclein is normally unfolded, but under certain conditions such as during oxidative damage it can produce protofibrills and aggregates and is found as a major component of Lewy bodies (Spillantini et al., 1997; Lee and Trojanowski, 2006). Animals with varying expression levels of a—synuclein have been created and have aided in our understanding of the functions of this protein and how it may relate to sporadic PD. 40 a—Synuclein K0 or KD Several lines of a-synuclein KO mice have been generated that are viable and show little phenotypic abnormalities. While one line of a-synuclein KO mice has a small decrease in ST DA levels and attenuated amphetamine—induced locomotor response (Abeliovich et al., 2000), others have no disruption in locomotor activity following amphetamine and normal ST DA levels (Specht and Schoepfer, 2001; Cabin et al., 2002; Schluter et al., 2003). A small upregulation of B-synuclein was observed in the ST in some but not all KO mice, suggesting possible genetic compensation (Schluter etaL,2003) a-Synuclein KO mice are resistant to NSDA cell death after either acute or chronic exposure to MPTP (Dauer et al., 2002; Drolet et al., 2004b). The axon terminal regions of NSDA neurons are also partially resistant to MPTP treatment compared to WT littermates (Drolet et al., 2004b). These findings are consistent with the hypothesis that higher levels of a—synuclein are associated with increased risk of PD. While gene transfer approaches have been developed to silence the expression of a-synuclein, this has not been combined with other methods in order to determine if transient knock down of the gene can be protective against various toxins or in PD. Overexpression of WT or Mutant a-Synuclein Several a—synuclein transgenic mice have been also described to date, most utilizing a cDNA construct with a heterologous promoter. Overexpression of WT gene recapitulates some features of PD including translocation of a-synuclein into the cell 41 body from its normal axonal and pre-synaptic location (Kahle et al., 2000; Masliah et al., 2001; Matsuoka et al., 2001; Rockenstein et al., 2002), accumulation of fibrillar accumulation of a-synuclein (Kahle et al., 2000; van der Putten et al., 2000; Masliah et al., 2001), reduced dopaminergic nerve terminal density in the ST and some motor abnormalities (Masliah et al., 2000; van der Putten et al., 2000; Masliah et al., 2001; Fleming et al., 2004). However, loss of NSDA neurons does not occur in a—synuclein overexpressing mice (Matsuoka et al., 2001), making this model less than ideal for studying the etiology of PD. Mice expressing the human A30P mutant a—synuclein, where an alanine is replaced with a proline, also display mislocation of a-synuclein to the cell body (Kahle et al., 2000; Matsuoka et al., 2001; Lee et al., 2002a; Gomez-Isla et al., 2003) and with the exception of one model, do not display fibrillar inclusions (Kahle et al., 2000). These mice have also been shown to have altered short-term hippocampal synaptic plasticity (Steidl et al., 2003), increased gliosis, and motor dysfunction (Gomez-Isla et al., 2003). However, none of these transgenic lines display NSDA neuron loss, even in aged animals. Mice expressing the human A53T a-synuclein mutation, where an alanine is replaced with a threonine, seem to be the most affected (Kahle et al., 2000; Matsuoka et al., 2001; Lee et al., 2002b; Gomez-Isla et al., 2003). The AS3T transgenics show the abnormal translocation of a—synuclein to the cell bodies (van der Putten et al., 2000; Giasson et al., 2002; Lee et al., 2002b; Gispert et al., 2003) and show drastic and progressive motor abnormalities (van der Putten et al., 2000; Giasson et al., 2002; Lee 42 et al., 2002b). These mice also have accumulation of non-fibrillar and fibrillar a- synuclein and ubiquitin (van der Putten et al., 2000; Giasson et al., 2002; Lee et al., 2002b). Other deficits observed in these mice include mitochondrial DNA damage including reduced Complex IV activity (Martin et al., 2006). As with other a—synuclein transgenics, even high expression of AS3T a-synuclein does not induce NSDA cell death. Interestingly, mice that express both A53T and A30P mutations do not show exacerbated pathological or motor phenotypes in comparison to the single mutant models described above (Richfield et al., 2002). Transgenic mice expressing the A53T or A30P mutation are more sensitive to lower doses of MPTP (Nieto et al., 2006; Yu et al., 2008). Moreover, treatment with the pesticides paraquat and maneb increases inclusion formation in AS3T transgenics (Norris et al., 2007) and worsens behavioral defects without causing cumulative NSDA cell loss (Fernagut et al., 2007). These data suggest increased levels of mutated a—synuclein may render these neurons more susceptible to toxicity. However, both the WT and AS3T transgenic mice are resistant to paraquat toxicity (Manning-Bog et al., 2003). Viral gene delivery in adult animals has provided models that include NSDA neuronal cell loss. Adeno- or lenti-viral delivery of human WT and mutant (A30P or A53T) a-synuclein to the SNpc of rats and primates leads to DA neurons loss as well as neuritic pathology and a—synuclein inclusions (Kirik et al., 2002; Klein et al., 2002; Lo Bianco et al., 2002; Kirik et al., 2003; Lauwers et al., 2003; Yamada et al., 2004). Two rat models also demonstrated drug-induced rotational behavior (Kirik et al., 2002; Lauwers et al., 2003). WT and AS3T a—Synuclein delivery into the striatum and the 43 amygdala of mice using a lentivirus causes changes similar to those seen after nigral delivery, suggesting that the Lewy-like pathology and neurodegeneration are not restricted to DA cells (Lauwers et al., 2003). The fact that this degeneration is not specific to neurons that degenerate in PD suggests the expression levels of a-synuclein may be too high to be physiologically relevant. LRRK2 Over 20 LRRK2 mutations have been identified, and these are the most common known cause for familial PD (Farrer, 2007) and are clinically indistinguishable from sporadic PD. LRRK2 encodes for a large protein of 2052 amino acids with several functional domains including a kinase, GTPase, W040, and leucine rich repeat domains. These various domains suggest that the protein has many functions, which include kinase and GTPase activity as well as vesicular trafficking and membrane recycling (Biskup et al., 2007). It is, however, unclear how the normal function of the protein would relate to PD, as the mutations may cause gain of function rather than a disruption in the normal role of the protein. While LRRK2 K0 and transgenic mice have been recently generated, there has only been one report of a transgenic mouse line that has been published in peer- reviewed journals (Melrose et al., 2007). The non-peer reviewed data presented at professional meetings suggests that mice overexpressing WT do not have nigral abnormalities or phenotype. There have been no reports of toxin treatment of LRRK2 transgenic or KO mice. Parkin Mutations in parkin are currently recognized as one of the most common causes of early onset Parkinsonism. Parkin aids in the UPS by acting as an E3 ubiquitin ligase. Recent data, however has demonstrated that parkin also has the ability to alter the function of proteins via mono-ubiquination. The ability of parkin to assemble both K48- and K63-linked chains under different conditions is apparently governed by members of the E25 it recruits (Lim et al., 2005). Parkin KO Several spontaneous and targeted parkin KO mice have been described (Goldberg et al., 2003; Lorenzetti et al., 2004; Perez et al., 2005). Homozygous ’quaking mice’ with spontaneous deletion of parkin and parkin co—regulated gene (PACRG) show locomotor abnormalities, although these motor abnormalities have been attributed to the neighboring locus (Lockhart et al., 2004; Lorenzetti et al., 2004). Interestingly, targeted KO mice deficient in parkin have normal DA neuron numbers and lack major phenotypic or pathological abnormalities. Subtle abnormalities include increased levels of extracellular DA, reduced synaptic excitability in ST (Goldberg et al., 2003) and changes in energy metabolism, synaptic function and protein handling (Periquet et al., 2005) have been observed. Parkin KO mice are not more sensitive to treatment with 6- OHDA, methamphetamine (Perez et al., 2005), or MPTP (Thomas et al., 2007b). 45 Overexpression of Parkin Adenoviral delivery of parkin cDNA ameliorates a—synuclein-induced DA neuron loss and consequent motor dysfunction (Yamada et al., 2005). Furthermore, viral delivery of parkin or DJ1 protected against MPTP-induced NSDA cell death (Paterna et al., 2007). Pinkl Mutations in pinkl cause autosomal recessive P0 with a similar phenotype as parkin mutants including early onset and prolonged disease duration. The serine/threonine kinase domain and a mitochondrial localization signal in the pinkl protein make it relatively clear that it is a mitochondrial associated kinase (Cookson et aL,2007) Pinkl KO mice have impaired mitochondrial respiration in the ST (Gautier et al., 2008), abnormal DA release and synaptic plasticity in ST (Kitada et al., 2007) but no loss of DA neurons or axon terminals. Furthermore, silencing pinkl by conditional RNA interference does not produce DA neuronal loss in mice (Zhou et al., 2007). Pinkl silencing by RNA interference in SNpc of mice increased neuronal toxicity induced by MPP+ (Haque et al., 2008), suggesting a combination of genetic and environmental factors may contribute to NSDA cell death. Furthermore, WT Pink1, but not the G309D mutant which is linked to familial P0 or a kinase—dead mutant, protect neurons against MPTP (Haque et al., 2008), suggesting mutated pink1 loses its protective properties. 46 011 PD patients with mutations in the DJ-1 gene are phenotypically similar to those with parkin or pink1 mutations, with generally early onset (30—50 yrs) and long disease duration. The normal function of the gene is unknown, but is thought to play a role in maintaining cell survival after exposure to oxidative stress (Bonifati et al., 2003b). DJ1 has been shown to bind to RNA targets (e.g. mitochondrial genes, genes involved in glutathione metabolism and members of the PTEN/PI3K cascade) under normal, but not oxidative stress conditions (van der Brug et al., 2008). DJ-1 KO mice have been generated and have age-dependent and task- dependent motor deficits. While these behavioral deficits are accompanied by changes in ST DA reuptake rates and elevated DA content, no loss of DA neurons was observed (Chen et al., 2005). DJ-1 KO mice treated with MPTP showed increased DA neuron loss and striatal terminal density compared to WT littermates (Kim et al., 2005). This increased sensitivity is mitigated by adenoviral delivery of DJ-1. Interestingly, WT mice also receiving DJ-1 vector were resistant to MPTP damage. Overall, genetic models of PD have not been successful at reproducing the NSDA cell loss observed in PD, even with high expression levels of mutated forms of dominant or complete KO of recessive genes. This lack of cell loss may be due to differential aging processes between mice and humans (i.e. an aged mouse may not necessarily represent an old person). Another pitfall may be that these transgenic mice, which are kept in very controlled laboratory environments, are not exposed to 47 environmental toxins and stressors. Indeed, the few models that have combined genetic and toxin models have been most successful at reproducing most of the pathology seen in PD. G. Dissertation Objective The experiments described herein utilize neurochemical, histological, biochemical and molecular biology techniques to test the specific hypotheses listed under the following specific aims with the central overarching hypothesis that discovering factors that protect resistant neurons will lead to development of therapeutic approaches to halt or slow the progressive loss of NSDA neurons in PD. 1. Does the differential susceptibility among NSDA, MLDA and TIDA neurons exist in the chronic MPTP mouse model? Hypothesis: NSDA neurons are most susceptible, MLDA neurons are partially susceptible and TIDA neurons are unaffected by chronic treatment with MPTP. 2. Do NSDA, MLDA and TIDA neurons all initially respond to MPTP treatment with loss of DA and the resistant neurons recover? If so, is this recovery dependent on protein synthesis? Hypothesis: All DA neuronal populations lose DA in response to a single injection of MPTP, but TIDA neurons depend on synthesis of new proteins that allow them to recover. 3. Are the genomic responses of NSDA and TIDA neurons different when exposed to MPTP? Hypothesis: Several genes possibly coding for neuroprotective 48 proteins are upregulated in TIDA neurons but not NSDA neurons following treatment with MPTP. Is increased expression of parkin responsible for recovery of TIDA neurons from MPTP- induced DA loss? Hypothesis: Parkin is at least partially responsible for allowing TIDA neurons to recover from MPTP-induced DA loss. 49 Chapter 2. Materials and Methods A. Animals All experiments were conducted in male C57Bl/6J mice purchased from Jackson Laboratories (Bar Harbor, MA) unless otherwise indicated. Animals were housed two to four per cage, maintained in a light-controlled (12 h light/dark cycle) and temperature-controlled (22 i 1°C) room, and provided with food and tap water ad libitum. The Michigan State University Institutional Animal Care & Use Committee approved all experiments using live animals (AUF #s 08/08-123-00 and 01/08-004-00.) Generation of Parkin Knock-Out Mice Heterozygous parkin knock-out (KO) mice were obtained in breeding pairs from Jackson Laboratories and were bred to yield homozygous parkin KOs, heterozygotes (HT), and homozygous wild type (WT) mice (F1). Heterozygous mice were crossed and the offspring of this cross (F2) and further heterozygous (F2 x F2 and F3 x F3) crosses were performed to expand and maintain the colony for the experiments described herein. Parkin K0 and WT littermates were used for experiments after the F6 generation. The viability, fertility and basic biochemical features of these mice have been previously described (Goldberg et al., 2003). Genotyping Genotypes for parkin KO mice, HT and WT littermates were confirmed by polymerase chain reaction (PCR) analysis of genomic DNA. Mouse tail snips were dissolved in 600 pl of nuclei lysis buffer with proteinase K (25 mM EDTA, 50 mM NaCl, 0.8 mg/ml proteinase K) at 55°C for 4 h. RNA contamination was removed by 50 incubation with RNase (0.05 mg/ml) for 30 min. The protein was precipitated with 200ul of protein precipitation solution (4.2 M NaCl, 0.63 M KCL, 10 mM Tris base, pH 8.0) and the mixture was incubated on ice for 5 min, and centrifuged (15,000 x g 10 min) to pellet the protein. The supernatant containing the DNA was placed into a fresh microcentrifuge tube containing 600 pl isopropanol at room temperature. The solution was gently mixed by inversion until white strands of DNA precipitate were visualized. The DNA strands were pelleted by centrifugation at 15,000 x g for 1 min. The supernatant was removed, and the DNA-containing pellet was washed twice with 70% ethanol and resuspended in 100 (II of DNA rehydration solution (10 mM tris-HCL, 1 mM EDTA, pH 7.4). DNA concentration and purity in each sample was determined by measuring the absorbance using a Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). The concentration of each sample was adjusted to 50 ng/ul with DNA rehydration solution. For the PCR reaction, 5 ul of the DNA sample was added to 20 ul of PCR reaction solution (1 mM PCR buffer, 200 mM dNTP, 2 mM MgCl, 0.18 pm/ul oligo primers, 0.4 u/ul taq polymerase; Invitrogen Carlsbad, CA), mixed and placed into the PCR machine. PCR run parameters were 94°C for 5 min, 30 cycles of 94°C, 65°C, 72°C of 1 min each, followed by 72°C for 5 min. The PCR primers used were CCTACACAGAACTGTGACCTGG, GCAGAATTACAG CAGTTACCTGG, and ATGTI'GCCGTCCTCCTTGAAGTCG and have been previously described (Goldberg et al., 2003). Subsequent gel electrophoresis (75 mV, 1 h) on a 1.0% agarose gel was used to separate amplified DNA (10 pl) from each sample. As depicted in Figure 2-1, animal 51 genotype was confirmed for all animals through visualization of the amplified DNA using a UV transilluminator (UVC co., San Gabriel, CA). Only homozygous parkin KO mice or homozygous WT mice were used in the described experiments. 52 (- Parkin Figure 2-1. Genotyping of wild-type (WT), homozygous parkin knock-out (KO), heterozygous (HT) mice. Ethidium bromide stained 1% agarose gel following electrophoresis of PCR amplification products the Parkin gene and the green fluorescent protein (GFP) construct. 53 B. Drugs All drugs were purchased from Sigma-Aldrich (St. Louis, MO). Doses were calculated from the free base of the respective drug. 1-methyl, 4-phenyl, 1,2,3,6- tetrahydropyridine (MPTP), GBR-12909 (GBR), and cycloheximide were dissolved in 0.9% sterile saline. Dipropylsulfamoyl-benzoic acid (probenecid) was dissolved in 0.1 N NaOH and the pH was adjusted to 7.4 using hydrochloric acid (HCL). Chronic MPTP Male 8—12 week old mice received injections of either vehicle (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) every 3.5 days, for a total of 10 injections over 5 weeks. Probenecid (250 mg/kg; i.p.) was administered 1 h before each MPTP injection to increase the plasma and central nervous system half-life of MPTP and MPP+, respectively (Petroske et al., 2001). Probenecid alone does not have an effect on NSDA neurons (Lau et al., 1990). All experiments using MPTP were performed using previously published safety guidelines (Przedborski et al., 2001). Animals treated with MPTP were kept in a separate room and conditions (e.g. light/dark cycle, temperature, etc) were monitored and were similar between the rooms. Experiments were terminated 3 weeks after the last MPTP injection. giggle Injection MPTP Male 8-12 week old mice received a single injection of either vehicle (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.). For the time course study, the experiment was terminated 4, 8, 16 or 32 h after the MPTP injection. Saline treated animals were killed 8 h after injection and were used as zero time controls. 54 Cycloheximide Male 8-12 week old mice were treated with 4 injections of vehicle (10 ml/kg; i.p.) or the protein synthesis inhibitor cycloheximide (120 mg/kg; i.p.) every 2 h, with the first cycloheximide injection 15 min prior to a single injection of MPTP. GBR-12909 Male 8-12 week old mice were treated with an injection of either vehicle (10 ml/kg; i.p.) or the DAT blocker GBR (10 mg/kg; i.p.) 30 min prior to a single injection of MPTP. The experiment was terminated 4 h after MPTP treatment. Lentiviral shRNA Aqainst Mouse Parkin Transduction-ready lentiviral particles expressing 1 of 3 parkin shRNA sequences under the control of a U6 promoter were purchased (106 TU/ml; Sigma) and were stereotaxically injected bilaterally into the ARC. A non-target shRNA sequence ' was used as control. Animals were treated with a single injection of MPTP 3-5 weeks after injection with the lentivirus and killed 16 h later. Table 2-1 lists the shRNA sequences for parkin and non-target shRNA. 55 Lentiviral Particles Catalog ii Sequence ParkZ shRNA TRCN0000041143 CCGGCGTTTCATTATCTGCAACTTTCTCGA GAAAGITG CAGATAATGAAACGTTTTTG Park2 shRNA TRCN0000041145 CCGGCGGAGGATGTATGCACATGAACT C GAGTTCATGTG CATACATCCTCCGTTI'TTG Park2 shRNA TRCN0000041147 CCGGGACCT GGAACAACAGAGTATTCT C GAGAATACT CT GTI'GTTCCAGGTCTTITT G Non-target sh RNA SHC002 CCGGCAACAAGATGAAGAGCACCAACT C GAGTTGGTG CTCTTCATCI'TGTTG‘ITI'I'I' Table 2-1. Sequence and catalog information for parkin and non-target shRNA lentiviral particles purchased from Sigma. 500 nl of each virus containing parkin shRNA was bilaterally injected into the ARC of mice in order to knock down expression of parkin. Non-target shRNA lentivirus was bilaterally injected into ARC of mice that were used as controls. 56 C. Tissue Preparation At the appropriate time following drug administration, mice were killed by decapitation and their brains were rapidly removed from the skull and placed on a glass dissecting stage over ice. Under a dissecting microscope, ME was collected. For the chronic MPTP experiments, the brain was bisected along the mid-sagittal axis and half of the brain was drop-fixed for immunohistochemical analysis, while the other half was frozen rapidly on dry ice for neurochemical and Western blot analyses. In all other experiments, the whole brain was quickly frozen on dry ice after the dissection of ME. Consecutive coronal sections (500 um) were prepared from the frozen brains using a cryostat set at -10 °C (CTD-Model Harris, International Equipment Co., Needham, MA). Sections were collected through the rostro-caudal extent of the brain beginning approximately 2.5 mM anterior to Bregma (Paxinos and Watson 1986), thaw-mounted onto glass slides, and immediately refrozen. The regions of interest were dissected using a modification of the method described by Palkovits (Palkovits, 1973, 1978). Figures 2-2 and 2-3 demonstrate the location where tissue samples were obtained. Using a dissecting microscope, bilateral tissue punches from NA and ST were removed using 22- and 18—gauge round punch tools, respectively (Figure 2-2). One punch from ARC was obtained using an 18-gauge oval punch tool, and bilateral tissue punches from VTA and SNpc were collected using 18-guage round and oval punch tools, respectively (Figure 2-3). These tissue samples were used for neurochemical, Western blotting or RNA analysis and were processed according to the appropriate protocols described below. 57 Figure 2-2. Diagrams of coronal brain sections illustrating the location of micropunches used to dissect NA and ST. The brown circles indicate the location of the Palkovits micropunch. NA was dissected (top) from a 500 uM section 1.42 mm rostral to Bregma and ST was microdissected (bottom) from a 500 uM section 0.98 mm rostral to Bregma (Paxinos and Watson, 1986). 58 Figure 2-3. Diagrams of coronal brain sections illustrating the sections used to dissect ARC (top), VTA and SNPC (bottom). The brown circles indicate the location of the Palkovits micropunch. ARC was dissected (top) from a 500 (Ml section 1.94 mm caudal to Bregma and SNpc and VTA were microdissected (bottom) from a 500 (M section 3.08 mm caudal to Bregma (Paxinos and Watson, 1986). 59 D. Neurochemical Analyses Microdissected brain regions were placed into cold tissue buffer (0.1M phosphate-citrate buffer pH 2.5) for neurochemical analyses and stored frozen at -20 C. On the day of assay, samples were thawed and sonicated with 3 one second bursts (Heat Systems Ultrasonics, Plainview NY). Protein was pelleted by centrifugation at 16,000 x g (Beckman Coulter Microfuge, Palo Alto, CA) for 1 min. The supernatant containing the neurochemicals of interest was removed using a 100 pl Hamilton syringe, placed into afresh tube, and used for high pressure liquid chromatography with electrochemical detection (HPLC—EC) analysis. The pellet from the initial centrifugation containing the protein was resuspended and dissolved in 100 pl of 1 N sodium hydroxide (NaOH) by sonication. The Lowry method was used to determine protein concentrations of the tissue pellets (Lowry et al., 1951). Protein samples were transferred from the microcentrifuge tube to 12 x 75 glass culture tubes. Standard solutions with 6.25, 12.5, 25 or 50 ug of bovine serum albumin (BSA; Sigma) were dissolved in 100 pl of 1 N NaOH and transferred to individual 12 x 75 culture tubes. To each tube, 1.0 ml of Reagent A (0.2 M sodium carbonate, 40 mM cupric sulfate, 70 mM sodium potassium tartrate) was added and mixed by vortexing. The mixture was incubated at room temperature for 10 min, after which 100 (II of Reagent B (Folin reagent diluted 1:2 with dHZO) was added to the culture tubes. The mixture was vortexed and incubated at room temperature for 30 min. Protein concentrations were quantified by comparing the absorbance of each 60 sample, read at 700 nm on a Gilford spectrophotometer (Gilford Instruments, Oberlin, OH), to the absorbance of the known protein standards assayed concurrently. The content of the neurochemicals norepinephrine (NE), DA, DOPAC, HVA, 5- HT, and 5-HIAA were determined with HPLC-EC using a Waters 515 HPLC pump (Waters Corporation, Milford, MA) with a flow rate of 1.0 ml/min and an ESA Coulochem 5100A electrochemical detector with an oxidation potential of +0.4V. Neurochemical standards containing 1.0 ng of the desired neurochemicals and experimental samples were injected onto a C18 reverse phase analytical column (Bioanalytical Systems, West Lafayette, IN). The HPLC-EC mobile phase (0.5M sodium phosphate, 0.03M citrate, 0.1 mM EDTA, sodium octylsulfate, 15-20% methanol, pH 2.5) was adjusted by altering the concentrations of sodium octylsulfate and methanol to optimize neurotransmitter peak resolution. Neurotransmitter content was quantified by comparing the peak heights of each sample to the peak heights of standards. To correct for differences in sample size the neurotransmitter content was normalized to the amount of protein in each sample and expressed as a concentration in ng/mg protein. E. Western Blot Analyses Protein Extraction Microdissected brain samples were placed into cold homogenization buffer (320 mM sucrose, 5.0 mM HEPES, with Complete Mini Protease Inhibitor Cocktail Tablets, Roche Diagnostics, Mannheim, Germany, pH 7.4), homogenized by sonication 61 and centrifuged (1000 x g 10 min). The supernatants containing total cytoplasmic protein were removed and placed into fresh microcentrifuge tubes. The samples were assayed for protein content using the bicinchoninic acid protein method (BCA, Sigma). Ten ul of loading buffer (250 mM Tris base, pH 6.8, 20% glycerol, 5% 505, 0.01% bromophenol blue) was added to the appropriate amount of sample and the mixture was vortexed, boiled at 95°C for 10 min to denature the proteins, and cooled to 4°C. E lectroghoresis/T ransfer Conditions Two 15% polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were placed in the inner chamber of a mini-protean-3 electrophoresis cell (BioRad). The inner chamber was filled with Laemli running buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) and samples were loaded onto the gel. The volume was adjusted to load 20 ug of protein for each sample. A protein ladder of known molecular weights was also loaded onto each gel. Proteins were separated by applying a 100 mV current for approximately 1 h. Proteins were transferred to 0.45 pm nitrocellulose membranes (Fisher Scientific, Pittsburgh, PA, USA) by electrophoresis at 30 mV for 12 h. Protein Detection and Quantification Nitrocellulose membranes containing the proteins of interest were washed in 25 mM Tris buffered saline (TBS; 4 x 5 min), incubated in blocking buffer (Li Cor, Inc., Lincoln, NE, USA) for 1 h and reacted with primary antibody in blocking buffer overnight at 4 C. The source, dilution, and name of the company supplying each primary antibody used in the experiments are listed in Table 2-2. 62 Antibody Dilution Source Company TH 1:2000 Rabbit Chemicon, Parkin 1:1000 Rabbit Cell Signaling Bill tubulin 1:5000 Mouse Chemicon GAPDH 1:10000 Mouse Chemicon Table 2-2. Description of primary antibodies used in the experiments in this dissertation. Antibody characterization is based on the standard dilution used for Western blotting, animal used to generate the antibody (Source) and the company the antibody was purchased from (Company). TH: tyrosine hydroxylase, GAPDH = glyceraldehyde 3-phosphate dehydrogenase. 63 Nitrocellulose membranes were washed with TBS (3 x 5 min) and incubated with the appropriate IRDye 800-conjugated goat anti-rabbit (Rockland, Gilbertsville, PA, USA) or Alexa Fluor 680—Iabeled goat anti-mouse (Molecular Probes, Eugene, OR, USA) secondary antibodies (1:15,000 dilution in blocking buffer) for 1 h at room temperature. Membranes were washed in TBS (4 x 5 min) and bound antibodies were visualized with the Odyssey infrared imager (Li-Cor Biosciences, Lincoln NE). The density of each band was quantified by measuring the infrared absorbance using the Odyssey infrared imager and Odyssey software (version 3.0, Li-Cor Biosciences). Relative density was obtained by normalizing the band density of the protein of interest to that of the control protein used to account for variations in loading of samples onto the gel. GAPDH was used as the control protein and its detection and visualization was linear. Figure 2-4 demonstrates that expression levels of GAPDH are similar amongst the compared regions (ARC and SNpc) and do not change with treatment (Control and MPTP-treated). Each nitrocellulose membrane contained representative samples from all experimental conditions. 64 14° ' DSaline IMPTP 120 - 100 - J2 g’ 80 - 3 I E 60 < - ID 40 - 20 - 0 ARC SNpc Figure 2-4: GAPDH levels in ARC and SNpc of control and MPTP treated mice. Mice were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. ARC and SNpc were microdissected and expression of GAPDH protein was determined using Western blot analysis. 65 F. Immunohistochemistry Immunohistochemical staining was performed to visualize and count the number of TH immunoreactive cells in ARC, SNpc and VTA. Following removal from the skull, brains were dissected along the mid-sagittal axis and drop-fixed in 4% paraformaldehyde for 7 days and cryoprotected in 20% sucrose. Coronal sections (60 um) through the hypothalamus and midbrain (beginning at Bregma —2.5 through — 3.7mm; Franklin and Paxinos, 1996) were prepared with a -19°C cryostat using Multiblock'“ processing (Neuroscience Associates, Knoxville, TN). Immunohistochemistry was performed on free floating sections using a primary rabbit anti-TH antibody (Chemicon, Temecula, CA), followed by a biotin-conjugated, goat anti- rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA). Bound peroxidase was visualized with 0.05% 3-3’-diaminobenzidine tetrahydrochloride with 0.01% hydrogen peroxide using an ABC Elite kit (Vector Laboratories, Burlingame, VT). Alternate sections were stained with Nissl to determine if decreases in TH- immunoreactive (IR) cell counts corresponded with loss of neurons. G. Unbiased Stereological cell counting Using the Stereolnvestigator software (version 4.03, MicroBrightfield, Inc. 2000), sections were viewed on the screen at low magnification (4X), ARC, SNpc and VTA were delineated from the rostral through the caudal extent of each nuclei. The first plane was a randomly chosen section within 180 pm of the most rostral plane of 66 the ARC, SNpc or VTA as determined by a mouse atlas (Franklin and Paxinos, 1996). The delineated sections were evenly spaced apart (240 um). TH-IR and Nissl stained neurons were counted using the Optical Fractionator method, an unbiased stereological technique that employs a systematic, random strategy in which cells in a defined region are sampled. This method results in a quantifiable estimate of the total population of cells within a given region (West et al., 1991; Schmitz, 1997; Schmitz and Hof, 2000, 2005). The 3-dimentional probe used to sample the entire ARC, SNpc or VTA yields counts that are independent of cell shape and size or conformational changes in the tissue. A counting frame (60x60um) was utilized and a fraction of the delineated cells were sampled. Cell counts were made through 78% (14 um) of the total depth of the tissue (18 um) allowing for a 2 pm guard zone at the top and bottom of each section. TH-IR and Nissl stained cells were identified with a consistent distribution over the depth of the counting frame, thus confirming sufficient reagent penetration throughout the thickness of the tissue slice. TH-IR or Nissl counts were performed using a 60X objective and cells were counted only if the top of the nucleus came into focus within the virtual counting frame and within the delineated region of interest. Neurons were counted only if they showed TH-IR or Nissl staining within the cell body. For Nissl stained sections, cells that were larger in size (>15 um in diameter) and had a defined nucleus were counted as neurons. The smaller cells (<15 pm in diameter) that were amorphous in shape and did not have a well-defined nucleus were considered glia and were not counted. For estimates of number of neurons per half brain, the section periodicity was 4 as every 67 fourth section was selected for analysis (West et al., 1991, Schmitz, 1997). The coefficient of error for each estimate was calculated and was less than 0.1 (Gundersen, m=1) (Gundersen and Jensen, 1987). H. Rapid Immunofluorescent staining A rapid staining technique was used to visualize TH-IR neurons so that they could be selectively dissected by laser capture microscopy for RNA analysis. Coronal 10 um frozen sections through the midbrain and hypothalamus were obtained in a cryostat set at -10°C, mounted on slides and kept frozen on dry ice. Sections were rapidly fixed in ice-cold 70% acetone for 1 min and rinsed with PBS with 0.1% Triton X- 100 (PBS-Tx). Sections were incubated for 2 min in mouse anti-TH antibody (1:40; Zymed, CA. Cat # 32-2100) containing 400 U/ml of RNasin RNase inhibitor (Promega, WI. Cat # N2115), rinsed twice with TBS-TX and incubated for 2 min in Alexa Fluor 488 goat anti mouse secondary antibody (invitrogen, CA. Cat # A-11001). Slides were rinsed in PBS, dipped in cresyl violet for Nissl stain, and dehydrated in a 70%, 95% and 100% ethanol gradient followed by xylene. Sections were dried in a fume hood and laser capture microscopy (LCM) was performed within 2 h after staining. Figure 2-5 demonstrates the time course of RNA integrity using this rapid staining technique. This time course demonstrates that leaving section in staining solution for 10 min yields optimal staining without compromising RNA quality. 68 Staining Quality RNA Quality 0 Quality 8 .----—-----—-----------------------—-- l l j l 1 J 15 20 25 30 35 40 Minutes in Staining solution O U" .5 0 Figure 2-5: Time course of RNA degradation during rapid Immunofluorescent staining for TH. Coronal 10 um frozen sections through the midbrain and hypothalamus were rapidly fixed in ice-cold 70% acetone for 1 min and rinsed with PBS with 0.1% Triton X-100 (PBS-Tx). Sections were incubated in primary and secondary antibodies for a total of S, 10, 20 or 40 min. The sections were viewed under a microscope to determine staining quality. RNA was extracted from these sections and its quality was determined using a picochip (BioAnalyzer, AGILENT Technologies). 69 I. Laser Capture Microscopy (LCM) In LCM, a near-infrared power laser microbeam melts a thermoplastic ethyl vinyl acetate membrane which overlays the tissue of interest. The melted membrane sticks to the selected cells, which can be lifted and secured in a microfuge tube containing the RNA extraction solution. The surrounding tissue remains unchanged. This technique, coupled with rapid immunofluorescent TH staining allows for dissection of DA neurons without major contamination from other neuronal types and glia. LCM was performed using a PixCelI II Laser Microdissection System (Arcturus Engineering Incorporated; Mountain View, California, USA). The following parameters were used for dissection of TH-immunoflurescent cells: spot size = 7 pm; power = 45- 85 mW; and duration = 750-1100 us (Greene et al., 2005). After rapid staining, slides containing the sections were cleaned using a prep strip and placed onto the microscope. Capsure HS caps (Arcturus) were loaded onto the LCM and after focusing on the appropriate sections an HS cap was positioned on top of the tissue so that the cells of interest were in the center of the cap. TH-positive cells were collected from specific brain regions as depicted in Figure 2-6. The same HS cap was used to collect 300-500 cells from each brain region per animal. The cap was placed into the alignment tray and 10 ul of extraction buffer was placed into the fill port of the ExtractSure'". The tube was placed into the port, covered with a heating block (preheated to 42 C) and incubated for 30 min at 42 C to extract the cells. The cells were collected at the bottom of the tube by centrifugation for 2 min at 800 x g and were stored frozen at -80 C until RNA extraction. 70 Figure 2-6: Laser capture of tyrosine L , ‘ ,'_- ’ -..-t' - (TH-IR) cells of SNpc. A. TH- IR cells before capture, B. section after cells were captured, C. Cap with captured cells. 71 J. RNA isolation from LCM samples Total RNA was extracted using the Extractsure adapter and PicoPure Isolation Kit (Arcturus) with DNase digestion (Qiagen RNase-free DNase Set). RNA purification columns were incubated with 250 pl of preconditioning buffer for 5 min at room temperature. The cell extract was mixed with an equal volume (50 ul) of 70% ethanol and loaded onto the purification column. The mixture was centrifuged at 100 x g for 2 min to facilitate nucleic acid binding to the column, followed by centrifugation at 16,000 x g for 30 sec to remove flowthrough. The column containing the nucleic acids was washed using Wash Buffer 1. DNase mix solution (5 pl DNase and 35 ul DNase buffer) was added to the column and incubated at room temperature for 15 min to remove column bound DNA. After DNase treatment, the column was washed with Wash Buffers 1 and 2 and transferred to a new 1.5 ml tube. The column was incubated at room temperature with 12 ul of Elution Buffer and the eluted RNA was collected at the bottom of the tube by centrifuging at 1000 x g for 1 min. RNA quality was determined for representative samples from each group using a Bioanalyzer Picochip (Agilent Technologies, Santa Clara, CA). The Bioanalyzer uses a microfluidics-based platform to analyze RNA. The individual samples were examined, a pseudo gel image is created and bands were sized and quantified. Total extracted RNA was stored frozen at -80 C. 72 K. RNA Amplification Total RNA was amplified for each sample using the MessageAmp II aRNA Amplification Kit (Ambion, Austin, TX). First strand cDNA was synthesized using an oligo—dT primer containing the T7 promoter. E-coli DNA polymerase and RNase H were employed to simultaneously degrade the RNA and synthesize second strand cDNA. cDNA was purified to remove RNA, primers, enzymes and salts. In vitro transcription was performed to create multiple copies of aRNA. aRNA was purified to remove unincorporated nucleotides, salts, enzymes and inorganic phosphate. A second round of synthesis was performed as described above. For the in vitro transcription step, biotin labeled UTP (provided with the kit) was used to synthesize biotin labeled aRNA. The samples yielded approximately 400 ng of aRNA after the first round of amplification and approximately 100 ug of aRNA after the second round of amplification. RNA quality was assessed by absorbance readings on a Nanodrop (Thermo Scientific, Wilmington, DE) and RNA was determined to be of good purity (2602280 ratio of around 2.0-2.2). RNA integrity was also determined by running the samples on an agrose gel as well as on a Bioanalyzer picochip. A smear of aRNA bands that extended from 200 - 1000 bases was observed. The RNA was centered on 600 bases, which is consistent with other published records of aRNA after 2 rounds of amplifications (Van Gelder et al., 1990). 73 L. Microarray Hybridization and Initial Analysis In order to determine the differences in mRNA levels in ARC and SNpc of control and MPTP treated animals, amplified RNA from each sample was hybridized to an Affymetrix Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA). Microarray and preliminary analyses were performed by the Michigan State University genomics facility according to the methods specified by Affymetrix. Hybridization solution contained 0.05 ug/ul fragmented cRNA, 50pM control oligonucleotide 82, eukaryotic hybridization controls (bioB, bioC, bioD, 1,5, 5; 25 pM, respectively; Affymetrix, Santa Clara, CA), 0.1 mg/ml of herring sperm DNA, 0.5 mg/ml acetylated bovine serum albumin (BSA; Sigma, St. Louis, MO) in hybridization buffer (100 mM MES, 1M NaCl, 20 mM EDTA, 0.01% Tween20). Before being applied to the microarray the hybridization solution was heated to 95 C, cooled to 45 C, and clarified by centrifugation. Hybridization was carried out at 45°C in a hybridization oven for 16 h. Subsequent washing and staining of the arrays was performed using the GeneChip fluidics station protocol EukGE—WSZ. The GeneChip probe arrays were washed 10X at 25°C with Non Stringent Wash Buffer (0.9 M NaCI, 0.06 M NaHzPO4, 6 mM EDTA, 0.01% Tween20). The second wash consisted of 4 cycles of 15 mixes per cycle with Stringent Wash Buffer (100 mM MES; 2—N-MorpholinoethanesuIfonic acid, Sigma, St. Louis, M0), 0.1 M NaCl, 0.01% Tween-20) at 50 C. The arrays were stained for 10 min in streptavidin-phycoerythrin (SAPE) solution (10 ug/ml SAPE; Molecular Probes, Eugene, OR), 2 ug/ul acetylated BSA (Sigma, St.Lous, MO) in 100 mM MES; 1M NaCl, 0.05% TweenZO at 25 C. The post stain wash consisted of 10 cycles at 25 C in the 74 fluidics station using the non-stringent solution. The probe arrays were treated for 10 min in antibody solution (2 ug/ul acetylated BSA, 0.1 ug/ul normal goat lgG; Sigma), 3 ug/ml goat-anti-streptavidin, biotinylated antibody (Vector Laboratories, Burlingame, CA); in 100 mM MES; 1M NaCl, 0.05% Tween20) at 25 C. The final wash consisted of 15 cycles of 4 mixes per cycle at 30°C in fluidics station using the non-stringent solution. Following washing and staining, probe arrays were scanned twice at 3 pm resolution using the GeneChip System confocal scanner (Hewlett-Packard, Santa Clara, CA). Visualization of all arrays is included in Figures 2-7 and 2-8. Data analysis was performed using Microarray Suite Version 5.0 software and Data Mining Tool Version 2.0 software (Affymetrix). The Affymetrix arrays also contain probes to control for RNA degradation. These probes target different parts of mRNA from 3’ to 5’ ends. RNA degradation usually occurs starting from the 3’ or 5’ ends so RNA degradation can be visualized if probes that target the ends of mRNA bind fewer signals than the probes that target the center of the mRNA. Figure 2-9 demonstrates that there was equal hybridization to different parts of the probes, indicating good quality RNA with little to no degradation. 75 Figure 2-7: Visualization of individual arrays hybridized with amplified mRNA from ARC and SNpc of control animals. All arrays are visualized in order to rule out any artifacts. These pictures show an apparently random ditribution of biotin labled probes. The black bar and white block at the center of each array also demonsrtate proper hybridization. Figure 2-8: Visualization of individual arrays hybridized with amplified mRNA from ARC and SNpc of MPTP treated animals. All arrays are visualized in order to rule out any artifacts. These pictures show an apparently random ditribution of biotin labled probes. The black bar and white block at the center of each array also demonsrtate proper hybridization. 77 l . l Mean Intensity : shifted and scaled Figure 2-9: Assessment of RNA quality in individual arrays. Each line represents expression of genes hybridized to the 3’ through 5’ ends of RNA from each array. Flat lines demonstrate lack of RNA degradation in the samples. 78 M. Microarray Data Analysis and Statistics Normalization, Background Correction and Data Summarization Normalization, background correction and data summarization were performed in collaboration with Dr. Marianne Huebner at Michigan State University. GeneChip Robust Multi-array Analysis (GCRMA) was used for normalization of the gene expression data to correct for background noise. Starting with the probe-level data from a set of Gene Chips, the perfect-match (PM) values were background-corrected, normalized and summarized resulting in a set of expression measures. Background correction is arguably the most crucial step for probe level processing. The non-linear background correction used in GCRMA is done in a per-chip basis. The GCRMA function summarizes probe intensities, adjusts for optical effect and non-specific binding, background-corrects, and generates an expression set all in one step. With GCRMA, the base types (A,T,G or C) at each position (1—25) along the probe determine the affinity of each probe. The parameters of the position specific base contributions to the probe affinity are estimated in an NSB experiment in which only NSB but no gene-specific binding is expected. Normalization is necessary so that multiple chips can be compared to each other during analysis. The GCRMA normalization procedure is aimed at making the distributions identical across arrays and is quantile, which results in very sharp normalizations. All the arrays are used and no chip is discarded on the basis of extreme value considerations. Once the probe-level values were background-corrected and normalized, they were summarized into expression measures so that the result is a 79 single expression measure per probe-set, per chip. Figure 2-10 depicts mean expression levels for each array before and after normalization using GCRMA. The summarization used is motivated by the assumption that observed log- transformed PM values follow a linear additive model containing a probe affinity effect, a gene specific effect (expression level) and an error term. For GCRMA, the probe affinity effects are assumed to sum to zero, and the gene effect (expression level) is estimated using median polishing. Median polishing is a robust model fitting technique, which protects against outlier probes 80 Unnormalized Data :l A —-------.-vo.‘---—-a ---------.-.OOO‘C I I I I I I I l I I I I I I I I I I I 8 1012 14 l5 I ' . I —‘- I... I ' l l I ' I ' | ' I I I ' I ' I ' I D I ' 0 I I I I i ' I 0 I ' I I | ' I I I ' I I I ' I ' I ' I I I ' I I I ' I I I ‘ I I I : D I | I g | O 0 I I I L"-- -- ’III-I- II IU‘IIIIIIUUCIUI. ‘i-‘.. }-W -.----ccnuuo--- A. I —L— _L_ i T l i T T I r T I T I l' d Norma Ize Data In T ... T T -~ T -r- ...- ...- T T T T ....- T T H - - : : ' - . - - ' I u I : l I : . g I : : - : I ' ' ' : i i I ' E ' : i z : : ' i ' : I i I . ' l o g . . I O U Q .... - 3 : ' ' . I ' = ' I . : I ' I ' I i T I I T I i T T I T I i T T O —b I C e O -J— I Figure 2-10: Mean expression levels (Y-axis) of all genes in each array (X-axis) before (top) and after (bottom) normalization with GCRMA. The GCRMA method is used to ensure all arrays have similar distribution. 81 The Use of Updated Annotation Files Most of the commonly used Affymetrix platforms were designed before the respective genomes were fully sequenced. Therefore, these platforms have many probes that were designed after consensus sequences of clusters of Expressed Sequence Tags were established. In the original Affymetrix definitions many probe sets often map to the same gene (i.e. target different transcript isoforms) and some integrative microarray studies use ad hac heuristics such as the average or maximum to integrate these values into a single expression estimate. To solve these problems, updated probe set definitions have been generated by re-annotating the existing probes on Affymetrix platforms to better reflect the transcript information and gene annotations currently available (Wang et al., 2002; Dai et al., 2005). These pioneering studies have shown that updated probe set definitions will affect approximately 20— 30% of all probe sets, thus affecting a large portion of the gene estimates. As a consequence, the genes identified as differentially expressed using the original and updated probe set definition only show 50% overlap (Dai et al., 2005). Updated probe set definitions that map probes to transcript annotations, such as ensEMBL transcripts, Refseq and Entrez GenelD are now available (http://brainarray.mbni.med.umich.edu/Brainarray/default.asp) and were integrated into bioconductor packages such as GCRMA. Updated probe annotations have also been shown to improve the cross-platform reproducibility of microarray experiments (Wang et al., 2002; Dai et al., 2005). 82 §t_atistical Analyses To identify differentially expressed genes a linear model was fit for all genes based on a experimental design comparing two brain regions and two treatments. These estimators exhibit robust behavior even for a small number of arrays and allow for incomplete data arising from spot filtering or spot quality weights. For each gene a moderated t-statistic is calculated using posterior standard deviations to extract those with maximally differential expression (Smyth 2004). This analysis was carried out using the R software (v.2.6.1, http://cran.r-project.org/) and the linear model package Limma available in the R Bioconductor software suite (http://www.bioconductor.org). P-values were adjusted for multiple testing following Storey’s approach of controlling the false discovery rate (FDR). FDR of a set of predictions is the expected percent of false predictions in the set of predictions (Benjamini and Hochberg 1995, Storey 2002). QIgster Analysis After obtaining model-based expression values, high-level hierarchical clustering analysis was performed (Eisen et al. 1998) in dCHIP (Li and Wong 2001) to identify groups of genes with similar expression patterns. The clustering algorithm of genes was as follows: the distance between two genes is defined as 1 - r where r is the Pearson correlation coefficient between the standardized expression values of the two genes across the samples used. Two genes with the closest distance are first merged into a super-gene and connected by branches with length representing their distance, and are excluded for subsequent merging events. The expression value of the newly 83 formed super-gene is the average of standardized expression values of the two genes (centroid-linkage) across samples. The next pair of genes (super-genes) with the smallest distance is chosen to merge and the process is repeated n — 1 times to merge all the n genes. These standardization and clustering methods follow previously documented methods (Eisen et al., 1998; Golub et al., 1999). Centroid linkage can produce branch inversion when the distance between two clusters is smaller than the height of either cluster, dChip truncates the distance to be the larger of the two heights, which prevents the branch inversion in visualization, but the further distance computation is still based on the averaged profile. Figure 2-11 depicts an example of hierarchial clustering performed in dChip. 84 ARC SNpc Control MPTP Control MPTP Figure 2-11. Cluster analysis of genes significantly higher in ARC than SNpc after treatment with a single injection of MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300- 500 laser-captured TH immunofluorescent cells from ARC and SN of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and cluster analysis was performed to find genes with similar expression patterns. Each row represents a gene and each column represents a treatment group. Genes close to each other have high similarity in their standardized expression values across the 4 treatment groups. On the bottom of the clustering picture is the color scale: the red color represents expression level higher than mean of a gene across all samples, the white color represents mean expression, and the blue color represents expression lower than the mean. Since the expression level for each gene is standardized to have mean 0 and standard deviation 1, the standardized expression values most likely fall within the range of -3 to 3. dChip uses pure white to represent 0, pure red to represent 3 or higher, and pure blue to represent -3 or lower (Golub et al., 1999). Pathway Analysis with DAVID The Database for Annotation, Visualization and Integrated Discovery (DAVID) provides a comprehensive set of functional annotation and classification tools for understanding the biological meaning behind a large list of genes. The functional annotation tool suite was used to provide typical batch annotation and Gene Ontology (GO) term enrichment analysis in order to highlight the most relevant GO terms associated with a given gene list including GO terms, protein- protein interactions, protein functional domains, disease associations, bio-pathways, sequence general features, homologies, gene functional summaries, gene tissue expressions, literatures, etc. The functional Classification Tool was used to generate a gene—to-gene similarity matrix based upon shared functional annotation using over 75,000 terms from 14 functional annotation sources. The clustering algorithms classified highly functionally related genes into groups, which provided a rapid means to organize large lists of genes to help unravel the biological content determined by the microarray. Analysis of Candidate KEGG Pathways KEGG (Kyoto Encyclopedia of Genes and Genomes) PATHWAYTM is a collection of manually drawn pathway maps representing current knowledge of molecular interaction and reaction networks. Candidate KEGG pathways relating to PD were determined and a list of all genes involved in each pathway was generated. These genes were located in our microarray database in order to determine general changes in candidate pathways. 86 N. RNA isolation from microdissected tissue punches Total RNA from microdissected tissue was extracted using the MELT total nucleic acid isolation with DNase digestion (Ambion, Austin, TX). Tissue was digested with 100 ul of MELT digestion mastermix (4 pl of MELT cocktail and 96 ul of MELT buffer) and vigorous vortexing for 10 min. The lysate was centrifuged at 10,000 x g for 3 min and transferred to a 96 well plate containing 210 pl of binding bead master mix (10 pl binding beads, 100 pl binding solution and 100 pl of 80% ethanol). The mixture was incubated at room temperature for 2 min, which allowed the RNA to bind to the magnetic binding beads. The beads containing the RNA were magnetically captured during a 3 min incubation and the supernatant was discarded. The beads were washed with 300 pl of Wash Solutions 1 and 2 and were air dried for 5 min at room temperature. DNA contamination was removed from the beads by incubation with 100 pl of TURBO DNase digestion master mix (2 pl TURBO DNase 2U/ul and 98 ul of DNase buffer) for 10 min at room temperature with gentle agitation. The beads were captured again using the magnetic stand and washed twice with 300 pl of Wash Solution 2. The beads containing the RNA were air dried for 5 min and RNA was eluted by incubation in 20 ul of elution buffer. The beads were captured using the magnetic stand and the purified RNA was collected and placed into a fresh elution tube. RNA concentration was determined using a Nanodrop and quality was assessed using a picochip (BioAnalyzer, AGILENT Technologies). 87 0. Reverse transcription RNA was incubated with oligo(dT) primers and dNTP mix at 65 C for 5 min to promote primer annealing. The mixture was incubated with 0.1M DTT, 3 reverse transcriptase (Superscript ll, Invitrogen) and RNase inhibitor (RNaseln, Ambion) at 42 C for 1 h to allow for reverse transcription. The mixture was heated to 70 C for 15 min in order to destroy the reverse transcriptase. And resulting cDNA was stored in -80 C until use with real time PCR. P. Real Time Quantitative PCR Primers were identified using Primer3 and synthesized by the macromolecular and structural facility (Michigan State University, East Lansing, MI) and are listed in Table 2-3. All primer sets were tested to ensure a single product as assessed by 2% agarose gel electrophoresis and melt-curve analysis. GAPDH was used as the reference gene and Figure 2-12 demonstrates that GAPDH mRNA levels were similar across regions (ARC and SNpc) and did not change with treatment (Control vs. MPTP). No template reactions were used as negative controls. Total RNA was reverse transcribed as described above and used as template in a 10 pl PCR reaction with 400 nM primers, and SYBR Green PCR Master Mix (Applied Biosystems). Forty cycles of PCR were performed and samples from 8 mice were run in triplicate. Threshold cycle (C) were determined and compared to C for the reference genes. 88 Gene Name Forward Primer Reverse Primer GAPDH AAC'ITTGGCATTGTGGAAGG ACACATTGGGGGTAGGAACA Parkin TTCTGACACCAGCATCTTGC CTT TCCTCCGTGGTCTCTG UCH-Ll CCCTI'GG'ITTGCAGCTIT AG ACACACAGGAGGGAAAGTGC Pinkl GAGGGCGTGCACCATCTG AGGATGTTGTCGGACTTGAGA TC SNCA AGGCAGCTGGAAAGACAAAA CAGCT CCCT CCACT GTCTI' C LR RK2 GATGTCAGTACG CCCCTGAT CTGCCAGCG CTATGATGTTA DJ-l GCTI' CCAAAAGAGCT CT GGTCA GCTCTAGTCTTTGAGAACAAGC Table 2-3. Forward and reverse primers used for real time PCR analysis. All primers were identified using the Primer3 software and used for real time PCR amplification. 89 l'_'l Control 120- IMPTP 100- $80- > 2 < 2 0:50- E I O O. <40- L9 20- o ARC SNpc Figure 2-12. GAPDH mRNA levels in ARC and SNpc of control and MPTP-treated mice. Mice were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed by decapitation 8 h later. ARC and SNpc were microdissected and expression of GAPDH mRNA was determined using real time PCR. 90 Fold change between regions was determined using the (SAC: method, which compares the C values of the samples of interest with a control or calibrator such as a non—treated sample or RNA from normal tissue. The C values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene. The AACt method is also known as comparative Ct method where AACt = Act’samp.e - ACLreference. Here, Act'sample is the C value for any sample normalized to the endogenous housekeeping gene and ACWerenm is the C value for the calibrator also normalized to the endogenous housekeeping gene. For the MC calculation to be valid the amplification efficiencies of the target and the endogenous reference must be approximately equal. PCR efficiencies of all primer sets were therefore tested to ensure similarity to the GAPDH primer (SS-100%). This was established by determining how Ac. varies with template dilution. If the plot of cDNA dilution versus delta C is close to zero, it implies that the efficiencies of the target and housekeeping genes are very similar. Significant differences between SNPC and ARC were determined using a one- way ANOVA with one correlated measure (p s 0.05). 91 Q. Stereotaxic Lentiviral Injections Mice were injected bilaterally into the ARC with 500 nl of lentiviral particles expressing a Parkin or non-target shRNA. Stereological injections (David Kopf Instruments, Tujunga, CA) were performed under ketamine : xylazine anesthesia (330 mg/kg : 20 mg/kg ; s.c.). A Hamilton Syringe (Hamilton, Reno, NV) with a 30 gauge blunt-tip needle was used to inject 500 nl of viral particles into the left and right ARC. Each mouse received bilateral injections of lentiviral vectors at a 10 degree angle in the following coordinates from Bregma: rostro-caudal -1.85, latero-medial 1.25 and -1.25, and dorso-ventral -S.8 from the skull surface. The virus was injected over a period of 5 min at a rate of approximately 100nl/min, and the needle was left in place for an additional 10 min. Figure 2-13 depicts the location of the bilateral stereotaxic injections. Figure 2-14 shows ARC injected with a lentivitus encoding for Green Fluorescent Protein demonstrating successful transfection of ARC neurons with the lentivirus. Figure 2-15 demonstrates a lack of microglial activation and inflammatory response with IBA1 staining. Activated microglia become round and detract processes when they become activated under inflammatory conditions. This is not the case in lentivirus treated animals as microglia retain their normal inactive morphology. 92 Interaunl l 98 mm Bregma -1.82 m I I I I I I I I. I . I I I I \- . 1.: I I . I I I I ... . ., S A J 2 l (I 1 Figure 2-13. Bilateral stereotaxic injection of lentiviral vectors into ARC of control and MPTP- treated mice. Animals received bilateral stereotaxic injections of Parkin shRNA or non-target shRNA lentivirus into the ARC. Three to five weeks later, animals were injected with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed by decapitation 16 h later. Concentrations of parkin mRNA were determined in ARC of all animals. 93 3V Figure 2-14. Stereotaxic injection of lentiviral vector encoding for green fluorescent protein (GFP) into ARC of mice. Animals received stereotaxic injections of GFP lentivirus into the ARC. Three weeks later, animals were killed and brains were viewed under the microscope for presence of GFP protein. 94 Figure 2-15. IBA1 staining for brain sections from mice that received stereotaxic injection of lentiviral vector encoding for green fluorescent protein into ARC. 3V = third ventricle. 95 P. Statistical Analysis Power analyses were conducted to determine optimal sample size required for each experiment. One-way analysis of variance (ANOVA) tests were used to detect statistical significance between two or more groups on a single independent variable. Two-way ANOVA’s were used to detect statistical significance between two or more groups when there were two independent variables in the study. Repeated measures ANOVA was used when comparing brain regions within the same animal. A p value of less than or equal to 0.05 was considered statistically significant. If the ANOVA revealed an interaction of statistical significance Tukey’s test was used for multiple comparisons among groups. 96 Chapter 3: Characterization of Difierential Susceptibility of TIDA, NSDA and MLDA Neurons in the Chronic MPTP Mouse Model A. Introduction: It has been hypothesized that abnormal DA metabolism underlies the selective degeneration of NSDA neurons in PD (Lotharius and O'Malley, 2000; Lotharius and Brundin, 2002; Vergo et al., 2007). However, it is unclear why all DA neuronal populations are not affected to the same extent in P0; with TIDA neurons remaining fully intact while NSDA neurons are severely damaged and MLDA neurons less severely affected (Braak et al., 1996; Braak and Braak, 2000; Braak et al., 20063; Braak et al., 2006b). In order to study this differential susceptibility, an animal model for PD that mimics this differential susceptibility needs to be established. The ideal model should recapitulate the severe retrograde NSDA neuronal loss, less severe retrograde damage to MLDA neurons and complete preservation of TIDA neurons. The MPTP mouse model has been commonly used to study PD and its strengths over other toxin-based models have been highlighted in Chapter 1 (lntrOduction, Section F. Animal Models of PD). Chronic treatment with the toxin mimics the long duration of retrograde degenerative insult to NSDA neurons observed in PD (Petroske et al., 2001; Drolet et al., 2004b). The differential susceptibility of DA neuronal populations, particularly of TIDA neurons, has not been well characterized in the chronic MPTP mouse model. If differential susceptibility exists amongst these DA neuronal populations in response to MPTP administration, then this model can be 97 utilized to discern the intrinsic properties of these neuronal populations and identify protective and/or deleterious factors that underlie their responsiveness to neurotoxin insult. B. Hypothesis This experiment tests the hypothesis that TIDA, NSDA and MLDA neurons are differentially susceptible in the chronic MPTP mouse model, with TIDA neurons being fully spared while NSDA neurons are severely damaged and MLDA neurons are moderately affected. C. Experimental Design The chronic MPTP mouse model consisted of injections of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks for a total of 10 injections (Figure 3-1). All animals also received probenecid (250 mg/kg; i.p.) with each MPTP/saline injection. Probenecid prevents the transport of organic acids across epithelial membranes and therefore blocks the clearance of MPP+ from the brain (Petroske et al., 2001). Animals were decapitated 3 weeks after the last MPTP injection and the brains were quickly removed. Following the dissection of ME from the ventral surface of the brain, the brain was bisected along the mid-sagittal axis. One half of the brain was frozen for neurochemical analysis and the other half was drop-fixed in 4% paraformaldehyde for immunohistochemical analysis. The details of animal use, drug administration, tissue preparation, neurochemical and immunohistochemical analyses were described previously in Chapter 2 (Methods, Sections. A-F). 98 it at at It a: lit: 4:- In: an: Kill animals C C C C C C C C C C .9 .9 .9 .9 .9 .9 .9 .9 .9 .9 ,3 e e .23. e. e; e; .3 £91 ,3; 3 weeks with no injections s\.s s 5\ 5 \s s\s\ s\£\ A l 1 1 1 1 1 r V 'V T T " T Y V V V I“ “L‘ ‘ ““A :‘r‘ A ‘A A‘ “Wild“ ‘ ‘ ‘ A" I l I l l l l l 1 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Figure 3-1. Time course of chronic MPTP administration. Male C57bl mice (n=8/group) were injected with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. Animals also received probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. Mice were killed by decapitation 3 weeks after the last MPTP or saline injection. 99 The primary endpoints used to determine axon terminal loss were DA concentrations in ME, ST and NA; the axon terminal regions of TIDA, NSDA and MLDA neurons, respectively. DA concentrations in these regions primarily reflect vesicular, rather than cytoplasmic or extracellular DA (Carlsson, 1975b; Carboni et al., 1992; Di Chiara et al., 1996), as DA not sequestered inside vesicles is rapidly metabolized. DA concentrations reflect the ability of neurons to synthesize, and store neurotransmitter in their axon terminals. Because axon terminals have had 3 weeks to recover from MPTP, DA concentrations in this model reflect the relative density of the remaining functional axon terminals. The concentrations of DA after chronic MPTP are consistent with other indices of axon terminal integrity such as TH and VMAT—Z levels (Wilson et al., 1996; Kilbourn et al., 2000; Reveron et al., 2002). Cytosolic DA is converted in the presynaptic terminals to DOPAC and therefore the concentrations of DOPAC and ratio of DOPAC to DA are commonly used to estimate the activity of central DA neurons (Westerink and Spaan, 1982; Lookingland et al., 1987b; DeMaria et al., 1999). The endpoint used to determine DA cell body loss was unbiased stereological cell counts of TH positive neurons in the ARC, SNpc and VTA; the cell body regions of TIDA, NSDA and MLDA neurons, respectively. In order to confirm that lower TH positive cell counts were due to loss of neurons rather than down regulation of TH, stereological cell counts were also performed on alternating Nissl stained cells in ARC, SNpc and VTA. For Nissl stained sections, morphological criteria were used to differentiate between neurons and glia. These morphological criteria are described in detail in Chapter 2 (Methods, Section G. Unbiased Stereological Cell Counting). 100 In order to demonstrate that the dose of MPTP used in these experiments did not damage non-DA neuronal populations, concentrations of NE, as well as S-HT and its metabolite S-hydroxyindoleacetic acid (S—HIAA) were determined. 101 Endpoint Method Measure of Indicates DA HPLC-EC Vesicular DA DA axon terminal density DOPAC HPLC-EC DA metabolite DA metabolism DOPAC/DA HPLC-EC Relative amount of DA DA neuron activity metabolized to that stored in axon terminals NE HPLC-EC Vesicular NE NE axon terminal density S-HT HPLC-EC Vesicular S-HT S-HT axon terminal density S-HIAA HPLC-EC S-HT metabolite 5-HT metabolism TH cell counts Stereology Cells expressing TH DA cell number Nissl cell Stereology All cells Total neuron number counts Table 3-1. The experimental endpoints used for indices of neuronal function in the chronic MPTP mouse model. DA = dopamine, DOPAC = 3,4-dihydroxyphenylacetic acid, NE = norepinephrine, S-HT = serotonin, S-HIAA = S-hydroxyindoleacetic acid, TH = tyrosine hydroxylase and HPLC-EC = high performance liquid chromatography coupled with electrochemical detection. 102 D. Results Following chronic treatment with MPTP the concentrations of DA were measured in ME, ST and NA. MPTP did not alter DA concentrations in ME while inducing significant DA loss (”70%) in ST. MLDA neurons were less severely, but significantly, affected with only "45% DA loss in NA (Figure 3-2). Similarly, following treatment with MPTP, DOPAC concentrations remained unchanged in ME, but decreased significantly in ST, and NA (Figure 3-3). The ratio of DOPAC/DA was determined and was unchanged in ME, but significantly increased in ST (Figure 3-4). There was no change in DOPAC/DA ratio in NA following MPTP administration, suggesting either that these neurons do not have D2 receptor mediated compensatory activation or that insufficient amounts of MLDA axon terminals have been lost following this dose of MPTP to produce compensatory activation unlesioned MLDA neurons. Following chronic treatment with MPTP, concentrations of NE (Table 3-2), as well as 5-HT and its metabolite 5-HIAA (Table 3-3) were determined in ME, ST and NA. Concentrations of NE, 5-HT and 5-HIAA were unchanged in all regions examined, demonstrating that MPTP is specific to DA neuronal populations. 103 120 - El Saline IMPTP 100 - I I § 80 - E * * U "5 so - g M 6 g 40 - 20' - o ME ST NA Figure 3-2. The effects of chronic MPTP administration on DA levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. DA levels are presented as % of saline treated. Actual DA concentrations (rig/mg protein) are as follows in saline treated mice: ME: 1471135, ST: 14016.5 and NA: 4312.9 , and MPTP treated mice: ME: 151115, ST: 4015 and NA: 20.4122. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 104 140 F El Saline I MPTP 120 - 100 '- 80- 60- DOPAC as % of Control 40- 20- 0L— ME ST NA Figure 3-3. The effects of chronic MPTP administration on DOPAC levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. DOPAC levels are presented as % of saline treated. Actual DOPAC concentrations (ng/mg protein) are as follows in saline treated mice: ME: 3.610], ST: 11.0105 and NA: 7210.8, and MPTP treated mice: ME: 3.610], ST: 6.6109, and NA: 5110.9. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values in MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 105 250 F a saline IMPTP * 200 - E E 8 '05 150 P 33 8 a 100 _ L r l o < n. O a 50 - o — ME ST NA Figure 3-4. The effects of chronic MPTP administration on the ratio of DOPAC/DA in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. Ratios are presented as % of saline treated controls. Actual DOPAC/DA ratios are as follows in saline treated mice: ME: 0.031001, ST: 0.061001 and NA: 0.141001, and MPTP treated mice: ME:0.0210.01, ST: 0.111001, and NA: 0.191002. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 106 Concentrations of NE (ng/mg ME ST NA Control 17.9 1 1.6 ND 3.0 1 0.5 MPTP 18.5 1 4.2 ND 3.9 1 0.3 Table 3-2. Norepinephrine (NE) concentrations in median eminence (ME), striatum (ST) and nucleus accumbens (NA) of control and MPTP treated mice. Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. ND = Not Detectable. 107 A. Concentrations of 5-HT (ng/mg protein) ME ST NA Control 8.5 1 1.2 7.5 1 1.3 13.2 1 1.5 MPTP 8.0 1 1.4 7.4 1 0.6 15.2 1 2.7 8. Concentrations of S-HIAA (ng/mg protein) ME ST NA Control 16.2 1 1.3 7.0 1 2.1 3.4 1 0.5 MPTP 13.4 1 4.1 7.5 1 1.1 4.1 1 0.6 Table 3-3. Serotonin (5-HT; A) and 5-hydroxyindoleacetic acid (S-HIAA; 8) concentrations in median eminence (ME), striatum (ST) and nucleus accumbens (NA) of control and MPTP treated mice. Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. 108 Paraformaldehyde fixed brains were sectioned at 60 uM and every third section was dual-stained for TH (Figure 3-5) and Nissl. Unbiased stereological cell counts were performed on TH immunoreactive cells in ARC, SNpc and VTA, the cell body regions of TIDA, NSDA and MLDA neurons, respectively. MPTP administration failed to induce loss of TH cells in ARC or VTA, while treatment with this neurotoxin caused a significant decrease in TH immunoreactive neurons in SNpc (Figure 3-6). Alternate sections were stained for Nissl and the number of neurons were counted to determine if lower TH immunoreactive cell counts in SNPC were due to cell loss rather than down regulation of TH. There was a similar "30% loss of Nissl stained neurons in SN PC following treatment with MPTP (Figure 3-7) . 109 Control Wig-1’ “ ’«K‘ " ‘4 .- Figure 3-5. Tyrosine hydroxylase immunoreactive (TH-IR) cells In the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and arcuate nucleus (ARC) of control and MPTP treated mice. Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. Half brains were drop-fixed in paraformaldehyde, 60 pM sections were collected and dual-stained for TH and Nissl. Arrows indicate localization of brown stained TH immunoreactive cells and their processes within each brain region. 110 140 - El Saline I MPTP 120 - 100 - 80- 40- TH-IR Cell number as % of Control . 20- ARC SNpc VTA Figure 3-6. The effects of chronic MPTP administration on tyrosine hydroxylase immunoreactive (TH-IR) cell numbers in the arcuate nucleus (ARC), substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. Half brains were drop-fixed in paraformaldehyde, 60 (1M sections were collected and dual-stained for TH and Nissl. Numbers of TH-immunoreactive cells are presented as % of saline treated controls. Actual unilateral TH-immunoreactive cell numbers are as follows in saline treated mice: ARC: 12051237, SNpc: 26871365 and VTA: 22511130, and MPTP treated mice: ARC:11171121, SNpc: 18921195 and VTA: 2146176. Columns represent means of groups and vertical bars represent +1.0 Standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 111 140 - USaline IMPTP 120 - l 5 100 - T U u— o as .. m 80 *- o E E 60 ' :l c 3 4o - '5 .2 2 20 . 0 ARC SNpc VTA Figure 3-7. The effects of chronic MPTP administration on Nissl positive cell numbers in the arcuate nucleus (ARC), substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Male mice (n=8/group) were treated with either MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.) every 3.5 days for 5 consecutive weeks. All animals received an injection of probenecid (250 mg/kg; i.p.) with each MPTP or saline injection. Half brains were drop-fixed in paraformaldehyde, 60 micron sections were collected and stained for Nissl. Neurons were counted using unbiased stereological methods and are presented as % of saline treated controls. Actual unilateral cell numbers are as follows in saline treated mice: ARC: 94101907, SNpc: 49181228 and VTA: 38261907, and MPTP treated mice: ARC: 90161470, SNpcz3151184 and VTA: 38551470. Columns represent means of groups and vertical bars represent +1.0 Standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. ' 112 E. Discussion: These experiments demonstrate that chronic MPTP treatment can successfully model the differential loss of DA neuronal populations observed in PD. Specifically, TIDA neurons are completely spared in the chronic MPTP mouse model of PD, while NSDA neurons are markedly depleted, and MLDA neurons are less severely damaged. These results show that the MPTP mouse model recapitulates the pattern of differential susceptibility seen in PD. This makes the MPTP mouse model an attractive model study mechanisms that contribute to susceptibility in DA neurons. In accordance with previous reports (Petroske et al., 2001; Drolet et al., 2006), chronic systemic MPTP treatment leads to loss of DA and DOPAC concentrations in ST. This long-term loss of neurotransmitter levels has been consistently mirrored by loss of DA neuron-specific proteins such as TH, DAT and VMAT-2 (Wilson et al., 1996; Kilbourn et al., 2000; Reveron et al., 2002; Drolet et al., 2004b, 2006) and can be reliably used to represent loss of NSDA axon terminal density and integrity. The activation of the remaining axon terminals, as demonstrated by the increased DOPAC/DA ratio, suggests the remaining axon terminals compensate for the severe DA loss. This loss of NSDA axon terminal density and compensatory activation of remaining neurons is also present in the brains of PD patients (Ribeiro et al., 2002; Sossi et al., 2002). Consistent with previous studies employing the chronic mouse model (Petroske et al., 2001; Drolet et al., 2004a), the loss of axon terminals ("70%) is accompanied by significant, but less severe ("30%) loss of TH immunoreactive cell bodies in SNpc after treatment with MPTP. This loss of TH positive neurons was mirrored by loss of Nissl 113 stained cell bodies, demonstrating that lower cell counts were due to loss of entire cell bodies, and not transient down-regulation of TH, as previously reported in some MPTP mouse models (Kuhn et al., 2003). The more severe loss of axon terminals after MPTP is supportive of other reports that chronic treatment with the toxin causes retrograde damage to NSDA neurons, affecting the axon terminals before killing the cell bodies (Petroske et al., 2001; Drolet et al., 2006). This retrograde pattern of toxicity of NSDA neurons is also present in the brains of PD patients with 70—80% loss of ST nerve terminals and 50—60% loss of SNpc neurons when clinical symptoms appear (Bernheimer et al., 1973; Agid, 1991). Unlike NSDA neurons, TlDA axon terminals have unchanged DA and DOPAC levels after chronic treatment with MPTP. This novel finding suggests that TIDA neurons are completely resistant to chronic MPTP treatment with full preservation of axon terminal density and integrity. Stereological cell counts also demonstrate that the cell bodies of these neurons also remain fully intact, which suggests that TIDA neurons remain completely spared. This differential susceptibility between TIDA and NSDA neurons mimicks PD, where no lesion of TIDA neurons, as measured indirectly using circulating prolactin levels or directly using post-mortem cell counts, is accompanied by severe damage to NSDA neurons (Agnoli et al., 1981; Eisler et al., 1981; Matzuk and Saper, 1985; Laihinen and Rinne, 1986; Franceschi et al., 1988). DA and DOPAC levels in the NA also decrease after treatment with chronic, systemic MPTP, demonstrating that there is some damage to the axon terminals of these neurons. However, the degree of terminal loss in NA ("50%) is less than that 114 observed for NSDA terminals ("70%). Furthermore, MPTP treatment does not cause loss of MLDA cell bodies demonstrating that these neurons are less susceptible to treatment with MPTP than NSDA neurons. The ratio of DOPAC/DA is not significantly increased in NA following treatment, suggesting that the degree of axon terminal loss may not be sufficient to cause a compensatory increase in activity. These findings reveal that MLDA neurons are relatively resistant to the neurotoxic effects of Complex | inhibition when compared with NSDA neurons, but these neurons are not completely spared. This partial susceptibility following neurotoxic exposure may be due to the presence of a heterogeneous population of cells in the VTA; some that display resistance and some that are vulnerable to neurotoxic insult. In high doses, MPP+ has an affinity for the norepinephrine (NE) and serotonin (5-HT) transporters and can cause nonselective toxicity to these neuronal populations. Furthermore, lipophillic MPTP may be bioactivated in NE or S-HT neurons via MAO in sufficient amounts to damage these neuronal types. The cell bodies and some terminal regions of these DA pathways are innervated by NE and 5-HT neurons, originating from the locus coeruleus and raphe nuclei, respectively (Alex and Pehek, 2007). Thus, NE or S-HT can regulate the function of DA neurons via their actions on DA cell bodies or terminals and complicate findings due to the direct action of MPTP on DA neurons. It is therefore important to determine the effect of MPTP on NE and 5-HT neurons in order to separate direct and indirect effects of MPTP on DA neuronal populations. While this model has been previously described (Petroske et al., 2001; Drolet et al., 2004b), it has not been determined whether the dose of MPTP is low enough to be 115 specific only to DA neurons. The experiments in this chapter demonstrate that following treatment with chronic MPTP, concentrations of NE, as well as S-HT and its metabolite S-HlAA remain unchanged in all measured regions. This suggests that the MPTP treatment regimen employed in this study does not have any deleterious effects on NE or 5-HT neuronal populations. Accordingly, any toxic effects may be attributed to direct effect of the MPP+ on the DA neurons. A number of hypotheses have been proposed to explain the preferential susceptibility of NSDA neurons to neurotoxic insult (Hirsch, 1992; Hirsch et al., 1997). These hypotheses include variations in pharmacokinetic factors such as toxin distribution and bio-activation of MPTP to MPP+ by astrocytes. Others have postulated that differential entry of MPP+ into the cell via DAT may play a role in the differential susceptibility of these neurons. Finally, differences in cell body size, length of projecting axons and density of innervation of these axons have been proposed to play a role in the differential demise of these neuronal populations. Several studies have demonstrated that MPTP is readily distributed and converted to MPP+ in SNpc, VTA and hypothalamus, suggesting that differences in toxin distribution or bio-activation is not a likely explanation for differential sensitivity (Del Zompo et al., 1993; Hung et al., 1995; Speciale et al., 1998). Moreover, exposure to MPP+ markedly increases apoptosis in TH-IR midbrain primary cultures, but not TH-IR mediobasal primary cultures (Behrouz et al., 2007). Since these cells were exposed to the same concentration of the active toxin for the same amount of time, the 116 differential sensitivity of NSDA and TIDA neurons cannot be due to bioactivation, distribution or clearance of the toxin. Differential uptake of MPP+ via DA transporter (DAT) and intracellular compartmentalization into vesicles via VMAT-2 have also been proposed to explain differential susceptibility to neurotoxins (Uhl, 1998). Variations in transporter expression, however, do not address the selective NSDA neuronal toxicity following exposure to rotenone (Behrouz et al., 2007). Rotenone is a lipophillic molecule that, unlike MPP+, does not require DAT to enter neurons and is not compartmentalized into vesicles (Le et al., 1999; Betarbet et al., 2000; Masliah et al., 2000; Bywood and Johnson, 2003). Rotenone diffuses into all cells and chronic, systemic rotenone administration decreases Complex l activity uniformly throughout the entire brain (Greenamyre et al., 2001). Differential susceptibility amongst TIDA, NSDA and MLDA neurons can be seen following chronic systemic rotenone treatment (Betarbet et al., 2000; Behrouz et al., 2007), demonstrating this differential susceptibly is not dependent on entry of the toxin into the cells. These in vivo results are complemented by data from primary culture studies that demonstrate differential susceptibility of DA neuronal populations to rotenone (Behrouz et al., 2007). Differences in cell body size, length of projecting axons and density of innervation of these axons have also been proposed as explanations for differential toxicity (Braak and Braak, 2000). The somata of TIDA neurons have a similar diameter and contain the same organelles as those seen in the midbrain cell groups (Van den Pol et al., 1984). NSDA and MLDA neurons have long projecting axons while TIDA neurons 117 have short axons that terminate locally within the MBH (Bjorklund A, 1984). The short axons of TIDA neurons are unlikely to be a key resistance phenotype since MLDA neurons, which do have long projecting axons, also show some resistance to these toxins. Density of innervation is also unlikely to explain the differential sensitivity of discrete DA neuronal systems since the resistant TIDA and highly affected NSDA neurons have virtually identical innervation densities to their target neurons and the mildly affected MLDA neurons have a lower density of innervation, as demonstrated by DA concentrations in regions containing axon terminals. Taken together, the present findings suggest that inherent properties of DA neurons related to cell body size, axonal length, and density of innervation of target cells are unlikely explanations for the differential sensitivity of these neurons to neurotoxins. Differences in intrinsic response of TIDA, NSDA and MLDA neurons, such as expression of protective or deleterious genes may be responsible for the differential susceptibility of these neuronal populations to MPTP. The MPTP mouse model can therefore be used to examine the differential responses of these neuronal populations and analyze intrinsic properties that may infer susceptibility or protection. 118 Chapter 4: Characterization of the Immediate Neurochemical Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of MPT P A. Introduction Chronic systemic treatment with MPTP causes a similar pattern of DA neuron damage observed in symptomatic PD patients. In this model, TIDA neurons remain unaffected with intact cell bodies, while NSDA neurons are extensively and retrogradely lesioned and MLDA neurons are moderately affected. This differential susceptibility of DA neuronal populations is likely dependent upon their intrinsic properties. The long term response of these neuronal populations after chronic MPTP is an excellent model for studying advanced PD when cell death has already occurred. On the other hand, examination of responses to the first injection of MPTP should mimic early PD, when the most susceptible neurons undergo degeneration. This will allow for assessment of the intrinsic properties of the most susceptible midbrain neurons as they undergo degeneration as compared with resistant TlDA neurons. The immediate response of NSDA neurons to MPTP has been well characterized. By 1.5 h after a single systemic injection of MPTP, DA is displaced from synaptic vesicles and lost from the axon terminals. Retrograde neurodegeneration proceeds such that by 24 h after treatment with MPTP DA levels in ST remain low, and by 72 h cell bodies in the SNpc express markers of apoptosis (Pileblad et al., 1984; Perry et al., 1985; Pileblad et al., 1985). TlDA neurons are intact and function normally after 119 chronic treatment with MPTP, but it is possible that these neurons respond to MPTP in a fashion similar to NSDA neurons and later recover. If this is the case, their properties during the period of recovery (when they may express protective factors) can be examined. Determination of the time course of events immediately after MPTP treatment can also address the possibility that MPP+ may not enter these neurons due to lower DAT expression (Demarest and Moore, 1979a). lf TIDA neurons respond to treatment with MPTP in a similar fashion to NSDA neurons, it is possible that MPP+ enters these neurons via a DAT-independent mechanism. The results from chronic MPTP experiments described in the Chapter 3 demonstrated that MLDA neurons are partially resistant to the toxic effects of MPTP when compared with NSDA neurons. It would therefore be insightful to learn about the response of this neuronal population as well. It has been proposed that NSDA neurons produce deleterious proteins in response to toxin exposure that cause their demise (Kuhn et al., 2003). Alternatively, it is possible that TIDA or MLDA neurons may respond to toxin treatment by production of protective proteins. Determining whether the differential susceptibility of NSDA, MLDA and TIDA neurons to toxins is due to mechanisms already in place under normal conditions or through inducible response mechanisms to toxin treatment would allow for identification of intrinsic mechanisms that could determine neuronal fate. The objectives of the experiments described in this chapter are to examine and compare the short-term neurochemical responses of TIDA, NSDA and MLDA neurons to MPTP administration and determine whether these responses are DAT dependent. 120 Experiments also determine whether the neurochemical responses of these DA neuronal populations are dependent on synthesis of proteins in response to MPTP treatment. B. Hypothesis The experiments in this chapter test the general hypothesis that TIDA and MLDA neurons initiate intrinsic protective mechanisms in response to MPTP. C. Experimental Design Time Course of Responses of TIDA, NSDA and MLDA Ngrons to a Single Injection of MPTP A single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) was administered (Figure 4-1) and animals were killed by decapitation either 4, 8, 16 or 32 h later. Saline treated animals were killed 8 h after saline injection and were used as zero time controls. Immediately after decapitation brains were removed, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for neurochemical analyses. The details of methods for animal use, drug administration, tissue preparation, and neurochemical analyses are described in Chapter 2 (Methods, Sections. A-D). 121 Klll MPTP injection Klll Kill Kill Hours Figure 4-1. Paradigm for the single injection MPTP time course experiment. Male C57bl mice (n=8/group) were injected with MPTP (20 mg/kg; s.c.) and killed by decapitation either 4, 8, 16 or 32 h later. Saline (10 ml/kg; s.c.) treated animals were killed 8 h post-injection and were used as zero time controls. 122 The primary endpoints (Table 4-1) used to determine vesicular DA loss from axon terminal regions were DA concentrations in ME, ST and NA; the axon terminal regions of TIDA, NSDA and MLDA neurons, respectively. Under basal conditions, DA concentrations in these regions primarily reflect vesicular DA (Carlsson, 1975a; Carboni et al., 1992; Di Chiara et al., 1996). Since MPP+ converted from MPTP causes displacement of DA out of vesicles and axon terminals (Chiueh et al., 1994), DA concentrations following a single injection of MPTP reflects a combination of vesicular, cytoplasmic and extracellular DA (see Chapter 1; Introduction, Section E). Accordingly, decreased concentrations of DA shortly after treatment with MPTP should be interpreted as loss of DA stores rather than loss of axon terminal integrity. Only after MPP+ has cleared from the brain and neurons recover in the absence of MPTP (by 32 h post-injection) can the loss of DA be attributed to the loss of axon terminal integrity. Moreover, since MPTP and MPP+ bind to and inhibit the activity of MAO in DA neurons (Takamidoh et al., 1987), changes in DOPAC and DOPAC/DA should not be interpreted solely as DA neuronal activity, but may be contributed partially to MAO inhibition. In order to demonstrate that the dosage used in these experiments does not directly affect non-DA neuronal populations, concentrations of NE, as well as 5-HT and its metabolite S-HIAA were measured. If these neurons are directly affected by MPP+, there should be a decrease in neurotransmitter levels. Other changes in NE or S-HT/S- HIAA levels may be attributed to the response of these neurons to the DA neuronal populations. 123 Endpoint Method Measure of Indicates DA HPLC-EC Vesicular, cytoplasmic or DA neuron function extracellular DA DOPAC HPLC-EC DA metabolite DA metabolism, MAO activity NE HPLC-EC Vesicular NE NE axon terminal density S-HT HPLC-EC Vesicular S-HT 5-HT axon terminal density S-HIAA HPLC-EC S-HT metabolite 5-HT metabolism Table 4-1. The experimental endpoints used for neuronal function following treatment with a single injection of MPTP. DA = dopamine, DOPAC = 3,4-dihydroxyphenylacetic acid, NE = norepinephrine, S-HT = serotonin, S-HIAA = S-hydroxyindoleacetic acid, TH = tyrosine hydroxylase and HPLC-EC = high performance liquid chromatography coupled with electrochemical detection. 124 Effects of Blockade of DAT on the response of TIDA, NSDA and MLDA Neurons to a Sinale Injection of MPTP This experiment tests the hypothesis that the mode of MPP+ entry into TIDA neurons, unlike NSDA and MLDA neurons, is DAT-independent. Mice were pretreated with DAT inhibitor GBR-12909 (GBR, 10 mg/kg; i.p.) or saline (10 ml/kg; i.p.) 30 min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed by decapitation 4 h after MPTP administration (Figure 42) and the brains were quickly removed. Immediately after decapitation, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for neurochemical analyses. The details of methods for animals, drug administration, tissue preparation, and neurochemical analyses are described in Chapter 2 (Methods, Sections. A-D). The primary endpoints (Table 4-1) used in this experiment are similar to the previous experiment. 125 mm Injection GBR Injection -30 min 0 4 hrs Figure 4-2. Experimental paradigm for the combination of GBR pretreatment with a single injection MPTP. Male C57bl mice (n=8/group) were pretreated with GBR-12909 (GBR; 10 mg/kg; i.p.) or saline (10 ml/kg; i.p.) 30 min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed by decapitation 4 h after MPTP. 126 Effects of Protein Synthesis Inhibition on the Responses of TIDA, NSDA and MLD_A Algyrons to a Single Injection of MPTP This experiment tests the hypothesis that the recovery from MPTP-induced DA loss in TIDA neurons is dependent on de novo synthesis of proteins. Mice were treated with 4 or 2 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) every 2 h, with the first cycloheximide injection 15 min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). This dose of cycloheximide has been shown to block protein synthesis in the brain for at least 2 h (Squire and Barondes, 1973; Squire et al., 1973; Squire and Barondes, 1976). Animals receiving 4 cycloheximide injections were decapitated 14 h after MPTP and animals that received 2 cycloheximide injections were decapitated 7 h post MPTP (Figure 3-4). Immediately after decapitation, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for neurochemical analysis. The details of methods for animals, drug administration, tissue preparation, and neurochemical analyses are described in Chapter 2 (Methods, Sections. A-D). The primary endpoints (Table 4-1) used in this experiment are similar to the previous experiment. 127 Cycloheximide Cycloheximide Cycloheximide (Ndoheximide -15 min 1h 45min 3h 45min 5h 45min Qdoheximide Kill -15 min 1h 45min Figure 4-3. Experimental paradigm for the combination of cycloheximide pretreatment with a single injection of MPTP. Male C57bl mice (n=8/group) were treated with 4 (A) or 2 (8) injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) every 2 h, with the first cycloheximide injection 15 min M to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/ kg; s.c.). Animals receiving 4 cycloheximide (A) injections were killed 14 h after MPTP and animals receiving 2 cycloheximide injections (8) were killed 7 h post MPTP. 128 Effects of Immediate vs. Delayed Protein Synthesis Inhibition on the Responses of TIDAI NSDA and MLDA Neurons to a Single Injection of MPTP This experiment tests the hypothesis that de novo protein synthesis at different times prevents recovery of TIDA neurons from MPP+ induced DA loss. Mice were treated with 2 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) 2 h apart starting either 15 min prior to or 3 h 45 min after a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed by decapitation 16 h after MPTP (Figure 3-5) and the brains were quickly removed. Immediately after decapitation, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for neurochemical analysis. The details of methods for animals, drug administration, tissue preparation, and neurochemical analyses are described in Chapter 2 (Methods, Sections. A-D). The primary endpoints (Table 4-1) used in this experiment are similar to the previous experiment. 129 Cycloheximide MPTP Cycloheximide Kill . ° . 16h -15 min 1h 45mm Cycloheximide Cycloheximid e 3h 45min 5h 45min Figure 44. Experimental paradigm for the combination of cycloheximide treatment with a single injection MPTP Male C57bl mice (n=8/group) were treated with 2 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.), 2 h apart, starting either 15 min m to (A) or 3 h 45 min fig (B) a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed by decapitation 16 h after MPTP. 130 D. Results Time CflJrse of Responses of TIDA, NSDA and MLDA Neurons to a Single Injection of ME Figure 4-5 demonstrates the time—course of the responses of TIDA, NSDA and MLDA neurons to a single systemic injection of MPTP. ME, ST and NA DA levels were significantly decreased by 4 h after MPTP treatment. ME DA levels increased at 8 h, and were similar to controls at 16 and 32 h post-injection. In contrast, DA levels in ST decreased further at 8 h post-MPTP and remained low (”25% of controls) for the remainder of the time. Decreased levels of DA (”70% of control) were maintained in the NA at 8 and 16 h post-MPTP injection, but returned to control levels by 32 h. As demonstrated in Figure 4-6, DOPAC levels mirrored DA levels after treatment with a single injection of MPTP. DOPAC levels in ME, ST and NA decreased significantly by 4 h after MPTP treatment. DOPAC levels returned to control levels in ME by 16 h after treatment, but remained low in ST and NA. No changes in NE concentrations were observed in NA or ME at any time after treatment with MPTP. Concentrations in ST were not detectable even in zero time controls (Table 4-2). S-HT levels increased in ST 4 h after MPTP treatment (Figure 4-7), but returned to normal by 8 h post-MPTP and did not change at 16 or 32 h post- treatment. 5-HT levels in ME or NA remained unchanged following MPTP treatment. 5-HIAA levels increased in ST by 4 and 8 h after MPTP, but returned to normal at 16 and 32 h after treatment (Figure 4-8). 131 -O-ST 120 _ -D- NA 100 80 .................... 60 40 DA as % of CODtrol 20. 0 4 8 12 16 20 24 28 32 Figure 4-5: The time course of effects of a single injection of MPTP on DA levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with MPTP (20mg/kg; s.c.) and killed by decapitation either 4,8,16 or 32 h later. Saline (10 ml/kg; s.c.) treated animals were killed 8 h post-injection and were used as zero time controls. DA levels are presented as % of these saline treated controls. Actual DA concentrations (rig/mg protein) are as follows in ME: zero h control : 176112, MPTP 4 h:5117, MPTP 8 h: 112110, MPTP 16 h: 153111, MPTP 32 h: 172120; NA: zero h control: 7113 , MPTP 4 h: 4517, MPTP 8 h: 4816, MPTP 16 h: 5116, MPTP 32 h: 6216 ; and in ST: zero h control: 17217 , MPTP 4 h: 94115, MPTP 8 h: 6315, MPTP 16 h: 5318 and MPTP 32 h: 5114 . Geometric symbols represent means of groups and vertical bars represent 1 1 standard error of the mean. Filled in symbols represent MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 132 -O-ST -Cl- NA 120 e -/_\.- ME 100 80 60 40 DO PAC (% of control) 20~ l l l l I 0 l l l 0 4 8 12 16 20 24 28 32 Time(h) Figure 4-6: The time course of effects of a single injection of MPTP on DOPAC levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with MPTP (20mg/kg; s.c.) and killed by decapitation either 4, 8, 16 or 32 h later. Saline (10 ml/kg; s.c.) treated animals were killed 8 h post-injection and were used as zero time controls. DOPAC levels are presented as % of saline treated. Actual DOPAC concentrations (ng/mg protein) are as follows in ME: zero h control: 1211, MPTP 4 h: 612, MPTP 8 hr: 612, MPTP 16 hr: 911, MPTP 32 hr: 1012, in NA: zero h control: 1611 , MPTP 4 h:511, MPTP 8 h: 711, MPTP 16 h: 610, MPTP: 1012 and in ST: zero h control: 24.212 , MPTP 4 h:1413, MPTP 8 h: 811, MPTP 16 h: 1212, and MPTP 32 h: 912 . Geometric symbols represent means of groups and vertical bars represent 1 1 standard error of the mean. Filled in symbols represent MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 133 Concentrations of NE (ng/mg protein) Time (h) after MPTP ST NA ME 0 ND 7.3 1 1.4 21.8 1 4.5 4 ND 9.9 1 1.3 26.3 1 3.6 8 ND 7.8 1 1.9 20.9 1 1.8 16 ND 9.4 1 1.1 21.8 1 2.3 32 ND 8.3 1 1.2 27.2 1 5.1 Table 4-2. Norepinephrine (NE) concentrations in median eminence (ME), striatum (ST) and nucleus accumbens (NA) of saline control and MPTP treated mice. Male mice (n=8/group) were treated with MPTP (20mg/kg; s.c.) and killed by decapitation either 4, 8, 16 or 32 h later. Saline (10 ml/kg; s.c.) treated animals were killed 8 h post-injection and were used as zero time controls. ND = Not Detectable. 134 250 l -O-ST -l:l - NA -A- ME 200 :2‘ E' 150 8 q. 0 33 ________ :100 :.‘.:.".:_‘ 'LI.’_"...:T —————— -- 55 —o 50 - 0 l n 1 L J A 1 I O 4 8 12 16 20 24 28 32 Time(h) Figure 4-7: The time course of effects of a single injection of MPT P on S-HT levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with MPTP (20mg/kg; s.c.) and killed 4,8,16 or 32h later. Saline (10 ml/kg; s.c.) treated animals were killed 8h post-injection and are considered 0 time controls. S-HT levels are presented as % of saline treated. Actual S-HT concentrations (ng/mg protein) are as follows in ME: zero h control: 1412, MPTP 4 h:1613, MPTP 8 hr:1712, 16 hr:1311, MPTP 32 hr: 1412,in NA: zero h control: 1312 , MPTP 4 h: 1312, MPTP 8 h: 1212, MPTP 16 h: 1212, MPTP 32 hr:1313 and in ST: zero h control: 1211 , MPTP 4 h: 2013, MPTP 8 h: 1011, MPTP 16 h: 911, MPTP 32 hr: 811. Geometric symbols represent means of groups and vertical bars represent 11.0 standard error of the mean. Filled in symbols represent MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 135 200 - -O-ST -Cl-NA -A- ME 150 ”‘2“ +4 . C O 1 U .................. "5 100 I2 ‘ ”'- .33. < E I L0 50 ' 0 l I n L . l J J 0 4 8 12 16 20 24 28 32 Time(h) Figure 4-8: The time course of effects of a single injection of MPTP on SHIAA levels in median eminence (ME), striatum (ST) and nucleus accumbens (NA). Male mice (n=8/group) were treated with MPTP (20mg/kg; s.c.) and killed 4,8,16 or 32h later. Saline (10 ml/kg; s.c.) treated animals were killed 8h post-injection and are considered 0 time controls. SHIAA levels are presented as % of saline treated. Actual SHIAA concentrations (ng/mg protein) are as follows in ME: zero h control: 611, MPTP 4 h: 911, MPTP 8 h: 911, MPTP 16 h: 511, MPTP 32h: 612, in NA: zero h control: 611 , MPTP 4 h: 711, MPTP 8 h: 711, MPTP 16 h: 511, MPTP 32 h: 612 and in ST: zero h control: 611 , MPTP 4 h: 911, MPTP 8 h: 911, MPTP 16 h: 511, MPTP 32 h: 611. Geometric symbols represent means of groups and vertical bars represent 11.0 standard error of the mean. Filled in symbols represent MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 136 Effects of Blockade of DAT on the Responses of TIDA, NSDA gnd MLDA Negrons to a Single Injection of MPTP: As demonstrated in Figure 4-9, DA concentrations decreased in ME 4 h following MPTP administration in both control and GBR-treated mice. In ST, 3 significant loss of DA was observed 4 h after MPTP-treatment, and this DA loss was blocked by GBR-pretreatment (Figure 4-10). Figure 4-11 demonstrates that there was a loss of DA in NA 4 h after treatment with MPTP, but GBR pre-treatment blocked MPTP induced DA loss in NA. In all three regions, GBR treatment alone did not alter DA levels. Figure 4-12 demonstrates that DOPAC levels decreased in ME 4 h after MPTP in both control and GBR-treated mice. Moreover, DOPAC levels remained unchanged in ME after GBR treatment alone. MPTP treatment also caused a loss of DOPAC in ST of control and GBR-treated mice (Figure 4-13). ST had decreased levels of DOPAC following GBR treatment alone. DOPAC levels also decreased in NA following MPTP treatment in both control and GBR-treated mice (Figure 4-14) and GBR treatment alone did not alter DOPAC concentrations in this region. 137 140 - ' El Saline IMWP 120- l —100- I 8 4.! C 8 .880- 32 * * 360- < O 40- 20y o Saline GBR Figure 4-9. The effects of GBR pre-treatment and single injection MPTP administration on DA levels in median eminence (ME). Male mice (n=8/group) were treated with the DAT inhibitor GBR (10 mg/kg; i.p.) or saline (10 ml/kg; i.p.). Thirty min after GBR administration, mice received a single injection of MPTP (20mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 4 h after MPTP or saline treatment. DA levels are presented as % of saline treated. Actual DA concentrations (ng/mg protein) are as follows: Saline/saline: 13118, Saline/MPTP: 60111, GBR/saline: 144113, and GBR/MPTP: 6915. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 138 Saline 120 - D IMPTP 100b I 7.3 gsoe * U a. O 3,360- (I < O 40. 20- o Saline GBR Figure 4-10. The effects of GBR pre-treatment and single injection MPTP administration on DA levels in striatum (ST). Male mice (n=8/group) were treated with the DAT inhibitor GBR (10 mg/kg; i.p.) or saline (10 ml/kg; i.p.). Thirty min after GBR administration, mice received a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 4 h after MPTP or saline treatment. DA levels are presented as % of saline treated. Actual DA concentrations (ng/mg protein) are as follows: Saline/saline: 18217, Saline/MPTP: 122112, GBR/saline: 16216, and GBR/MPTP: 16018. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 139 [J Saline IMPTP 140 { 120 - _9 E 100 r ..‘L’ o o 80 - 3;, * N < O i- 20 - 0 Saline GBR Figure 4-11. The effects of GBR pre-treatment and single injection MPTP administration on DA levels in nucleus accumbens (NA). Male mice (n=8/group) were treated with the DAT inhibitor GBR (10 mg/kg; i.p.) or saline (10 ml/kg; i.p.). Thirty min after GBR administration, mice received a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 4h after MPTP or saline treatment. DA levels are presented as % of saline treated. Actual DA concentrations (ng/mg protein) are as follows: Saline/saline: 80119, Saline/MPTP: 3317, GBR/saline: 69112, and GBR/MPTP: 5714. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 140 140 r E] Saline IMPTP 120 - TC.) 100 - ...: C 8 .._ 80 - O *3. _ * * 8 60 E O O 40 _ 20 - 0 Saline GBR Figure 4-12. The effects of GBR pre-treatment and single injection MPTP administration on DOPAC levels in median eminence (ME). Male mice (n=8/group) were treated with the DAT inhibitor GBR (10 mg/kg; i.p.) or saline (10 ml/kg; i.p.). Thirty min after GBR administration, mice received a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 4 h after MPTP or saline treatment. DA levels are presented as % of saline treated. Actual DOPAC concentrations (ng/mg protein) are as follows: Saline/saline: 411, Saline/MPTP: 211, GBR/saline: 411, and GBR/MPTP: 110. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 141 E] Saline 120 F IMPTP 100 - I _o g 80 - A o u “5 I , a": 60 - * * (O U E o 40 - o 20 - 0 Saline GBR Figure 4-13. The effects of GBR pre-treatment and single injection MPTP administration on DOPAC levels in striatum (ST). Male mice (n=8/group) were treated with the DAT inhibitor GBR (10 mg/kg; i.p.) or saline (10 ml/kg; i.p.). Thirty min after GBR administration, mice received a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 4 h after MPTP or saline treatment. DA levels are presented as % of saline treated. Actual DOPAC concentrations (rig/mg protein) are as follows: Saline/saline: 1711, Saline/MPTP: 610, GBR/saline: 1111, and GBR/MPTP: 611. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 142 E] Saline IMWP 140- 120- E a 5100- ..‘L’ o 3280i- * U! m U E50' * o o 40. 20- O Saline GBR Figure 4-14. The effects of GBR pre-treatment and single injection MPTP administration on DOPAC levels in nucleus accumbens (NA). Male mice (n=8/group) were treated with the DAT inhibitor GBR (10 mg/kg; i.p.) or saline (10 ml/kg; i.p.). Thirty min after GBR administration, mice received a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 4 h after MPTP or saline treatment. DA levels are presented as % of saline treated. Actual DOPAC concentrations (ng/mg protein) are as follows: Saline/saline: 811, Saline/MPTP: 311, GBR/saline: 812, and GBR/MPTP: S11. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 143 The Impact of Protein Synghesis Inhibition on the Neurochemical Responses of NSDA, MLDA and TIDA Neurons to a Single Injection of MPTP: Figure 4-15 demonstrates the effects of cycloheximide treatment on DA concentrations in the ME in MPTP treated mice. Treatment with MPTP alone decreased DA levels in ME by 7 h but there was complete recovery of DA to control levels by 14 h post-MPTP. Treatment with cycloheximide prevented MPTP induced loss of ME DA concentrations at 14 h, but not 7 h. In contrast, DA concentrations in ST were significantly decreased 7 and 14 h after treatment with MPTP in both control and cycloheximide treated mice (Figure 446). Similarly, DA levels in NA decreased 7 and 14 h following MPTP treatment in both control and cycloheximide treated mice (Figure 4— 17). Cycloheximide treatment alone did not alter DA levels in ME, ST or NA. DOPAC levels mirrored DA levels following treatment with cycloheximide and MPTP (Table 4-3). DOPAC levels decreased in ME by 7 h after MPTP treatment alone and returned to control levels by 14 h post-MPTP, whereas DOPAC levels did not change in the ME of mice treated with cycloheximide and MPTP. ST and NA DOPAC concentrations decreased with MPTP treatment in control and cycloheximide treated mice. Cycloheximide alone did not alter DOPAC levels in ME, NA, or ST. 144 12° ' -o-Saline ’ -CI-Cycloheximlde * l, 100 mi 91 so a a 8 U ‘8 so - at 3 < D 40 - 20 P 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Figure 4-15. The effects of cycloheximide and MPTP administration on DA levels in median eminence (ME). Male C57bl mice (n=8/group) were treated with 2 or 4 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) every 2 h, with the first cycloheximide injection 15 min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). Animals receiving 2 cycloheximide injections were decapitated 7 h after MPTP and animals receiving 4 cycloheximide injections were decapitated 14 h post MPTP. DA levels are presented as % of saline treated. Actual DA concentrations (ng/mg protein) in ME are as follows in saline treated mice: zero h control: 132118, MPTP 7 h: 2212 and MPTP 14 h: 124114, and Cycloheximide treated mice: zero h control: 118118, MPTP 7 h: 2916 and MPTP 14 h: 3816. Geometric shapes represent means of groups and vertical bars represent +1.0 standard error of the mean. Filled in symbols indicate values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. (*) indicates values for cycloheximide treated mice that were significantly different (p< 0.05) from saline treated controls. 145 120 —o—Sallne -CJ -Cyclohexlmlde 100 - 80- 60- 40- DA as $6 of Control 20- 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (h) Figure 4-16. The effects of cycloheximide and MPTP administration on DA levels in striatum (ST). IVIale C57b| mice (n=8/group) were treated with 2 or 4 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) every 2 h, with the first cycloheximide injection 15min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). Animals receiving 2 cycloheximide injections were decapitated 7 h after MPTP and animals receiving 4 cycloheximide injections were decapitated 14 h post MPTP. DA levels are presented as % of saline treated. Actual DA concentrations (ng/mg protein) in ST are as follows in saline treated mice: zero h control: 18718, MPTP 7 h: 88115 and MPTP 14 h: 5215, and Cycloheximide treated mice: zero h control: 203123, MPTP 7 h: 114113 and MPTP 14 h: 63 1 9. Geometric shapes represent means of groups and vertical bars represent +1.0 standard error of the mean. Filled in symbols indicate values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. (*) indicates values for cycloheximide treated mice that were significantly different (p< 0.05) from saline treated controls. 146 120 - -<>-Sallne .. -CI-Cyclohexlmide 100- sue '5 :s: ----------i ‘5 so. 7 as 3 3 4o) 20 ‘- I I I 4 I I I I I I I J I L I 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (h) Figure 4-17. The effects of cycloheximide and MPTP administration on DA levels in nucleus accumbens (NA). Male C57bl mice (n=8/group) were treated with 2 or 4 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) every 2 h, with the first cycloheximide injection 15 min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). Animals receiving 2 cycloheximide injections were decapitated 7 h after MPTP and animals receiving 4 cycloheximide injections were decapitated 14 h post MPTP. DA levels are presented as % of saline treated. Actual DA concentrations (rig/mg protein) in NA are as follows in saline treated mice: zero h control: 94111, MPTP 7 h: 6418 and MPTP 14 h: 5819, and Cycloheximide treated mice: zero h control: 92110, MPTP 7 h: 69110 and MPTP 14 h: 6517. Geometric shapes represent means of groups and vertical bars represent +1.0 standard error of the mean. Filled in symbols indicate values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. (*) indicates values for cycloheximide treated mice that were significantly different (p< 0.05) from saline treated controls. 147 Saline 6.7 1 1.7 4.5 1 0.9 6.0 1 1.7 Cycloheximide 6.0 1 1.1 7.1 1 2.0 5.9 1 2.0 Saline 17.2 1 1.9 3.9 1 11* 7.0 1 1.1* | Cycloheximide 20.5 1 1.7 5.3 1 2.0* 6.4 1 2.0* Saline 21.7 1 1.5 7.1 1 0.7* 9.5 1 1.1* Cycloheximide 27.2 1 2.3 8.0 1 2.0* 11.5 1 2.0* Table 4-3. DOPAC concentrations (ng/mg protein) in ME, ST and NA after a single injection MPTP with protein synthesis inhibition using cycloheximide. Male CS7bl mice (n=8/group) were treated with 4 or 2 injections of cycloheximide (120 mg/kg; i.p.) or saline (10 ml/kg; i.p.) every 2 h, with the first cycloheximide injection 15 min prior to a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). Animals receiving 4 cycloheximide injections were decapitated 14 h after MPTP and animals receiving 2 cycloheximide injections were decapitated 7 h post MPTP. 148 The Impact of Immediate vs. Delayed Protein Synthesis Inhibition on the Neurochemical Responses at NSDA, MLDA and TIDA Neurons to a Single Injection of M_PT££ Figure 4-18 demonstrates the effects of immediate and delayed cycloheximide treatment on DA concentrations in the ME in MPTP treated mice. DA levels in ME were similar to controls 16 h after treatment with MPTP. Cycloheximide alone had no affect on DA levels, but immediate and delayed cycloheximide treatment significantly decreased DA levels in ME in MPTP treated mice. As depicted in Figure 4-19, ST DA levels decreased 16 h after treatment with MPTP. Immediate or delayed treatment with cycloheximide along with MPTP also caused significant loss of DA; however, the magnitude of DA loss was significantly less in early cycloheximide + MPTP treatment than with MPTP alone. DA levels in NA decreased after treatment with MPTP in the control and immediate or delayed cycloheximide treated animals (Figure 4-20). DOPAC levels mirrored DA levels following treatment with cycloheximide and MPTP (Table 4-4). DOPAC levels decreased in ST and NA after treatment with MPTP in control and immediate or delayed cycloheximide treated mice. Cycloheximide treatment alone did not alter DOPAC levels in NA, or ST. DOPAC levels were not determined due to experimental error. 149 120 p DSaline l IMPTP 100 - » . , * E 80 '- * E O U ‘8 60 r- a? U) (D < o 40 - 20 - 0 Saline Immediate Cycloheximide Delayed Cycloheximide Figure 4-18. The effects of immediate and delayed cycloheximide and single Injection MPTP on DA levels in ME. Male C57bl mice (n=8/group) were treated with 2 injections of cycloheximide (120 mg/kg) or saline (10 ml/kg; s.c.), 2h apart, starting either 15 min prior (A) to or 3 h 45 min after (B) a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 16 h after MPTP. DA levels are presented as % of saline/saline treated. Actual DA concentrations (ng/mg protein) in NA are as follows: saline/saline: 204138, saline/early cycloheximide: 161116, saline/late cycloheximide: 187133, MPTP/saline: 152122, MPTP/early cycloheximide: 114115 and MPTP/late cycloheximide: 1261 53. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 150 120 » Cl Saline IMPTP 00 O I DA as 96 of Control 8 b o I Saline Immediate Cycloheximide Delayed Cycloheximide Figure 4-19. The effects of immediate and delayed cycloheximide and single injection MPTP on DA levels in ST. Male C57b| mice (n=8/group) were treated with 2 injections of cycloheximide (120 mg/kg) or saline (10 ml/kg; s.c.), 2 h apart, starting either 15 min prior (A) to or 3 h 45 min after (B) a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 16 h after MPTP. DA levels are presented as % of saline/saline treated. Actual DA concentrations (rig/mg protein) in ST are as follows in saline treated mice: saline/saline 1551 6, saline/early Cycloheximide: 15019 and saline/late cycloheximide: 14714, MPTP/saline: 5015, MPTP/early cycloheximide: 7317 and saline/late cycloheximide: 5912. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 151 DSaline 120 e I MPT P 100- 80- DA as % of Control 8 8 N O I Saline Immediate Cycloheximide Delayed Cycloheximide Figure 4-20. The effects of immediate and delayed cycloheximide and single Injection MPTP on DA levels in NA. Male C57bl mice (n=8/group) were treated with 2 injections of cycloheximide (120 mg/kg) or saline (10 ml/kg; s.c.), 2 h apart, starting either 15 min prior (A) to or 3 h 45 min after (B) a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 16 h after MPTP. DA levels are presented as % of saline/saline treated. Actual DA concentrations (rig/mg protein) in NA are as follows in saline/saline: 6716, saline/early cycloheximide: 6717, saline/late cycloheximide: 67111, MPTP/saline: 5317, MPTP/early cycloheximide: 5614 and MPTP/late cycloheximide: 4918. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 152 DOPAC Concentrations in ME Cycloheximide Saline Immediate Delayed Saline ND ND ND MPTP ND ND ND DOPAC Concentrations in ST Cycloheximide Saline Immediate Delayed Saline 21.3 1 3.4 24.2 1 2.0 25.6 1 2.0 MPTP 13.8 1 0.8* 13.0 1 0.2* 16.7 1 20* DOPAC Concentrations in NA Cycloheximide Saline Immediate Delayed Saline 9.3 1 1.5 12.2 1 1.2 11.7 1 2.0 MPTP 6.5 1 0.8* 7.5 1 10* 5.4 1 20* Table 4-4. DOPAC concentrations (ng/mg protein) in ST, NA and ME after a single injection MPTP with protein synthesis inhibition using cycloheximide. Male C57bl mice (n=8/group) were treated with 2 injections of cycloheximide (120 mg/kg) or saline (10 ml/kg; s.c.), 2 h apart, starting either 15 min prior (Immediate) to or 3h 45min after (Delayed) a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were decapitated 16h after MPTP. 153 Discussion: The experiments in this chapter characterize the time course of neurochemical events following toxin entry into TIDA, NSDA and MLDA neuronal populations. These experiments also address the differential mode of toxin entry into the DA neuronal populations as well as the dependency of their MPTP-induced response on synthesis of new proteins. These results are discussed in detail below. Differential Response of TIDA, NSDA and MLDA News After a Single Injection of m_PT_P A novel temporal profile of neurotoxin-induced neurochemical changes is observed in TIDA, NSDA, and MLDA neurons after treatment with a single injection of MPTP. TIDA, NSDA and MLDA neurons respond in a similar fashion 4 h after treatment with MPTP exhibiting DA loss from their axon terminals. These responses, however, vary greatly after this time point and these differential responses may determine their fate. TIDA neurons respond to MPTP exposure with a substantial DA loss from axon terminals 4 h after a single injection. This rapid loss of DA likely represents MPP+- induced displacement of vesicular DA from the axon terminals rather than a reduction in density and integrity of terminals as observed in the chronic model. In agreement, the initial loss of DA in TIDA neurons is followed by a rapid recovery by 8 h that continues to up to 16 h after MPTP treatment. DOPAC levels mirror DA levels in this model and this loss of DOPAC is likely due to a combination of displacement of DA from vesicles and diffusion out of axon terminals, loss of ATP due to Complex I inhibition, as 154 well as inhibition of MAO by MPP+ (Takamidoh et al., 1987). The transient DA and DOPAC loss from this region correlates with the short half life of MPP+ in the brain (Langston and Irwin, 1986; Irwin et al., 1989; Cosi and Marien, 1999; Saporito et al., 2000). The rapid recovery of TIDA neurons from MPTP-induced DA loss provides a temporal window that may be examined to determine underlying mechanisms that allow for this recovery. Consistent with previous reports (Pileblad et al., 1984; Pileblad et al., 1985), NSDA axon terminals also lose DA 4 h after MPTP treatment, but unlike TIDA neurons, they do not recover from MPTP toxicity for up to 32 h after toxin exposure. In fact, NSDA neurons have ongoing DA loss between 4 and 8 h post MPTP-injection, during which time TIDA neurons are rapidly recovering. DOPAC levels also mirror DA levels in ST and this loss of DOPAC, at least for up to 8 h after treatment, while MPP+ is present, is likely due to both loss of DA that can be converted to DOPAC and inhibition of MAO by MPTP/MPP+. These experiments also confirm that MLDA neurons are partially resistant to the toxic effects of MPTP. The initial response of MLDA neurons after 4 h is very similar to that of NSDA neurons, but NSDA terminals continue to lose DA between 4 and 8 h, whereas MLDA terminals maintain moderate DA levels. MLDA neurons, however, do not show the rapid recovery that TIDA neurons experience at 8 and 16 h after MPTP injection. Similar to that observed in TIDA and NSDA neurons, DOPAC levels mirror DA levels in the NA demonstrating lower metabolism of DA due to loss of DA as well as inhibition of MAO by MPTP/MPP+. These results support the hypothesis that MLDA 155 neurons may be composed of a heterogeneous group of neurons; some that display resistance and some that are vulnerable to neurotoxic insult and do not recover, whereas TIDA neurons are homogeneously resistant. There is some evidence for this hypothesis, as a group of MLDA neurons that express the protein calbindin are protected from MPTP-induced toxicity (Lavoie and Parent, 1991; Lavoie et al., 1991; Parent and Lavoie, 1993). NE levels remain unchanged in the ME and NA suggesting that MPP+ does not enter noradrenergic neurons via the NE transporter in sufficient quantities to disrupt neurotransmitter stores. This explains why noradrenergic neurons are not lesioned after chronic treatment with MPTP in experiments described in Chapter 3. 5-HT concentrations, on the other hand, increase by 4 h after treatment with MPTP, but only in ST. It is unlikely that this change in 5-HT concentrations is due to a direct effect of MPP+ on these neurons. If MPP+ entered these neurons in sufficient amounts to cause damage, the neurotransmitter levels would have decreased so it is likely that the increase in 5-HT levels is actually due to indirect effects of DA systems on the serotoninergic neurons. It is known that 5-HT release both in SNpc and in ST act on 5- HT ZB/C receptors on NSDA cell bodies and axon terminals to inhibit DA release (De Deurwaerdere et al., 1996; De Deurwaerdere and Spampinato, 1999). It is therefore not surprising that large displacement of DA from NSDA axon terminals would cause activation of this ”brake” system. 156 Mechanism of MPP+ uptake is different in TIDA neurons vs. NSDA and MLDA neurons: The time-course of toxin-induced neurochemical changes in TIDA neurons suggests that, even though DAT expression is low in TIDA terminals (Demarest and Moore, 1979a, 1979b), MPP+ entry into these cells is sufficient to disrupt neurotransmitter storage. Treatment with the DAT blocker GBR has no affect in MPTP- induced DA loss in TIDA axon terminals, revealing that MPP+ enters these cells via DAT- independent mechanisms. Furthermore, blocking DAT does not change DOPAC concentrations in the ME, consistent with the conclusion that TIDA neurons do not utilize DAT for uptake of extracellular DA under basal conditions (Demarest and Moore, 1979b, 1979a). One way that MPP+ could gain access inside TIDA neurons is through direct conversion of MPTP to MPP+ by MAO located inside these cells. However, this remains an unlikely explanation because NSDA and MLDA neurons also express MAO but do not show this DAT-independent response to MPTP. Furthermore, since TIDA neurons have a lower DOPAC/ DA ratio than NSDA or MLDA neurons, it is unlikely that MAO is more active or has higher expression in these neurons. It is also unlikely that MPP+ is entering these neurons via non-specific transporters, since other neuronal populations, including 5-HT or NE neuronal types, do not show neurotransmitter loss in response to MPTP treatment. It is possible that MPP+ enters TIDA neurons via a specific transporter that is unique to these neurons. Although not well characterized, a unique high volume, low affinity transporter has been described in TIDA neurons (Annunziato et al., 1980), supporting the hypothesis that a unique mode of MPP+ entry may exist in 157 these neurons. Lack of specific antibodies, agonists or antagonists to characterize this transporter makes it difficult to test this hypothesis. It is possible that the fate of molecules taken up via this unique transporter is different from those taken up by DAT. For example, molecules taken up via this transporter may be more or less likely to be packaged inside vesicles or metabolized. It is, however, unlikely that MPP+ sequestration into vesicles plays a role in the differential susceptibility of these neurons since a similar pattern of susceptibility can be observed with toxins such as rotenone that are not transported into synaptic vesicles (Betarbet et al., 2000; Behrouz et al., 2007) In accordance with previous reports (Pileblad and Carlsson, 1985), NSDA and MLDA neurons showed complete protection against MPTP-induced DA loss, suggesting that entry via DAT is the only way sufficient amounts of MPP+ can enter these neurons. This difference in response between TIDA neurons and NSDA or MLDA neurons suggests that MPP+ enters TIDA neurons via different mechanisms. Moreover, DOPAC levels in ST were significantly decreased after GBR alone, supporting previous data that demonstrates NSDA neurons normally utilize DAT to uptake extracellular DA (Bannon, 2005). On the other hand, DOPAC levels in NA do not decrease after GBR treatment alone, suggesting that either less DA is taken up through DAT in these neurons or that DA normally taken up through DAT in MLDA neurons may be more efficiently packaged. Previous reports that demonstrate lower levels of DAT expression in MLDA neurons than NSDA neurons support the former of the two possibilities (Freed et al., 1995;Haberetal,1995) 158 Recovery of TIDA Neurons is Dependent on Synthesis of New Proteins after MPTP Treatment: Since the axon terminals of TIDA neurons are outside the blood brain barrier, they are likely exposed to toxic chemicals and oxidative stress to a greater extent than NSDA or MLDA neurons. It is evolutionarily sensible that these neurons have a mechanism in place that protects them from extracellular stresses and allows recovery from neurotoxin-induced toxicity. The experiments in this chapter demonstrate that inhibition of protein synthesis blocks the ability of TIDA neurons to recover by 16 h post-treatment. This suggests that TIDA neurons may constitutively synthesize or upregulate expression of protective proteins in response to MPTP exposure. These experiments also demonstrate that blocking protein synthesis either immediately or 4 h after treatment with MPTP blocks the ability of TIDA neurons to recovery from MPTP- induced DA loss. Since low levels of DA in TIDA terminals at 4 h are likely to lead to increased prolactin secretion from the anterior pituitary, which in turn feed back to increase the activity of TIDA neurons, it would be plausible to hypothesize that prolactin may be responsible for the recovery of these neurons. It has been previous demonstrated, however, that the immediate prolactin control over TIDA neuron activity is not dependent on protein synthesis. Only after 16 h following prolactin changes this control become dependent on synthesis of new proteins. Since the recovery of these neurons is dependent on synthesis of new proteins, prolactin control is unlikely to be responsible for this recovery. 159 On the other hand, blocking protein synthesis in NSDA neurons does not alter their neurochemical profile 7 or 14 h after treatment with MPTP. This suggests that synthesis of new proteins following treatment with MPTP does not play a role in causing the deleterious effects observed at this time point. This is perhaps not surprising, since the immediate response of all neurons is to maintain homeostasis and perform their function. Instead, it is likely that NSDA neurons lack the neuroprotective mechanisms that are in place in TIDA neurons. General Conclusions The experiments in this chapter reveal that TIDA neurons have protein synthesis dependent protective mechanisms that are activated in response to treatment with MPTP. The timeline of this recovery has been established. Studying these neurons 8 h after treatment with MPTP should prove useful in identifying proteins that infer protection to these neurons. Indeed, 8 h after MPTP the susceptible NSDA neurons are disrupted but are still viable. This makes the sampling of neurons 8 h after MPTP treatment a very attractive model for further comparison of the properties of NSDA and TIDA neurons. MLDA neurons lie somewhere between the fully resistant TIDA and susceptible NSDA neurons. Based on the neurochemical profile of these neurons after treatment with a single injection of MPTP (or chronic repeated exposure as shown in Chapter 3) it is likely that there is a heterogeneous population of these neurons, one that is susceptible to neurotoxic insult (like NSDA neurons) and another that is resistant (like 160 TIDA neurons). Without knowing the phenotype that makes some of these neurons resistant and some susceptible, it would be difficult to assess their protective properties especially if these populations are heterogeneously distributed within the VTA rather than clustered into discrete groups. Gene expression differences in key cellular pathways may account for the differential susceptibility of TIDA and NSDA neurons to cell death. A recent study providing an initial analysis of gene expression profile comparisons of NSDA and MLDA neurons suggested several molecular pathways that may be involved in neuroprotection (Greene et al., 2005). These pathways include energy metabolism, neuropeptide neurotransmission, and aberrant kinase or phosphatase signaling pathways. Studying gene expression profiles of TIDA neurons would undoubtedly identify novel neuroprotective properties that are distinct from those observed in NSDA neurons. Identification of protective and deleterious properties in DA neuronal populations with divergent responses to neurotoxins can provide a method to discover targets that can subsequently be translated into neuroprotective treatments for patients with PD. 161 Chapter 5: Characterization of the Genomic Responses of TIDA and NSDA Neurons to a Single Injection of MPTP F. Introduction The previous chapter demonstrated that treatment with a single systemic injection of MPTP causes loss of DA from axon terminals of TIDA and NSDA neurons, but that only TIDA neurons are able to recover from this DA loss. Moreover, the recovery of TIDA neurons was demonstrated to be dependent on synthesis of new proteins. It’s unclear what proteins are responsible for the recovery of TIDA neurons. Identification of genes with increased expression following MPTP in TIDA neurons will allow for functional studies aimed at determining which proteins protect TIDA neurons from MPTP toxicity. The results from these studies may be exploited in the development of strategies to protect NSDA neurons from toxicity. The objective of the experiments outlined in this chapter is to determine the genomic profile of TIDA and NSDA neurons under normal basal conditions and 8 h after treatment with MPTP when NSDA neurons continue to lose axon terminal DA while TIDA neurons are rapidly recovering from DA loss. G. Hypothesis: The experiments in this chapter test the hypothesis that several genes are upregulated in the resistant TIDA neurons, but not susceptible NSDA neurons, in response to treatment with systemic MPTP. 162 H. Experimental Procedures Mice were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed by decapitation 8 h later. Immediately after decapitation brains were removed in RNase free conditions and frozen on dry ice. Sections (10 pm) through the hypothalamus and midbrain were obtained and rapidly stained for TH, and 300-500 TH-immunofluorescent cells were microdissected from ARC and SNpc of each animal using LCM. RNA was isolated for each region and amplified linearly. Amplified RNA for each sample was biotin labeled and hybridized to an Affymetrix Mouse 430 2.0 genechip. Data was analyzed using the software R. The details of methods for animal use, drug administration, tissue preparation, rapid immunofluorescent staining, LCM, RNA isolation and amplification, microarray hybridization and analysis, reverse transcription and quantitative real time PCR are described in Chapter 2 (Methods, Sections. A-D; H-M). For real time PCR experiments, mice were treated with a single systemic injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed by decapitation 8 h later. Immediately after decapitation brains were removed in RNase free conditions and frozen on dry ice. Sections (500 um) were obtained and the ARC and SNpc were microdissected and RNA was collected from each sample. mRNA was reverse transcribed and real time PCR was performed to assess expression levels of each gene. The details of methods for animal use, drug administration, tissue preparation, reverse 163 transcription and quantitative real time PCR are described in Chapter 2 (Methods, Sections. A-D; NO). I. Results Genes with Differential Expression in ARC and SNpc in Control and MPTP Treated Mice The number of genes with an adjusted p-value of 0.05 or less that were differentially expressed between ARC and SNpc by at least 2 fold (log; 1 fold) were determined (Figure 5-1). The details of these genes are available in the Appendix. Figure 5-2 demonstrates the number of genes significantly higher in ARC or SNpc under control conditions and 8 h after treatment with a single injection of MPTP. Overall, 348 genes had higher expression in ARC than SNpc in control animals, 254 of which also had higher expression in ARC 8 h after treatment with MPTP. There were 156 genes with higher expression in ARC when compared to SNpc after treatment with MPTP but not in saline treated controls. Under control conditions, 309 genes were expressed in higher levels in SNpc, and 253 of these genes had higher expression in SNpc even after treatment with MPTP. There were 153 genes with higher expression in SNpc when compared to ARC of MPTP treated but not saline treated controls. 164 ARC vs. SNpc in Controls: -..—... -..... ~—-.--. """ fig...» ...“. .-..-. ...---.... s 110 1‘5 20 Log Odds 0 -S -10 10 Log Fold Change ARC vs. SNpc after MPTP: 20 . ....L... 10 15 Log Odds 5 0 Ln —4 I T 10 40 Log Fold Change Figure 5-1: The pattern of expression of genes differentially expressed among regions in the control (top) and MPTP treated (bottom) animals. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed 8 h after MPTP or saline treatment. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each mouse were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that were significantly different between the two regions by at least 2 fold were identified. 165 Number of genes with higher expression in ARC | Control j ( MPTP I Number of genes with higher expression in SNpc l Control L MPTP ( / \ z z/ \ Figure 5-2: The number of genes with statistically higher expression in ARC (top) and SNpc (bottom) under control and MPTP treated conditions. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed 8 h after MPTP or saline treatment. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SN of each mouse were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that were significantly different between the two regions by at least 2 fold were identified. 166 Unbiased Pathway Analysis at Microarray Data with DAVID: Unbiased pathway analysis was performed for genes higher in ARC when compared to SNpc after treatment with MPTP using DAVID annotation tools. DAVID uses a modification of Fisher Exact P-value by comparing the ratio of the occurrence rate of each Gene Ontology (GO) pathway within the given gene list to the ratio of occurrence of the pathway within the entire genome by chance alone. The G0 pathways that had significantly higher occurrence than would be expected by chance alone were identified (P s 0.05) and genes within-that pathway were further analyzed. Table 5-1 depicts biological GO pathways in a set of genes that were significantly higher in ARC than SNpc after treatment with MPTP. These pathways include general cellular processes, RNA metabolic processes, apoptosis and regulators of gene expression. The number of genes that overlap within each biological pathway are indicated in Table 5- 1. Figure 5-3 demonstrates the relative expression levels of genes that overlap with the apoptosis pathway and have higher expression in ARC following treatment with MPTP. The genes apolipoprotein E, HSPA1A, Kainate 2 glutamate receptor, BCL-2 associated anthanogene 3, FC receptor, CD1D1 antigen, angiopietin-like 4, 5le a, mitogen activated protein kinase 7, and TAIL1 are all proteins that regulate apoptosis and had higher expression in ARC when compared with SNpc after treatment with MPTP but not in saline treated controls. DAVID annotation tools were also utilized to examine differences between SNpc and ARC levels in saline treated controls. Several general and specific pathways were 167 implicated and are listed in Table 5-2. These pathways include general synaptic transmission as well as neurotransmitter metabolism and secretion amongst others. The genes listed on this table that were specific to DA metabolism, transport and secretion are depicted in Figure 5-4. These results confirm previous findings that D2 receptors and DAT have higher expression in NSDA neurons when compared to TIDA neurons. Other DA related genes that had higher expression in SNpc when compared to ARC were synphilin, TH, nuclear receptor 4A2, synaptic vesicle glycoprotein 2C, complexin 1, VMAT-Z, islet cell autoantigen 1 and nicotinic cholinergic receptors (14 and (16. 168 Gene Ontology Pathway Ii of genes P-value in pathway GO:0065007~biological regulation 46 7.25E-05 GO:0050789~regulation of biological process 43 9.33E-05 GO:0050794~regulation of cellular process 39 2.87E-04 GO:0010467"‘gene expression 31 0.005062 GO:0016070"RNA metabolic process 26 0.011584 GO:0009987~cellular process 83 0.015522 GO:0048518"positive regulation of biological process 13 0.017751 GO:0006915~a poptosis 10 0.020681 GO:0012501"‘programmed cell death 10 0.022838 GO:0008380~RNA splicing 5 0.025273 GO:0008219~cell death 10 0.028062 GO:0009605"response to external stimulus 8 0.028087 GO:0016265"death 10 0.028496 GO:0042108"positive regulation of cytokine biosynthetic 3 0.029302 process GO:0006139~nucleobase, nucleoside, nucleotide and nucleic 30 0.029917 acid metabolic process GO:0010468"‘regulation of gene expression 23 0.030603 GO:0019222~regulation of metabolic process 24 0.036963 GO:0007268~synaptic transmission 5 0.036987 GO:0048522"positive regulation of cellular process 11 0.03955 GO:004S727"positive regulation of translation 3 0.039564 GO:0006350~transcription 22 0.039712 GO:OO45059~positive thymic T cell selection 2 0.040871 GO:0043368~positive T cell selection 2 0.040871 GO:0031323"regulation of cellular metabolic process 23 0.044313 GO:0031328"positive regulation of cellular biosynthetic process 3 0.045134 Table 54: DAVID Pathway analysis of genes expressed higher in ARC than SNpc after treatment with MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300-500 laser- captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and pathway analysis was performed for genes that were significantly higher in ARC than SNpc after MPTP treatment. The biological Gene Ontology (GO) pathways that had the most genes of interest were determined and are listed above. 169 DSaline MAP” * I MPTP Angptu - _ SIRTI - _ C0101 — chr3 * BAGS -3.5 -2.5 -1.5 -0.5 0.5 1.5 2.5 3.5 Log; FC higher In ARC Figure 5-3. Relative expression levels of genes that regulate apoptosis in ARC vs. SNpc of saline and MPTP treated mice. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300- 500 laser-captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and DAVID annotation tools were utilized to determine important biological pathways. Genes involved in regulation of apoptosis are depicted here. Horizontal bars represent means of groups and (*) indicates values of expression levels in ARC that were significantly (p S 0.05) higher than SNpc of the same animals. 170 Table 5-2: DAVID Pathway analysis of genes higher in SNpc than ARC only after treatment with MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and pathway analysis was performed for genes that were significantly higher in SNpc than ARC in saline treated controls. The biological Gene Ontology (GO) pathways that had the most genes of interest were determined and are listed above. 171 Gene Ontology Pathway It of genes in P-value pathway GO:0019226~transmission of nerve impulse 17 0.00 GO:0007268"synaptic transmission 15 0.00 GO:0001505"regulation of neurotransmitter levels 11 0.00 GO:0046928~regulation of neurotransmitter secretion 6 0.00 GO:0007267"'ceIl-cell signaling 17 0.00 GO:0007271"'synaptic transmission, cholinergic 5 0.00 GO:0007269"neurotransmitter secretion 7 0.00 GO:0042417"dopamine metabolic process 5 0.00 GO:0045055"regulated secretory pathway 7 0.00 GO:0044255"cellular lipid metabolic process 18 0.00 GO:0006584"catecholamine metabolic process 5 0.00 GO:0018958"phen0l metabolic process 5 0.00 GO:0014070~response to organic cyclic substance 4 0.00 GO:0042133"neurotransmitter metabolic process 5 0.00 GO:0030534"adult behavior 6 0.00 GO:0051046~regulation of secretion 6 0.00 GO:0043279~response to alkaloid 4 0.00 GO:0003001~generation of a signal involved in cell-cell signaling 7 0.00 60:0006629"lipid metabolic process 18 0.00 GO:0006776"vitamin A metabolic process 4 0.00 GO:0006066"alcohol metabolic process 11 0.00 GO:0007399"nervous system development 18 0.00 GO:0014059"regulation of d0pamine secretion 3 0.00 GO:0014046”dopamine secretion 3 0.00 GO:004504S"secretory pathway 10 0.00 GO:0050433~regulation of catecholamine secretion 1 3 0.00 GO:0035094~response to nicotine 3 0.00 GO:0006775~fat-soluble vitamin metabolic process 4 0.00 GO:0006836"neurotransmitter transport 5 0.00 GO:0042391"regulation of membrane potential 5 0.00 GO:0022008"neurogenesis 12 0.00 GO:0007628"adult walking behavior 4 0.00 GO:0007626"locomotory behavior 9 0.00 GO:0050432"catecholamine secretion 3 0.00 GO:0035264~multicellular organism growth 5 0.00 GO:0032940~secretion by cell 10 0.00 GO:0048699"generation of neurons 11 0.00 172 60:0048699Ngeneration of neurons 11 0.00 GO:0048666~neuron development 9 0.00 GO:OO48878”chemical homeostasis 9 0.01 6020051179~localization 48 0.01 GO:OO30182~neuron differentiation 10 0.01 GO:0050801~ion homeostasis 8 0.01 6020008202~steroid metabolic process 7 0.01 GO:0008610~lipid biosynthetic process 9 0.01 GO:0007417“central nervous system development 9 0.01 GO:0010033~response to organic substance 4 0.01 GO:0006694~steroid biosynthetic process 5 0.01 60:0051234”estab|ishment of localization 43 0.01 GO:0006576"biogenic amine metabolic process 5 0.01 GO:0006810~transport 42 0.01 GO:0048732"gland development 5 0.01 GO:OO30902~hindbrain development 4 0.01 GO:0001964”startle response 3 0.01 GO:0006725~aromatic compound metabolic process 6 0.01 ‘ GO:0007610~behavior 10 0.01 GO:0055082"cellular chemical homeostasis 7 0.01 GO:0006873~ce|lular ion homeostasis 7 0.01 G020046903~secretion 10 0.01 GO:0008344~adu|t locomotory behavior 4 0.01 GO:0048856"anatomical structure development 34 0.01 GO:0046879”hormone secretion 4 0.02 GO:0021983~pituitary gland development 3 0.02 GO:0050793~regulation of developmental process 9 0.02 GO:0006575~amino acid derivative metabolic process 5 0.02 GO:0051899”membrane depolarization 3 0.02 GO:OO35270~endocrine system development 4 0.02 GO:0042445”h0rmone metabolic process 5 0.02 GO:0021536~diencephalon development 3 0.02 G020009653~anatomical structure morphogenesis 22 0.02 G020040014~regulation of multicellular organism growth 4 0.02 GO:0022604”regulation of cell morphogenesis 4 0.02 GO:0022603~regulation of anatomical structure morphogenesis 4 0.02 173 GO:0008360~regulation of cell shape 4 0.02 GO:0042592~homeostatic process 10 0.02 GO:0007420~brain development 7 0.02 GO:0019725~cel|u|ar homeostasis 8 0.02 G020065008~regu|ation of biological quality 15 0.03 GO:0040018”positive regulation of multicellular organism 3 0.03 growth GO:0009058"biosynthetic process 23 0.03 GO:0000902”ce|l morphogenesis 12 0.03 G020032989“cel|ular structure morphogenesis 12 0.03 GO:OOO7270“nerve-nerve synaptic transmission 3 0.03 6020006766~vitamin metabolic process 4 0.04 GO:0042416”dopamine biosynthetic process 2 0.04 174 Chrna4 F : Chrna6 : lcal : VMAT2 : El Saline cplxl i I MPTP Sv2c P———J: Nr4a2 : sncaip — 1 0"” #3: . . . DAT l-—.-'"i . . . -6 —4 -2 0 2 4 6 8 Log2 FC higher in SNpc Figure 5-4: Expression levels of genes related to DA metabolism, transport and secretion in ARC vs. SNpc of Saline and MPTP treated Mice. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and DAVID annotation tools were utilized to determine important biological pathways. Genes involved in DA metabolism, transport and secretion are depicted here. Horizontal bars represent means of groups and (*) indicates values of expression levels in SNpc that were significantly higher (p s 0.05) than ARC of the same animals. 175 Cluster Analysis of Genes Significantly Higher in Mn SNpc in MPTP-Treated Mice Hierarchial cluster analysis was performed for genes with higher expression in ARC than SNpc after treatment with MPTP. Figure 5-5 depicts the pattern of expression of a small cluster of genes that increase expression in ARC but not SNpc after treatment with MPTP. Further analysis of this small cluster using DAVID demonstrated that at least 2 of these 15 genes, HSP1A and HSP 8, are chaperones that respond to cellular stress and are involved in pathways such as responding to protein stimulus or unfolded (or misfolded) proteins (Table 5-3). 176 ARC SNpc COMFO' MPTP Control MPTP anti Lite: 9130404d14 - cdSBG 8antigen = nsm 4 supe erfma riken 2610507k20 Solute carrier 3farn 14 - FUSE bi ndln ng3 Rec Inltoek rin alpha 6 1inase Tla l1 ewvmv-vm Zinc finger imprintedl Figure 5-5: Cluster Analysis of genes up—regulated in ARC but not SNpc after treatment with MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and cluster analysis was performed to identify genes with similar expression patterns. A cluster was identified with genes that have higher expression in ARC after MPTP than in Controls. Blue = low expression, white = moderate expression, and red = high expression. 177 GO pathway Genes implicated P-value GO:0051789~response to protein stimulus HSP1A, HSP8 0.04 GO:0006986"response to unfolded protein HSP1A, HSP8 0.04 Table 5-3: DAVID Pathway analysis of genes higher in SNpc than ARC only after treatment with MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and cluster analysis was performed using dCHIP. Pathway analysis was performed for a cluster of genes that increased expression in ARC but not SNpc after treatment with MPTP. The biological Gene Ontology (GO) pathways that had the most genes of interest were determined and are listed above. 178 Analysis of Candidate Genes There have been several genes linked to PD. The relative expression levels of these candidate genes were determined in SNpc and ARC of control and MPTP treated animals and are depicted in Figure 5-6. Synphilin had higher expression in SNpc under both saline and MPTP treatment and a-synuclein had higher expression in SNpc only after treatment with MPTP. None of the genes analyzed were significantly higher in ARC when compared to SNpc of control or MPTP-treated animals. 179 Snca erk2 Ucth CH1 Phwkl DSaline I MPTP Stxla Gpr37 SeptS Calbl Sncaip Pitx3 Null _ H: 4 -7.00 -5.00 -3.00 -1.00 1.00 3.00 5.00 7.00 Log2 FC higher in SNpc Figure 5-6: Expression levels of Parkinson's disease related genes In ARC vs. SNpc of Saline and MPTP treated Mice. Male mice (n=8/gr0up) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each animal were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that have been implicated in Parkinson’s disease were identified. Horizontal bars represent means of groups and (*) indicates values of expression levels in SNpc that were significantly different (p< 0.05) from ARC of the same animals. SNCA = a-synuclein, LRRK2 = Leucine rich repeat kinase, UCH-L1 = Ubiquitin carboxyI-terminal esterase L1, D11 = oncogene Dll, Pink1 = PTEN induced putative kinase 1, pitx3 = Paired-like homeodomain transcription factor 3, and Nurr1 = Nuclear receptor subfamily 4, group A, member 2. 180 Real Time PCR Analysis of Candidate Genes The mRNA expression levels of candidate genes were analyzed in tissue punches containing ARC and SNpc of control and MPTP-treated mice using real time PCR. Figure 5-7 demonstrates that parkin mRNA was significantly increased after treatment with MPTP in ARC, but not in SNpc. UCH-L1 levels also increased following treatment with MPTP in ARC, but remain unchanged in SNpc (Figure 5-8). Several other genes including LRRK2, synuclein, D11 and Pinkl had no change in expression after treatment with MPTP (Table 5-4). All of the examined genes had similar expression levels between ARC and SNpc in saline treated controls. 181 3.5 e * ElControl IMPTP 3 . C a 2.5 - 1 D. u".. '0 2 b .3 a E 1.5 - O z .E f 1 ~ & 0.5 - 0 ARC SNpc Figure 5-7: RT-PCR analysis of normalized parkin expression levels in ARC and SNpc in controls and MPTP treated mice. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. ARC and SNpc were microdissected and mRNA was isolated from each region. Real time PCR analysis was performed and expression levels of parkin were normalized to GAPDH expression levels in each region. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 182 3 I E] Control I MPTP 2.5 - 1.5 - UCH-Ll Normalized Expression 0.5 - ARC SNpc Figure 5-8: RT-PCR analysis of normalized UCH-L1 expression levels in ARC and SNpc in controls and MPTP treated mice. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. ARC and SNpc were microdissected and mRNA was isolated from each region. Real time PCR analysis was performed and expression levels of UCH-Ll were normalized to GAPDH expression levels in each region. * indicates significant difference from control. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 183 A. Normalized LRRK2 Expression ARC SNpc Saline 1.3 1 0.1 1.0 1 0.1 MPTP 1.1 1 0.1 0.8 1 0.1 8. Normalized d-synuclein Expression _ ARC SNpc Saline 0.7 1 0.1 1.0 1 0.2 MPTP 0.7 1 0.1 1.0 1 0.2 C. Normalized D11 Expression ARC SNpc Saline 1.3 1 0.1 1.0 1 0.1 MPTP 1.4 1 0.1 1.4 1 0.1 Normalized Pinkl Expression ARC SNpc Saline 0.8 1 0.0 1.0 1 0.0 MPTP 0.8 1 0.1 1.0 1 0.1 Table 5-4: Normalized mRNA expression levels of LRRK2, synuclein, D11, and Pinkl in ARC and SNpc of mice injected with saline or MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. ARC and SNpc were microdissected and mRNA was isolated from each region. Real time PCR analysis was performed and expression levels of LRRK2, synuclein, D11 and Pink1 were normalized to GAPDH expression levels in each region. 184 Discussion: The experiments in this chapter demonstrate the genomic response of TIDA and NSDA neurons under normal control conditions and after treatment with MPTP. These genomic responses were analyzed based on two general hypotheses; that TIDA neurons may upregulate expression of protective genes that allow them to recover from neurotoxic insult, and that NSDA neurons may have mechanisms in place under basal conditions that do not allow these neurons to recover from MPTP treatment. Evidence for the first hypothesis is particularly strong as the recovery of TIDA neurons from DA loss is dependent on synthesis of new proteins following MPTP. Several methods, including unbiased pathway analysis, cluster analysis and examination of candidate genes were utilized to identify these potentially protective or deleterious genes. Analysis of Pathways using DAVID Annotation Tools: Genes that have higher expression in ARC compared with SNpc after MPTP but not after saline treatment are parts of several general and specific biological pathways. Interestingly, one of the pathways is regulation of apoptosis. Certain genes involved in regulation of apoptosis delay cell death and thereby protect the cells from self- destruction in response to MPTP. Some of these genes have been well studied whereas others are relatively unknown. For example, HSPA1A is a member of the HSP70 family of chaperones and can stabilize existing proteins against aggregation and mediate the folding of newly translated proteins in the cytosol and organelles. The 185 involvement of HSPA1A in the UPS and its ability to correct misfolded proteins has previously implicated this gene in PD pathogenesis (Bucci et al., 2000; Sittler et al., 2001; Auluck et al., 2002; Neckers, 2002; Shen et al., 2005). The HSP70 family has been shown to be protective in a variety of PD models, including a-synuclein toxicity and MPTP treatment (Auluck et al., 2002; Shen et al., 2005; Nagel et al., 2008). One property shared by the genes in Figure 5-3 is that they down regulate cell death and their increased expression in ARC following MPTP may be protecting these neurons. In depth functional analysis of these genes, separately and in concert, would be helpful in determining which may act to protect TIDA neurons from toxicity. Pathway analysis of genes that have higher expression in SNpc than ARC under control conditions may reveal pathways that could hinder the ability of NSDA neurons to recover following MPTP treatment. For example, several genes that regulate DA synthesis, transport and secretion are highly expressed in SNpc compared with the ARC under normal conditions (Figure 5-4). Interestingly, genes that are involved in breakdown of cytosolic DA, such as MAO B and mitochondrial AD had similar expression between the two regions. This may indicate that although NSDA neurons have a higher capacity to synthesize, package and secrete more DA, they may not be as efficient in metabolizing cytosolic DA. This may become a problem if large amounts of DA accumulate in the cytosol and is shunted into metabolic pathways that generate ROS or toxic DA adducts. On the other hand, the fact that the recovery of TIDA neurons is dependent on synthesis of new proteins suggests that differences in basal DA metabolism alone cannot underlie the differential susceptibility of NSDA and TIDA 186 neurons. It is possible that after MPTP treatment, NSDA neurons have both increased oxidative stress due to cytosolic DA, and lack the ability to upregulate anti-apoptotic factors and chaperones that can help alleviate oxidative damage. Further investigation into differences in the regulation of DA synthesis, metabolism and release would be required to examine the importance of these processes in determining the differential susceptibility of NSDA and TIDA neurons to acute MPTP. Identification at Candidate Clusters: Cluster analysis of genes with higher expression in ARC than SNpc after MPTP treatment implicates 16 genes with a similar pattern of upregulation following MPTP exclusively in the ARC. Pathway analyses of these genes demonstrate that at least two genes, HSPA1A and HSP 8 are chaperones that respond to stress by properly folding misfolded or damaged proteins. Chaperones are important in situations when oxidative stress can modify proteins and cause them to lose their function or aggregate (Beaucamp et al., 1998; Buzzard et al., 1998; Nagel et al., 2008). As expected, there is much overlap in the genes implicated with cluster analysis with those identified using pathway analysis. Cluster analysis, however, can also serve to implicate genes whose functions are unknown. In this cluster, there are at least 2 genes with unknown function, and it is likely that these genes work in concert with other genes that are also upregulated and may have protective properties. Further functional analysis of the genes in this cluster would be required to determine whether they are protective to 187 TIDA neurons and if so, whether these protective properties can be utilized to rescue NSDA neurons. Analysis of Candidate Genes: There are several genes that have been previously implicated in the pathogenesis of PD. Some of these genes, including Pitx3 and Nurr2, have been reported to have higher expression in the susceptible NSDA neurons versus the resistant TIDA neurons. The results from the microarray analysis confirm previous findings that Pitx3, and Nurr1 have higher expression in SNpc than ARC (Backman et al., 1999; Hwang et al., 2003; van den Munckhof et al., 2003; Chu et al., 2006; Katunar et al., 2008). Surprisingly, microarray analysis did not strongly implicate genes with causative PD mutations. The only gene significantly different amongst the two regions was a—synuclein after treatment with MPTP. While increased expression of a-synuclein has been associated with increased toxicity (Abeliovich et al., 2000; Singleton et al., 2003; Chartier-Harlin et al., 2004; Drolet et al., 2004b), this is unlikely the case in NSDA neurons following MPTP, since blocking protein synthesis does not alter their response to MPTP (Figure 4-16). Furthermore, Affymetrix microarrays provide a ’present’ call for genes that can be reliably detected beyond background noise. Due to this, sometimes genes with low signals are not detected. One of the candidate genes, parkin, which has been shown to be protective in several models of PD, did not show as present on the array. Follow up real time PCR analysis of the 6 genes directly involved in familial PD demonstrates that 188 parkin mRNA levels are similar between the two regions under control conditions but increase significantly only in ARC after treatment with MPTP. The levels of UCH-L1 also increase in ARC after treatment with MPTP, although the magnitude of this change is smaller than that seen with parkin. Interestingly, both parkin and UCH-L1 are components of the ubiquitin proteasome pathway, with parkin acting as an E3 ligase and UCH-Ll as a deubiquinating enzyme. Upregulation of these two genes along with the HSPA1A and HSP 8 chaperones implicate the ubiquitin proteasome pathway as important player in the differential response of NSDA and TIDA neurons to MPTP. Further examination of parkin, UCH-L1 and the chaperones may provide insight about their role in protecting TIDA neurons from toxicity. Genes and Pathways that Potentially Protect TIDA Neurons tram Toxicity. There are several genomic differences between NSDA and TIDA neurons under basal conditions, as well as in response to MPTP treatment that may explain differential susceptibility to the toxin. Basal DA synthesis and metabolism, upregulation of anti- apoptotic factors, as well as chaperones and proteins involved in protein breakdown may play a role in determining susceptibility. Examination of all of these factors is an extensive process that is beyond the scope of this dissertation. However, one factor that stands out after this genomic analysis of the two regions is parkin. Mutations in parkin cause autosomal recessive PD (Kitada et al., 1998; Sriram et al., 2005), which suggests this protein is essential for the survival of NSDA neurons. Furthermore, increased levels of parkin have been shown to protect against a variety of models 189 including a-synuclein toxicity (Petrucelli et al., 2002; Yamada et al., 2005), kainate excitotoxicity (Staropoli et al., 2003), and MPTP toxicity (Paterna et al., 2007). This suggests that the increased levels of parkin in response to MPTP may protect TIDA neurons and allow them to recover from MPTP toxicity. If parkin can protect TIDA neurons, either alone or in concert with other proteins, it can be utilized to protect NSDA neurons. 190 Chapter 6: The Involvement of Parkin in the Recovery of TIDA Neurons from MPTP- Induced DA Loss A. Introduction: Parkin is one of several genes upregulated in resistant TIDA neurons but not in susceptible NSDA neurons following treatment with MPTP. Mutations in this gene cause early onset autosomal recessive PD (Kitada et al., 1998) with largely similar pathology to sporadic PD, with the exception that most patients carrying the parkin mutation lack Lewy bodies (Cookson, 2005). Parkin protein has long been demonstrated to have protective properties in several models of neurodegeneration as well as tumorigenesis (Staropoli, 2008). Two general approaches have been utilized to determine the protective functions of parkin: genetic KO models and transient KD or overexpression. Models utilizing transient expression or KD of parkin have been particularly successful at showing its protective properties. Lentiviral delivery of parkin prevents degeneration of NSDA neurons caused by overexpression of a-synuclein in the rat brain (Petrucelli et al., 2002). Adeno-associated viral delivery of parkin in rat NSDA neurons can also protect these neurons from 6-OHDA toxicity (Vercammen et al., 2006; Manfredsson et al., 2007). Parkin also protects cells in vitro from MPP+, hydrogen peroxide, and serum withdrawal (Hyun et al., 2005). Transient knock down of parkin using RNA interference makes neurons more susceptible to kainate excitotoxicity (Staropoli et al., 191 2003) suggesting that increase in parkin is protective and decrease in the levels of this protein cause the cells to become more susceptible. On the other hand, none of the transgenic lines of mice with mutations in parkin show NSDA neuron pathology or hypersensitivity in response to toxin administration (Perez and Palmiter, 2005; Thomas et al., 2007b). This is interesting because the same mutations in humans cause autosomal recessive PD. There is considerable evidence for genetic compensation in parkin KO mice. For example, one would expect that loss of parkin would lead to accumulation of substrates that are normally tagged by this E3 ligase, however this is not the case in parkin KO mice suggesting other members of the UPS may be upregulated to compensate for loss of parkin (Periquet et al., 2005). Indeed upregulation of several members of the UPS, as well as chaperones and other proteins has been reported in these KO mice suggesting data gathered from these KO mice should be interpreted with caution (Goldberg et al., 2003; Perez et al., 2005; Perez and Palmiter, 2005). It should be mentioned that this genetic compensation seems to be limited to mouse models. Drosophila missing the parkin homologue have severe mitochondrial pathology and loss of DA neurons that can be rescued by expression of human parkin (Clark et al., 2006). In the mouse transient manipulation of gene expression has provided a better tool for studying the functions of parkin without confounding genetic compensations. The 12 exons making up the parkin gene encode a 465 amino acid protein with a pair of Really Interesting New Gene (RING) finger motifs, and in-between RING finger 192 (IBR) and ubiquitin-like domains (Figure 6-1). As with many other proteins with RING domains (Jackson et al., 2000), parkin can act as part of the UPS and tag substrates with poly-ubiquitin chains at their lysine residues. Parkin interacts with E2 conjugating enzymes and its many substrates include (but are not limited to) synphilin-1, o- glycosylated a-synuclein, C-terminus of the HSC70 interacting protein (CHIP), Parkin- associated endothelin receptor (PAELR), Cell division cycle related 1A (cdc-RellA), cyclin E, synaptotagmin XI, Septin 5 (septS) and PARK2 co-regulated (PACRG) (Cookson, 2005). Figure 6-1 demonstrates how E3 ligase activity of parkin may protect cells against build up of parkin substrates such as o-glycosylated a-synuclein. Of the known parkin substrates, synphilin has higher expression in NSDA neurons when compared with TIDA neurons (Figure 5-7) suggesting its accumulation may contribute to toxicity in this subset of neurons. 193 _‘m -(a <— / ubiqultlnatlon *2 . a f¥fi$ ' “A“ . ‘ ,1 .; ".Sf Ol-synucleln o-glyoosylatlon proteosome l' - fibril peptides ubiquitin formation? monomer Figure 6-1. Structure and E3 ligase activity of parkin. The parkin protein consists of the Ubiquitin-like Domain (UBL)/Unique Parkin Domain (UPD) region that binds substrates such as glycosylated a-synuclein and two Really Interesting New Gene (RING) finger domains with an In Between Ring domain (IBR) which can recruit specific E2 ubiquitin conjugating enzymes and add ubiquitin monomers to substrates. The UBL domain can also interact with the proteasome, targeting poly-ubiqutin tagged substrates for degradation. Figure from Haywood and Staveley (2004) 194 Recently, parkin has also been shown to modify proteins via mono- ubiquitylation (Hampe et al., 2006), which modifies the molecular chaperone HSP70 without tagging it for degradation. This mono-ubiquitylated HSP70 is likely to change its function (Moore et al., 2008) in ways that are yet to be elucidated Interestingly, HSP70 mRNA levels increase in TIDA neurons following MPTP treatment, suggesting the mono-ubiquitylating function of parkin may be important in protecting these neurons following MPTP treatment. Parkin has also been shown to be protective against mitochondrial dysfunction. In PC12 cells, increased expression of parkin delays mitochondrial swelling and cytochrome c release induced by the mitochondrial toxin ceramide (Darios et al., 2003). Furthermore, Drosophila lacking the parkin homologue have mitochondrial dysfunction that can be rescued by replacement with human parkin (Clark et al., 2006). Interestingly, pink1 KO flies have similar mitochondrial pathology as parkin KO flies. The Pinkl gene codes for a mitochondrial kinase and mutations in this gene lead to autosomal recessive PD with similar pathology to patients with parkin mutations. The functions of parkin and Pinkl have been connected because parkin overexpression can rescue the mitochondrial dysfunction and DA loss caused by loss of Pink1. On the other hand, Pinkl overexpression cannot rescue pathology seen in parkin KO flies, suggesting parkin’s protective function is downstream of Pinkl (Clark et al., 2006). The ability of parkin to be protective in so many different pathways implicated in the pathogenesis of PD is dependent on its ability to attach ubiquitin molecules to its 195 substrates, thereby tagging these proteins for breakdown or altering their function or localization within the cell (Cookson, 2005). As depicted in Figure 6-2, it has been proposed that selective vulnerability of DA neurons in PD is due to the attachment of DA adducts to cystenyl residues of parkin, which abolishes its E3 ligase activity (LaVoie et al., 2005). A major criticism of this hypothesis has been that parkin can be damaged by other stressors such as hydrogen peroxide or nitric oxide, and if DA plays a major role in PD pathogenesis, one would expect neurodegeneration of all DA neurons in PD (Cookson, 2005). Figure 5-7 suggests that parkin mRNA is upregulated in TIDA (but not NSDA) neurons following MPTP treatment. Upregulation of this molecule may be neuroprotective during a time when large amounts of DA are displaced into the cytoplasm, and in combination with MPP+-induced ROS, can damage the cells. This upregulation of parkin, alone or in concert with other protective proteins, may therefore explain the differential susceptibility of these DA neuronal populations to MPTP. 196 Parkin Resistant . neurons FRI-"IKZ mutatlons Absent, inactive 7 ~ , or misfolded 1°“ °' F3 9 ’ park'n PM" Ilgase actMty Recessive parki nsonism Triggering ..., Neuronal ...—q Vulnerable events 3“” neurons Loss of fifiuroprotection Sporadic Parkinson disease Figure 6-2. The proposed role of parkin in selective degeneration of DA neurons. Parkin may contribute to sporadic PD as well as familial forms caused by mutations in the gene. Auto- oxidized DA in the cytosol can react with parkin RING finger cystenyl residues and thereby inactivate this protective molecule. This loss of parkin function can lead to enhanced cell death in DA neuron populations. This hypothesis does not address differential susceptibility amongst DA neuronal populations in PD, as well as other factors known to inactivate parkin such as hydrogen peroxide and nitric oxide. Figure from Cookson (2005). 197 B. Hypothesis Induction of parkin protein expression following treatment with MPTP allows recovery of TIDA neurons from MPTP-induced DA loss. C. Experimental Design Parkin Protein Levels Following a Single Injection of MPTP A single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) was administered and animals were killed by decapitation 8 h later. Immediately after decapitation brains were removed, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for Western blot analyses. The details of methods for animal use, drug administration, tissue preparation, and Western blot analyses are described in Chapter 2 (Methods, Sections. A-F). Mrochemical Responses of TIDA negrons to MPTP in Parkin KO gnd WT Mice Parkin KO mice (F6) and WT littermates were injected with MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed by decapitation 16 h later. Immediately after decapitation brains were removed, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for neurochemical analyses. Immediately following decapitation, tail snips were collected and DNA isolated from these samples was used to confirm genotype. The details of methods for animal use, drug administration, tissue preparation, genotyping and neurochemical analyses are described in Chapter 2 (Methods, Sections. A-D). 198 Neurochemical Responses of TIDA Neurons to MPTP FollowinLLentivirgl Knock Dawn of ECLkifl. Lentivirus expressing one of 3 shRNA constructs against parkin or a non-target shRNA under the control of the U6 promoter was bilaterally injected into the ARC of C57bl/6 mice. A non injected control group was also included that did not undergo surgery. Animals were injected with MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) 20- 28 days following viral injection and animals were killed by decapitation 16 h later. Immediately after decapitation brains were removed, ME was dissected from the ventral surface of the brain, and the brain was frozen on dry ice for neurochemical and Western blot analyses. The details of methods for animal use, drug administration, tissue preparation, and neurochemical and Western blot analyses, as well as details of lentivirus injections are described in Chapter 2 (Methods, Sections. A-E and O). D. Results Lm‘kin Protein Levels Followinia Single Injection of MPTP Figure 6-3 demonstrates that 8 h following treatment with MPTP, there is a significant increase in parkin protein levels in the ME but not in ST of C57bl/6 mice. In saline treated controls, parkin protein levels were similar between ME and ST. 199 200 - El Saline * I MPTP 180 - 160 - 140 - 120 - 100 F Parkin RDU 60- 4o- ME ST Figure 6-3. The effects of a single injection of MPTP on parkin levels in median eminence (ME) and striatum (ST). Male mice (n=8/group) were treated with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 8 h later. Parkin levels were determined by Western blotting and normalized to GAPDH to get relative density units (RDU). Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 200 Neurochemical Responses of TIDA Neurons to MPTP in Parkin K0 and WT Mice Figure 6-4 demonstrates that no significant change can be detected in DA concentrations in ME 16 h following treatment with MPTP in parkin KO or WT littermate mice. ST DA levels were significantly lower 16 h following MPTP treatment in both parkin K0 and WT mice (Figure 6-5). 201 l:l Saline I MPTP 250 r 200 - 150 >- 100 F DA(ng/mg protein) Figure 6-4. The effects of a single injection of MPTP on DA levels in the median eminence (ME) of parkin knock out (K0) and wild type (WT) mice. Male parkin K0 and WT littermate (F6) mice (n=8/group) were treated with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 16 h later. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. 202 l:l Saline I MPTP 300 - 250 r 200 - 150 - 100 - DA(ng/mg protein) WT KO Figure 6-5. The effects of a single injection of MPTP on DA levels in the striatum (ST) of parkin knock out (K0) and wild type (WT) mice. Male parkin K0 and WT littermate (F6) mice (n=8/group) were treated with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 16 h later. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 203 Neurochemical Responses of TIDA Neurons to MPTP Following Lentiviral Knock Dawn of Beckie Figure 6-6 demonstrates that C57bl/6 mice that received lentiviral particles carrying one of 3 parkin shRNA constructs had significantly lower DA levels in ME 16 h following MPTP treatment. Non-injected control mice that did not undergo surgery and those injected with lentiviral particles carrying a non-target shRNA had unchanged DA levels after injection with MPTP. Stereotaxic injection of lentiviral particles carrying parkin or non-target shRNA alone did not alter DA levels in ME. DA levels in ME of MPTP-treated mice injected with parkin shRNA significantly correlates (r=0.61; p-value <0.01) with parkin levels in ARC of the same animals (Figure 6-7). There was no correlation between parkin levels and DA concentrations in animals that received parkin shRNA lentivirus (r= -0.32, p= 0.18), animals that received non-target shRNA and saline (r= -0.52 , p= 0.18) or MPTP (r= -0.33, p= 0.66) or animals that did not undergo surgery and received saline (r= -0.04, p= 0.93 ) or MPTP (r= 0.17, p= 0.78). No differences in NE, 5HT or S-HIAA concentrations were detected in ME of mice injected with MPTP, lentivirus or a combination of the two (Table 6-1). ST DA levels decreased following MPTP treatment in all groups, and the magnitude of DA decrease did not differ between animals that received stereotaxic lentiviral injections of scrambled non-target or parkin shRNA and non-injected controls (Figure 6-8). 204 250 F 200 - I 150 - 100 P DA (ng/mg protein) 50- Non—Injected Non-target 143 shRNA 14S shRNA 147 shRNA Figure 6-6. The effects of parkin shRNA administration and a single injection of MPTP on DA levels in the median eminence (ME). Male c57bl/6 mice (n=9/group) received bilateral stereotaxic injections of lentivirus (500 nl) containing non-target or one of 3 parkin shRNA sequences (143, 145 or 147). A non-injected group was also included in the study and did not undergo stereotaxic surgery. All mice were treated 20-28 days following surgery with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 16 h later. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 205 180 - 160 - I 140 - 120 - 100 - 80- DA concentrations 601 I 20- 0 l l l l I l 1 I I 0 5 10 15 20 25 30 35 40 45 Pa rkin Protein Levels Figure 6-7. The relationship between DA concentrations and parkin levels in ARC of MPTP treated mice injected with parkin shRNA (143, 145, or 147 sequences). Male c57bl/6 mice (n=9/group) received bilateral stereotaxic injections of lentivirus (500 nl) containing non-target or one of 3 parkin shRNA sequences (143, 145, or 147). A non-injected group was also included in the study and did not undergo stereotaxic surgery. All mice were treated 20-28 days following surgery with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 16 h later. Pearson’s correlation was performed to determine the relationship between DA concentrations and parkin levels in mice receiving parkin shRNA. 206 NE concentrations (ng/mg protein) in ME Non-Injected Non-Target 143 shRNA 145 shRNA 147 shRNA Control 48.5 1 10.8 48.3 1 4.0 53.9 1 10.7 53.1 1 4.2 53.1 1 4.2 MPTP 57.6 1 8.8 56.5 1 6.5 45.6 1 5.9 45.6 1 5.9 53.4 1 3.4 5-HT concentrations (ng/mg protein) in ME Non-Injected Non-Target 143 shRNA 145 shRNA 147 shRNA Control 13.5 1 1.7 12.7 1 1.6 14.4 1 6.6 13.0 1 1.2 13.0 1 3.4 MPTP 17.7 1 4.4 12.1 1 2.4 19.1 1 4.5 12.2 1 1.5 11.1 1 1.9 5-HIAA concentrations (ng/mg protein) in ME Non-Injected Non-Target 143 shRNA 145 shRNA 147 shRNA Control 8.7 1 0.9 7.3 1 0.4 8.4 1 2.9 8.6 1 0.7 7.7 1 1.0 MPTP 9.9 :l: 1.0 7.8 1 0.6 7.8 1 1.3 8.8 1 1.2 7.4 1 0.7 Table 6-1. Norepinephrine (NE), serotonin (S-HT) and (S-HIAA) concentrations in the median eminence (ME) following administration of shRNA to control and MPTP-treated mice. Male c57bl/6 mice (n=9/group) received bilateral stereotaxic injections of lentivirus (500 nl) containing non-target or one of 3 parkin shRNA sequences (143, 145 or 147). A non-injected group was also included in the experiment that did not undergo stereotaxic surgery. All mice were treated 20-28 days following surgery with either MPTP (20 mg/kg; s.c.) 0r saline (10 ml/kg; s.c.) and killed 16 h later. 207 250 - 200 ~ ' I ' l _ l .E 2 9 150 - Q * 00 e * ... g alt alt < 100 - Q 50 . 0 Nl NT 143 145 147 Figure 6-8. The effects of parkin shRNA administration and a single injection of MPTP on DA levels in the striatum (ST). Male c57bl/6 mice (n=9/group) received bilateral stereotaxic injections of lentivirus (500 nl) containing non-target or one of 3 parkin shRNA sequences (143, 145 or 147). A non-injected group was also included in the experiment that did not undergo stereotaxic surgery. All mice were treated 20-28 days following surgery with either MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.) and killed 16 h later. Columns represent means of groups and vertical bars represent +1.0 standard error of the mean. (*) indicates values for MPTP treated mice that were significantly different (p< 0.05) from saline treated controls. 208 Discussion: The experiments in this chapter demonstrate that mice that received lentiviral parkin shRNA had stunted recovery of MPTP-induced DA loss in TIDA neurons. These results are consistent with previously described protective properties of parkin (Petrucelli et al., 2002; Darios et al., 2003; Staropoli et al., 2003; Haywood and Staveley, 2004; Jiang et al., 2004b; Cha et al., 2005; Hyun et al., 2005; Yamada et al., 2005; Vercammen et al., 2006; Manfredsson et al., 2007; Poole et al., 2008) and suggest that upregulation of this protein in TIDA neurons following treatment with MPTP. On the other hand, TIDA neurons in parkin KO mice had no deficits in recovery following MPTP treatment, directly contradicting data from the K0 study. The lack of difference between KO mice and their WT littermates mirrors other studies showing no difference in susceptibility of other neuronal populations in parkin KO mice (Goldberg et al., 2003; Perez et al., 2005; Perez and Palmiter, 2005; Periquet et al., 2005; Thomas et al., 2007a). This is likely due to genetic compensation in parkin KO mice which makes this an inferior model to study of the functions of this protein (Periquet et al., 2005). Parkin protein levels are upregulated in the axon terminal regions of TlDA neurons 8 h following MPTP treatment. The upregulation of this protein likely plays a role in the recovery of TIDA neurons from MPTP-induced DA loss. Parkin protein levels are not upregulated in NSDA neurons following MPTP treatment, suggesting this difference may underlie the differential susceptibility of these neuronal populations. The results of experiments presented in this chapter confirm previous findings that NSDA neurons of parkin KO mice and WT littermates have a similar loss of DA 209 following treatment with MPTP (Thomas et al., 2007b). Interestingly, TIDA neurons of parkin K0 and WT littermates both recover from MPTP-induced DA loss by 16 h post- treatment. This leads to two possible hypotheses; 1) that parkin is not responsible for the DA recovery, or that 2) parkin KO mice have upregulation of other protective genes that compensate for the lack of parkin. Compelling evidence in the literature argues for the latter hypothesis since parkin KO mice are reported to have higher expression levels of chaperones as well as members of the UPS (Periquet et al., 2005). These upregulated proteins likely compensate for lack of parkin and explain why loss of parkin function causes PD in humans but does not cause pathology in aged KO mice or alter the sensitivity to toxins such as MPTP, 6-OHDA, and paraquat (Perez et al., 2005; Perez and Palmiter, 2005; Thomas et al., 2007a). Alternatively, the lack of difference could be due to species differences since several studies have demonstrated that disease causing mutations do not necessarily cause symptoms or altered sensitivity to toxins in mouse models, but do mimic disease pathogenesis in other species such as drosophila (Feany and Bender, 2000; Hashimoto et al., 2003; Clark et al., 2006; Thomas et al., 2007b) There is a general discrepancy about the protective role of parkin between studies utilizing parkin KO mice and those that transiently KD or overexpress the protein. Since the K0 and overexpression strategies utilize various delivery methods of the protein or shRNA, it is unlikely that these techniques are flawed and more likely that parkin KO mice have upregulation of other proteins that compensate for protective roles of parkin. As such, parkin KO mice are not the best model for studying 210 the protective role of parkin and it would be premature to dismiss the possibility that parkin may be protecting TIDA neurons based on negative results from KO mice. Viral delivery of 1 of 3 parkin shRNA sequences, but not a non-target shRNA sequence prevents the full recovery of TIDA neurons from MPTP-induced DA loss. This establishes a cause and effect relationship between parkin and DA recovery in TlDA neurons. While ME, 3 small region of less than 15 pg of total protein, was used to determine neurochemical content and therefore could not be used to confirm parkin K0 in this region, parkin protein levels in ARC and DA levels in ME demonstrate a statistically significant relationship in mice treated with any of the parkin shRNA sequences and MPTP. This positive correlation (r=0.61, p-value<0.01) demonstrates that mice with lower levels of parkin protein in ARC have lower DA concentrations in ME following MPTP administration. This correlation was absent in mice that received the non-target shRNA virus, or animals that did not undergo surgery but received MPTP. This correlation was also absent in all groups treated with saline, suggesting low parkin levels alone do not cause loss of DA. The fact that DA concentrations were not lower after MPTP treatment in mice receiving lentiviral delivery of non-target sh RNA suggests factors such as stereotaxic surgery, off-target effects of virus or shRNA, inflammatory response due to presence of virus did not contribute to lower DA levels in mice receiving parkin shRNA lentivirus. One pitfall of this experiment was that due to the time required for each stereotaxic surgery, animals had to be injected with virus over a period of 2 weeks, with the non- target shRNA lentivirus animals injected first, followed by 143, 145 and 147 parkin 211 shRNA constructs. It is unlikely that the short time between surgeries accounts for differences amongst non-target shRNA and parkin shRNA groups since lentiviral delivery provides non-transient expression of the shRNA due to incorporation into the host genome. Furthermore, the fact that parkin protein levels in ARC correlate significantly with DA levels in parkin shRNA treated animals suggests that the decreased DA levels in these animals is dependent on the efficiency of parkin KD. Other controls such as animals that did not undergo surgery confirm previous findings that TIDA neurons have full recovery from MPTP-induced DA loss by 16 h post injection. Comparison of other groups with non-injected mice also demonstrate that surgery, delivery of a non-target shRNA, or parkin shRNA alone do not alter DA levels in ME. ST DA concentrations demonstrate that similar levels of MPTP successfully penetrated the brain across different groups and that the differences in DA levels in ME are specific to TIDA neurons and not a global non-specific effect. Unchanged NE, 5-HT and S-HIAA levels in ME also demonstrate that the decrease in DA following MPTP in animals that received parkin shRNA is specific to DA containing neurons. Overall, the experiments in this chapter demonstrate that parkin plays an important role in the recovery of TIDA neurons from MPTP-induced DA loss. Further characterization of the time course of events following MPTP treatment in mice with low parkin levels will provide a temporal window and future mechanistic studies will determine how parkin exerts its protective effects. Examination of other upregulated factors may also bring into play the mechanisms by which parkin infers protection and whether its protective properties in this region rely on co-expression of other proteins. 212 Upregulation of parkin levels may be used as an approach to protect the susceptible NSDA neurons in PD models and eventually translated to a protective mechanism for NSDA neurons in PD. Furthermore, the exact mechanism by which parkin promotes the recovery of DA in the axon terminal region of TIDA neurons remains unclear. Parkin interacts with enzymes in the DA metabolism pathway and may affect the recovery of TIDA neurons following MPTP by directly interfering in this pathway. Parkin inhibits the expression of MAO (Jiang et al., 2006) and may therefore prevent DA from being broken down and allow for more packaging of DA, although this is counter- intuitive if cytosolic DA is harmful to cells. Parkin can also increase surface expression of DAT (Jiang et al., 20043) but it is unclear whether it can do the same for the low affinity high volume DA transporter. If so, more DA may be taken back up and repackaged following MPTP treatment in TIDA neurons accounting for the DA recovery. On the other hand parkin may act indirectly to diffuse oxidative stress thereby allowing the cell to return to a state where it can synthesize and package DA again in a normal fashion resulting in replenishment of DA stores. 213 Chapter 7. Concluding Remarks PD is a progressive and devastating disease causing motor abnormalities that disable patients who are forced to give up activities they enjoy, acquire considerable medical costs and have increased mortality (Wermuth et al., 1995; Schenkman et al., 2001; D'Amelio et al., 2006). Based upon 2004 estimates, each year PD costs $34 billion in direct health—related expenses, disability-related costs and lost productivity in the US alone (Whetten-Goldstein et al., 1997; Noyes et al., 2006). Yet symptomatic treatments are only effective for a short time and do nothing to slow the disease process. As the average age of the world’s population rises, more people are likely to be affected by diseases such as PD that have increased prevalence in the elderly. A therapeutic approach aiming to decrease or halt the progressive neurodegeneration in NSDA neurons is critical for improving the quality and duration of life for millions affected with PD. Once neuroprotective therapies have been developed, assays can be put into place to detect PD since half of NSDA neurons have already undergone degeneration at initial clinical presentation. DA Theory for Selective Vulnerability of NSDA Neurons in PD One property of PD is that while several groups of neurons show pathological abnormalities such as Lewy body formation, only a subset of neurons undergo selective degeneration in PD (Braak and Braak, 2000). This feature of the disease allows for 214 investigation of properties of the susceptible neurons in comparison with resistant neurons in hopes of discovering neuroprotective agents present only in the resistant neurons. Because the major characteristic of the susceptible neurons is synthesis and transmission of catecholamines such as DA and NE, deleterious properties of these neurotransmitters, particularly DA (which is also the precursor to NE) have been heavily studied and proposed to underlie degeneration. On the other hand, other DA neuronal populations including TIDA neurons do not degenerate in the PD brain. The major purpose of this dissertation was to examine differences between susceptible and resistant DA neurons in PD, with the overall goal of indentifying factors that could be utilized in development of therapies to protect the susceptible neurons. These studies characterize a toxin model that mimics the long progressive degeneration of NSDA, but not TIDA neurons in PD (Chronic MPTP), as well as examine the response of these neuronal populations that mimics early PD (single injection MPTP), before the most susceptible neurons have been lost. Basal Differences Between NSDA and TIDA NeuronaLI Populations Characterization of basal differences between NSDA and TIDA neurons, a goal of this dissertation, provides new insights about potential factors that may influence susceptibility. Interestingly, while ME is a much smaller region than ST, the concentration of stored DA within these regions is similar, suggesting disruptions in DA stores should affect both groups of neurons. On the other hand, genomic analysis of 215 these neuronal populations demonstrates that while both groups are DA neurons, the major differences between NSDA and TIDA neurons is in the way they synthesize, store, secrete and uptake DA. Metabolism of DA, however, is similar between these DA neuronal systems. It could therefore be argued that the ability of NSDA neurons to synthesize and store more DA, but not adequately metabolize this DA could contribute to their demise when exposed to neurotoxic insult. Conversely, since the concentration of DA stores is similar between these two groups of neurons, disruptions in DA should make both neuronal populations susceptible. Indeed, these neuronal populations both displace DA following MPTP administration, suggesting both are exposed to high levels of cytosolic DA that can cause damage by formation of DA quinones and ROS. Even more direct evidence against the role of DA as the only culprit comes when TIDA neurons become unable to recover from MPTP damage when protein synthesis is inhibited. This evidence strongly argues that the differential susceptibility of these neuronal populations to toxic stimuli is due to the evoked response of TIDA neurons to MPTP, in the form of synthesis of new proteins. Nevertheless, it is important to keep these basal differences in mind when comparing NSDA and TIDA neurons since a combination of basal differences (that may pre-dispose these neurons) and lack of neuroprotective responses may together underlie susceptibility. 216 Response of TIDA Neurons to MPTP The ME is a circumventricular organ, which means it lies in a region that has weakened blood brain barrier. These circumventricular organs serve to monitor the concentrations of toxins in the blood that cannot otherwise cross the blood brain barrier and communicate this to the rest of the brain, which adjusts its responses accordingly. Since these neurons are routinely exposed to stressors that other neuronal populations do not encounter, it is not surprising that they respond to toxic insult with synthesis of protective factors that allow for their recovery. Several genes only have higher expression in these protected TIDA neurons when compared with NSDA neurons following treatment with MPTP, but not under basal conditions. These genes encompassed chaperones, anti-apoptotic factors and components of the UPS. 1 Perhaps not surprisingly, positive regulators of transcription and translation were also upregulated in TIDA neurons following MPTP, suggesting proteomic, as well as genomic increase of protective properties may play a role in the ability of TIDA neurons to recover. One of the factors identified, parkin, is particularly interesting because not only has it been demonstrated to have protective effects in a wide array of degeneration models, mutations in this gene lead to autosomal recessive early onset PD, directly implicating the function of this gene in NSDA neuronal degeneration. One of the major findings in this dissertation is that parkin is upregulated in protected TIDA neurons following MPTP and transient downregulation of parkin via lentiviral delivery of parkin shRNA causes stunted recovery of the resistant TIDA neurons in response to MPTP. 217 This clearly demonstrates that presence and upregulation of parkin in TIDA neurons contributes to their recovery from MPTP-induced DA loss. Interestingly, parkin is not upregulated in the susceptible NSDA neurons following MPTP treatment, and this is accompanied by a lack of recovery of NSDA neurons from MPTP-induced DA loss and eventually cell death. It is important to determine if increased expression of parkin can alter the time course of response of NSDA neurons to MPTP. If parkin allows for recovery of TIDA neurons, it is likely that increased expression of parkin in NSDA neurons can also protect these neurons from toxicity. Hypotheses Regarding the Involvement of Parkin in Recovery of TIDA Neurons In furthering our knowledge of differential susceptibility of NSDA and TIDA neurons, the results from this dissertation have created additional questions regarding the exact mechanisms by which protective factors such as parkin may be functioning. It will be interesting to determine whether mice with KB of parkin can recover from MPTP-induced DA loss at a later time. A time course of the neurochemical and genomic responses of TIDA neurons following parkin KD would serve to determine whether these neurons can eventually recover. Alternatively, decreased parkin expression may lead to permanent damage including cell death. If TIDA neurons of parkin KD mice fully recover at a later time point it would suggest other factors are also involved in this recovery. Identification of other factors upregulated besides parkin may provide clues as to whether these factors work with or independently of parkin to promote cell survival and will serve to identify which pathways are essential for the 218 survival of the cell. The upregulation of these other protective factors in parkin KO mice may account for their lack of deficits when compared with WT mice. Other upregulated genes may use the same promoter or transcription factors to increase alongside parkin and may be important. While the parkin promoter has been identified (West et al., 2001), it is unclear whether any of the other genes that were co- upregulated with parkin in TIDA neurons following MPTP use the same promoter region. Overlap amongst genes upregulated alongside parkin and in parkin KO mice may suggest other proteins that work with parkin, perhaps with similar function, to protect the cells. The only gene that upregulates following MPTP microarray analysis from Chapter 5 and the proteomics data from parkin KO mice (Periquet et al., 2005) was HSPA1A, an inducible member of the HSP70 group. While this protein has been shown to be protective in PD models (Bucci et al., 2000; Auluck et al., 2002; Shen et al., 2005), it would be interesting to determine whether this protective chaperone can work in concert with parkin to protect cells from toxicity. On the other hand, if parkin KD alone can cause cell death, it will provide a model in which physiologically important and relevant mechanisms by which parkin provides protection can be determined. Whether parkin works alone or in concert with other protective factors, the mechanisms by which it exerts protection should be examined. These mechanisms likely include one or all of the pathways highlighted in Figure 7-1. Parkin has been shown to protect against proteolytic stress, oxidative damage, and recently some have suggested it may protect cells by mono-ubiquitylation of other proteins (Petrucelli et al., 2002; Darios et al., 2003; Greene et al., 2003; Staropoli et al., 2003; Haywood and 219 Staveley, 2004; Jiang et al., 2004b; Pesah et al., 2004; Cha et al., 2005; Hyun et al., 2005; Lim et al., 2005; Clark et al., 2006; Hampe et al., 2006; Haywood and Staveley, 2006; Vercammen et al., 2006; Yang et al., 2006; Manfredsson et al., 2007; Paterna et al., 2007; Deng et al., 2008; Moore et al., 2008; Olzmann and Chin, 2008; Poole et al., 2008). While much attention has been focused its E3 ligase activity due to its importance in protecting against proteolytic stress, it is unlikely to be parkin’s only critical function considering that some PD causing parkin mutations do not disturb its E3 ligase activity (Hampe et al., 2006; Matsuda et al., 2006). On the other hand, these mutations may change parkin’s tertiary structure and thereby preventing some of its substrates from being tagged. The fact that chaperones and other members of UPS are upregulated in TIDA neurons following MPTP suggests that pathways dealing with protein refolding or breakdown may be equally as important in the recovery of TIDA neurons from MPTP-induced DA loss. It would be interesting to examine if KD of parkin along with expression of E3 inactive parkin can still induce DA recovery in TIDA neurons. If so, it would suggest the E3 ligase activity of parkin is not the protective function. The ability of parkin to alter the functions of other proteins by lysine-63 linked mono-ubiquitylation may also be important for its neuroprotective effects. While this function of parkin has only been elucidated recently (Lim et al., 2005; Hampe et al., 2006; Moore et al., 2008), one of the few proteins already discovered to be mono- ubiquitylated by parkin, HSP70 is also upregulated in TIDA neurons following MPTP. It 220 would be interesting to determine if these proteins work in concert to protect TIDA neurons from toxicity. KD of HSP70 alone, parkin alone, or a combination of the two followed by MPTP treatment could determine whether these proteins work together to protect TlDA neurons. It would also be interesting to examine whether other proteins that are upregulated following MPTP are mono-ubiquitylated and if so, how this affects the function of these proteins. Finally, it would be interesting to determine whether parkin protects these neurons directly against mitochondrial induced oxidative stress via proteasome independent mechanisms. Parkin KO flies have mitochondrial pathology (Clark et al., 2006) and parkin has been proposed to work downstream of another PD related gene, Pinkl, to protect against mitochondrial damage. The loss of mitochondrial and tissue integrity in PINK1 and parkin mutant flies is proposed to derive from reduced mitochondrial fission (Deng et al., 2008; Poole et al., 2008) and independent of its proteasome activity. It would be important to determine if transient KD of parkin causes mitochondrial deficits in TIDA and NSDA neurons in a mouse model and determine whether parkin infers protection, partially by preserving mitochondrial function. Parkin’s involvement in a variety of pathways leads to the hypothesis that it protects the cells via several different mechanisms that are likely to occur in parallel all resulting in promotion of cell survival and inhibition of cell death (Figure 7-1). NSDA neurons may be particularly susceptible to toxicity because of their inability to upregulate this protein and the damage caused by cytosolic DA. ln PD patients carrying 221 the parkin mutations, one would expect other DA systems to be affected as well, since they lose the ability to upregulate this protective protein. Unfortunately no data has been published about TIDA neurons in these patients so far. 222 Cytosolic a-Synuclein, UCH-L1 DA . mutations Pinkl, D11 mutations Proteolytic Oxidative Stress Stress LEV. x parkin ), Other (chaperones and i anthapoptotlc factors) Cell Death Figure 7-1. The proposed involvement of parkin in pathways that lead to cell death in Parkinson's disease. Oxidative and proteomic stress have long been implicated in pathogenesis of PD and death of NSDA neurons. Cytosolic DA, as well as mutations in various genes can exacerbate the effects of abnormalities in these pathways. Chaperones and anti-apoptotic factors, on the other hand, are known to protect cells from proteolytic or oxidative stress. Here we propose that parkin likely functions by altering all of the above pathways. Parkin's E3 ligase activity can reduce proteolytic stress, it can rescue neurons from oxidative stress by unknown mechanisms and it can modify function of chaperones and anti-apoptotic factors to further protect the cells. The differences in the way parkin expression is regulated in neuronal types can contribute to their differential susceptibility in PD and models of PD. 223 Protection of NSDA Neurons in PD The studies in this dissertation shed light on several pathways and intrinsic factors that likely protect TIDA neurons in a toxin-based model of PD. The fact that these molecules are not upregulated in NSDA neurons may render these neurons susceptible. Experiments aimed at increasing the expression of these proteins, both individually and together, will determine whether these factors can protect the susceptible neurons. Further examination of these molecules and pathways should promote development of therapies, such as parkin overexpression in NSDA neurons of PD patients, which may halt or slow the progressive loss of these neurons in PD. Gene therapy approaches have been brought into light recently in hopes of providing more than just symptomatic treatment for PD patients. Before these models can be routinely employed in PD patients, clinical trials that test the safety and efficacy of gene delivery methods must be established (Lewis and Standaert, 2008). In animal models alone, gene therapy has provided many challenges with both safety and efficacy particularly with viral therapy (Hartman et al., 2008; Sakurai et al., 2008), although delivery of adeno-associated virus has been less problematic in terms of safety (Buch et al., 2008). These issues become even more challenging with clinical trials when the risks of viral therapy and need for sham-surgeries complicate matters greatly. Furthermore, trials of agents that are expected to slow the progression of the disease require longitudinal studies that can take decades. Nonetheless, clinical trials are being conducted with great care and show promise. One study utilizing an adeno- associated viral vector to deliver neurotrophic factors demonstrates some motor 224 improvements in patients but also that the virus is well tolerated for at least one year following surgery (Marks et al., 2008). Clinical studies utilizing delivery of parkin in PD patients may prove helpful in slowing the degenerative loss of NSDA neurons in the disease. 225 Appendix: 226 Figure A-1: Genes with statistically higher expression in ARC than SNpc of saline treated controls. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed 8 h after MPTP or saline treatment. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each mouse were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that were significantly higher in ARC than SNpc by at least 2 fold in saline treated controls were idenfified. 227 Table A-1. Genes with Higher Expression in ARC than SNpc in Control Mice Unigene ID Description Mm.277996 Pro-opiomelanocortin-alpha 9.62 Mm.154796 Neuropeptide Y 8.42 Mm.140765 Endothelin converting enzyme-like 1 8.15 Mm.57138 Sine oculis—related homeobox 6 homolog (Drosophila) 8.02 Mm.434306 Distal-less homeobox 1 7.28 Mm.75498 CART prepropeptide 7.26 Mm.2453 Somatostatin 7.19 Mm.42242 ISL1 transcription factor, LIM/homeodomain 6.80 Mm.336070 G protein-coupled receptor 101 6.44 Mm.414399 Transcribed locus 6.29 Mm.389327 Growth hormone releasing hormone 6.00 Mm.393566 Estrogen receptor 1 (alpha) 6.00 Mm.34797 Cellular retinoic acid binding protein I 5.95 Mm.4655 Galanin 5.89 Mm.2509 Procollagen, type VI, alpha 1 5.65 Mm.200916 Glutathione peroxidase 3 5.41 Mm.1528 Glypican 4 5.38 Mm.208287 FXYD domain-containing ion transport regulator 6 5.24 Mm.102196 RIKEN cDNA 2900001608 gene 5.22 Mm.22650 Serine (or cysteine) peptidase inhibitor, clade A, member 3N 5.21 Mm.41638 RIKEN cDNA 6330527006 gene 5.21 Mm.45054 Guanine deaminase 4.97 Mm.70980 Sortilin-related VPS10 domain containing receptor 3 4.92 Mm.277812 Villin 2 4.85 Mm.150401 Serine/threonine kinase 32A 4.78 Mm.42096 G substrate 4.76 Mm.429391 Transcribed locus 4.71 Mm.20108 Aldehyde oxidase 3 4.70 Mm.283194 Kelch domain containing BB 4.67 Mm.335953 Diacylglycerol kinase kappa 4.61 Mm.36006 Adenylate kinase 7 4.58 Mm.330975 G protein-coupled receptor 103 4.47 Mm.44305 RIKEN cDNA 1100001E04 gene 4.47 Mm.154093 Procollagen, type XXIII, alpha 1 4.47 Mm.405529 Transcribed locus 4.43 Mm.434981 Transcribed locus 4.41 Mm.1440 Tachykinin 1 4.38 Mm.186404 Coiled-coil domain containing 108 4.37 Mm.434527 Transcribed locus 4.36 Mm.2374 Tachykinin 2 4.34 Mm.332425 Corin 4.30 228 0" ° - - I I - e o ; Mm.402016 Transcribed locus 4.30 Mm.284102 Expressed sequence AI467606 4.23 Mm.4489 Potassium voltage-gated channel, subfamily H (eag-related), 4.22 member 1 Mm.397887 Unc-S homolog D (C. deans) 4.21 Mm.246573 RIKEN cDNA 9130014624 gene 4.20 Mm.296819 RIKEN cDNA 3100002J23 gene 4.19 Mm.41580 RABSB, member RAS oncogene family 4.18 Mm.383568 Adult male hypothalamus cDNA, RIKEN full-length enriched library, 4.18 clone:A230069K02 productzhypothetical protein, full insert sequence Mm.4672 G protein-coupled receptor 83 4.15 Mm.128861 Protocadherin 20 4.12 Mm.5137 Double C2, beta 4.09 Mm.64201 Neurotensin 4.08 Mm.77895 Chondrolectin 3.92 Mm.290693 G protein-coupled receptor 6 3.90 Mm.396927 Transcribed locus 3.90 Mm.435441 Laminin, beta 3 3.87 Mm.45207 Melanoma antigen, family L, 2 3.85 Mm.34980 Transcribed locus 3.80 Mm.31056 Coiled-coil domain containing 1098 3.79 Mm.126464 Transcribed locus 3.79 Mm.337409 Complement component 1, q subcomponent-like 2 3.79 Mm.6239 Prodynorphin 3.78 Mm.124728 AarF domain containing kinase 4 3.76 Mm.402920 Transcribed locus 3.75 Mm.273114 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 5 3.74 Mm.273285 Tescalcin 3.70 Mm.3896 Distal-less homeobox 2 3.69 Mm.392524 G protein-coupled receptor 83 3.62 Mm.416176 Transcribed locus 3.62 Mm.39617 Plexin domain containing 1 3.56 Mm.341593 Transmembrane protein 132E 3.54 Mm.262135 Protein kinase inhibitor beta, cAMP dependent, testis specific 3.53 Mm.329578 Neuropilin (NRP) and tolloid (TLL)-like 1 3.50 Mm.38674 Synaptotagmin-like 4 3.49 Mm.41395 Neuroglobin 3.49 Mm.42202 R-spondin homolog (Xenopus laevis) 3.49 Mm.101811 Dynein, axonemal, heavy chain 1 3.48 Mm.330649 Hypothetical LOC433485 3.46 Mm.285934 Monooxygenase, DBH-like 1 3.46 229 - - - n I - o o o; Mm.372587 RIKEN cDNA C730029A08 gene 3.43 Mm.395558 Forkhead box 01 3.43 Mm.435516 Synaptic nuclear envelope 1 3.42 Mm.1791 Dual specificity phosphatase 6 3.39 Mm.10299 Procollagen, type V, alpha 2 3.36 Mm.171378 Uncoupling protein 2 (mitochondrial, proton carrier) 3.36 Mm.287867 Neuronal PAS domain protein 4 3.36 Mm.70775 Cerebellin 2 precursor protein 3.31 Mm.266301 Double C2, alpha 3.24 Mm.413640 Transcribed locus 3.24 Mm.32622 G protein—coupled receptor 179 3.23 Mm.4629 Intercellular adhesion molecule 5, telencephalin 3.16 Mm.344095 Scavenger receptor class A, member 3 3.16 Mm.159995 RIKEN cDNA A930021C24 gene 3.16 Mm.188617 ATPase, Ca++ transporting, plasma membrane 4 3.15 Mm.51187 RIKEN cDNA 6030405A18 gene 3.14 Mm.347711 Procollagen, type IV, alpha 4 3.14 Mm.159201 RIKEN cDNA 4932442L08 gene 3.13 Mm.17993 RIKEN cDNA 1700027N10 gene 3.12 Mm.20355 Wingless—related MMTV integration site 4 3.12 Mm.74610 CDNA sequence BC023892 3.11 Mm.28385 Transmembrane protein 1768 3.07 Mm.27086 Reprimo, TP53 dependent GZ arrest mediator candidate 3.04 Mm.78373 Coiled-coil domain containing 19 3.02 Mm.423531 Neuropilin 1 2.99 Mm.23297 Retrotransposon gag domain containing 4 2.98 Mm.266341 Neuropilin 2 2.97 Mm.283777 Prokineticin receptor 2 2.97 Mm.281700 Huntingtin-associated protein 1 2.94 Mm.287187 Sphingomyelln phosphodiesterase, acid-like 3B 2.92 Mm.40173 Potassium voltage—gated channel, subfamily F, member 1 2.92 Mm.19316 GRAM domain containing 1C 2.91 Mm.296806 SEC14-like 3 (S. cerevisiae) 2.91 Mm.119 Nuclear receptor subfamily 4, group A, member 1 2.90 Mm.29891 Forkhead box 01 2.90 Mm.42257 Tektin 1 2.88 Mm.23114 Dynein light chain roadblock-type 2 2.88 Mm.38450 Septin 9 2.87 Mm.16347 Prepronociceptin 2.86 Mm.976 Flavin containing monooxygenase 1 2.86 Mm.238343 Annexin A2 2.86 230 Table A-1. Continued Unigene ID Description Mm.395657 Transcribed locus 2.83 Mm.4965 LIM homeobox protein 1 2.82 Mm.34901 GH regulated TBC protein 1 2.79 Mm.126193 TRAF2 and NCK interacting kinase 2.78 Mm.35708 Leprecan-like 2 2.75 Mm.329622 CDC42 binding protein kinase gamma (DMPK-Iike) 2.75 Mm.435439 Neuronal guanine nucleotide exchange factor 2.72 Mm.384976 Expressed sequence AW492955 2.71 Mm.20164 Glutathione peroxidase 7 2.68 Mm.401213 Transcribed locus 2.65 Mm.387697 Forkhead-associated (FHA) phosphopeptide binding domain 1 2.65 Mm.364830 Transcription elongation regulator 1-like 2.64 Mm.100144 $100 calcium binding protein A6 (calcyclin) 2.63 Mm.5304 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 2 2.62 Mm.4876 Reticulocalbin 1 2.61 Mm.4351 Arginine vasopressin receptor 1A 2.59 Mm.272814 Paraneoplastic antigen MA1 2.59 Mm.96057 RIKEN cDNA A730017D01 gene 2.56 Mm.433973 Protein tyrosine phosphatase, receptor type, K 2.55 Mm.336898 Ngfi-A binding protein 2 2.53 Mm.334207 RIKEN cDNA 4932409l22 gene 2.52 Mm.110505 lmmunoglobulin superfamily, member 1 2.48 Mm.275648 P02 and LIM domain 7 2.48 Mm.130952 Stam binding protein like 1 2.47 Mm.131530 Calcium/caImodulin-dependent protein kinase II alpha 2.47 Mm.39884 Receptor (calcitonin) activity modifying protein 3 2.44 Mm.20916 Wolfram syndrome 1 homolog (human) 2.44 Mm.268378 Calcium channel, voltajge-dependent, T type, alpha 1H subunit 2.44 Mm.100167 Phosphodiesterase 8B 2.43 Mm.281298 Growth arrest and DNA-damage-inducible 45 gamma 2.43 Mm.46662 Dapper homolog 1, antagonist of beta-catenin (xenopus) 2.42 Mm.2899 Preproenkephalin 1 2.41 Mm.315382 CDNA sequence BC040758 2.40 Mm.39600 RIKEN cDNA C030019l05 gene 2.38 Mm.3468 Suppressor of cytokine signaling 3 2.37 Mm.433558 Transcribed locus 2.37 Mm.356184 EF hand calcium binding protein 2 2.36 Mm.4352 Procollagen, type XVIII, alpha 1 2.36 Mm.132155 RIKEN cDNA C030009122 gene 2.35 Mm.39905 Copine V 2.33 Mm.220358 Glucokinase 2.31 Mm.45741 RIKEN cDNA 1110032F04 gene 2.30 231 Table A-1. Continued Unigene lD Description Mm.434783 Transcribed locus 2.29 Mm.208662 CDNA sequence BC029169 2.29 Mm.360398 Transmembrane channel-like gene family 4 2.29 Mm.275409 Transmembrane protein 46 2.27 Mm.400754 Transcribed locus 2.26 Mm.294225 Parathyroid hormone receptor 2 2.25 Mm.330890 Par-3 partitioning defective 3 homolog B (C. elegans) 2.24 Mm.3957 CDNA sequence BC034204 2.24 Mm.1451 Milk fat globule-EGF factor 8 protein 2.22 Mm.186678 CDNA sequence BC022687 2.21 Mm.308296 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- 2.21 acetylgalactosaminyltransferase-like 1 Mm.334569 RIKEN cDNA 4732435N03 gene 2.21 Mm.93193 RIKEN cDNA 4432405304 gene 2.20 Mm.51340 Ankyrin repeat and SOCS box-containing protein 4 2.20 Mm.23895 Tektin 2 2.19 Mm.32760 RIKEN cDNA 17mm9P1Lgene 2.15 Mm.398518 Calcium channel, voltage-dependent, gamma subunit 4 2.14 Mm.403263 Transcribed locus 2.13 Mm.332102 Pleckstrin homology domain containing, family G (with RhoGef 2.12 domain) member 5 Mm.435564 Ephrin A5 2.12 Mm.87051 Thymocyte selection-associated HMG box gene 2.12 Mm.33304 Potassium channel, subfamily K, member 2 2.11 Mm.40322 Slit homolog 1 (Drosophila) 2.11 Mm.158903 Hepatic leukemia factor 2.10 Mm.142856 LIM homeobox protein 2 2.10 Mm.36446 CDNA sequence BC007180 2.09 Mm.1433 Neuropeptide Y receptor Y2 2.09 Mm.251774 Peroxidasin homolog (Drosophila) 2.09 Mm.25785 LIM domain binding 2 2.07 Mm.3475 Retinoid X receptor gamma 2.06 Mm.28331 Zinc finger, MYND domain containing 10 2.06 Mm.434650 Transcribed locus 2.04 Mm.433168 Transcribed locus 2.01 Mm.283273 Inhibitor of DNA binding 4 2.01 Mm.40038 Apoptosis-inducing factor, mitochondrion-associated 3 2.00 Mm.359762 Peptidylprolyl isomerase (cyclophilin)-like 6 1.99 Mm.222496 Guanine nucleotide binding protein (G protein), gamma 7 subunit 1.99 Mm.80685 Ciliary rootlet coiled-coil, rootletin 1.99 Mm.332684 Kinetochore associated 1 1.96 Mm.390161 Apolipoprotein C-III 1.96 232 Table A-1. Continued Unigene ID Description Mm.284863 BMP and activin membrane-bound inhibitor, homolog (Xenopus 1.96 laevis) Mm.20615 Peroxisomal biogenesis factor 113 1.95 Mm.290207 E26 avian leukemia oncogene 2, 3' domain 1.92 Mm.261591 Insulin receptor substrate 4 1.91 Mm.22708 Serine (or cysteine) peptidase inhibitor, clade H, member 1 1.91 Mm.156919 Cathepsin Z 1.90 Mm.370334 Ataxin 2 binding protein 1 1.90 Mm.24005 Adhesion molecule with Ig like domain 2 1.89 Mm.27063 Thyroid hormone receptor interactor 6 1.89 Mm.121705 RIKEN cDNA A530088l07 gene 1.88 Mm.135129 ATPase, class V, type 10A 1.88 Mm.44561 RAS protein-specific guanine nucleotide-releasing factor 1 1.87 Mm.403636 Transcribed locus 1.86 Mm.425110 Hypothetical protein LOC620695 1.84 Mm.181021 Procollagen, type IV, alpha 2 1.84 Mm.391678 Odd Oz/ten-m homolog 4 (Drosophila) 1.83 Mm.342160 Carbonic anhydrase 10 1.82 Mm.394928 RIKEN cDNA 9430029N19 gene 1.82 Mm.348844 Transmembrane protein 1320 1.82 Mm.7821 Tumor protein D52-like 1 1.81 Mm.393388 0 day neonate thymus cDNA, RIKEN full-length enriched library, 1.81 clone:A430010E21 productzunclassifiable, full insert sequence Mm.4541 SRY-box containing gene 2 1.81 Mm.264210 Dynein, axonemal, heavy chain 9 1.81 Mm.275845 Synapsin II 1.81 Mm.32582 Early B-cell factor 4 1.81 Mm.392175 CREB regulated transcription coactivator 3 1.80 Mm.89201 Zinc finger protein 179 1.80 Mm.258746 RIKEN cDNA 1190002N15 gene 1.80 Mm.35324 Somatostatin receptor 4 1.80 Mm.31203 Glutathione S-transferase, mu 4 1.79 Mm.327654 LON peptidase N-terminal domain and ring finger 3 1.79 Mm.317947 Nuclear factor I/ B 1.78 Mm.158282 RIKEN cDNA 1700029107 gene 1.78 Mm.402837 Transcribed locus 1.78 Mm.28896 START domain containing 10 1.77 Mm.316890 RIKEN cDNA 5330417C22 gene 1.76 Mm.334108 Dachsous 1 (Drosophila) 1.76 Mm.155887 RIKEN cDNA 1600021P15 gene 1.75 Mm.193272 CDNA sequence BC068157 1.74 Mm.436351 Transcribed locus 1.71 233 Table A-1. Continued Unigene ID Description Mm.295397 DEP domain containing 6 1.71 Mm.337647 Zinc finger, BED domain containing 4 1.71 Mm.357108 Transmembrane protein 178 1.71 Mm.422738 WT1-interacting protein 1.69 Mm.386906 Hypothetical protein C430010C01 1.68 Mm.39691 15 days embryo head cDNA, RIKEN full-length enriched library, 1.63 , clone:0930025C07 productzunclassifiable, full insert sequence Mm.295230 Serine protease inhibitor, Kunitz type 2 1.62 Mm.295660 Tetratricopeptide repeat domain 28 1.62 Mm.28534 Leucine rich repeat containing 1 1.62 Mm.132391 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 1.62 Mm.17649 RIKEN cDNA 1700026008 gene 1.62 Mm.29389 Tensin like C1 domain-containing phosphatase 1.61 Mm.18889 Park2 co—regulated 1.59 Mm.390357 RIKEN cDNA 3010026009 gene 1.59 Mm.248445 Tripartite motif protein 25 1.59 Mm.248337 Vasorin 1.58 Mm.28244 CDNA sequence BC022623 1.58 Mm.276279 Zinc finger protein 667 1.58 Mm.87448 Fibronectin type 3 and ankyrin repeat domains 1 1.57 Mm.136238 Zinc finger and BTB domain containing 20 1.57 Mm.419530 Transcribed locus 1.56 Mm.33964 RIKEN cDNA 1110004B13 gene 1.55 Mm.379020 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 1.55 Mm.11545 Rhomboid family 1 (Drosophila) 1.54 Mm.435847 Expressed sequence AV025504 1.54 Mm.232525 Anthrax toxin receptor 1 1.54 Mm.328503 Retinoic acid induced 2 1.54 Mm.395887 SET domain containing 6 1.51 Mm.17964 KH domain containing, RNA binding, signal transduction associated 1.50 3 Mm.6442 Polycystic kidney disease 2 1.50 Mm.269088 Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A 1.50 Mm.434173 Transcribed locus 1.50 Mm.213204 Kv channel-interacting protein 2 1.48 Mm.40329 Calcium/calmodulin-dependent protein kinase I gamma 1.46 Mm.292567 Cysteine-rich with EGF-like domains 2 1.46 Mm.41033 Transformation related protein 53 inducible protein 11 1.45 Mm.236928 START domain containing 8 1.45 Mm.275547 Aristaless related homeobox gene (Drosophila) 1.45 Mm.84294 RIKEN cDNA 3110082l17 gene 1.45 Mm.130607 LY6/PLAUR domain containing 6 1.43 234 Table A-1. Continued Unigene ID Description Mm.204834 Solute carrier family 1 (glial high affinity glutamate transporter), 1.43 member 3 Mm.1786 Adenine phosphoribosyl transferase 1.41 Mm.1485 Ubiquitin-conjugating enzyme E2E 3, UBC4/5 homolog (yeast) 1.39 Mm.70979 G protein-coupled receptor 176 1.38 Mm.196464 Guanine nucleotide binding protein, alpha inhibiting 2 1.37 Mm.389552 F-box protein 10 1.36 Mm.378951 Kinesin family member C3 1.35 Mm.250731 Microtubule associated serine/threonine kinase 3 1.34 Mm.3390 Myosin IB 1.34 Mm.394224 Androgen receptor 1.34 Mm.28520 Sloan-KetteLng viral oncogene homolog 1.34 Mm.81916 Gene model 266, (NCBI) 1.33 Mm.434093 Transcribed locus 1.32 Mm.287163 Btg3 associated nuclear protein 1.31 Mm.331751 Syntaxin binding protein 5 (tomosyn) 1.31 Mm.400752 RIKEN cDNA 2900054C01 gene 1.29 Mm.408473 Transcribed locus 1.29 Mm.416039 Transcribed locus 1.29 Mm.393348 RIKEN cDNA 493OSGZDligene 1.29 Mm.320691 Potassium voltage-gated channel, Shal-related family, member 2 1.29 Mm.128580 DNA methyltransferase (cytosine-5) 1 1.28 Mm.76659 Secretion regulating guanine nucleotide exchange factor 1.28 Mm.193212 Hydroxypyruvate isomerase homolog (E. coli) 1.27 Mm.7852 RIKEN cDNA 2410008K0353ne 1.27 Mm.392845 Androgen receptor 1.27 Mm.424974 MARCKS-like 1 1.27 Mm.170515 Nuclear factor of kappa light chain gene enhancer in B-cells 1.26 inhibitor, alpha Mm.103477 Natriuretic peptide receptor 2 1.25 Mm.25181 Zinc finger, CCHC domain containing 11 1.24 Mm.152121 Recoverin 1.23 Mm.41702 SRY-box containing gene 11 1.23 Mm.87663 Calcium channel, voltage-dependent, gamma subunit 5 1.23 Mm.235590 Leucine rich repeat containing 8 family, member B 1.23 Mm.396682 RIKEN cDNA C030009012 gene 1.22 Mm.89564 Pregnancy upregulated non-ubiquitously expressed CaM kinase 1.22 Mm.252561 Heparan sulfate 6-O-sulfotransferase 2 1.19 Mm.279872 PRP3 pre-mRNA processing factor 3 homolog (yeast) 1.19 Mm.400798 Transcribed locus 1.19 Mm.46675 Kelch repeat and BTB (POZ) domain containing 11 1.18 Mm.388573 Tubulin tyrosine ligase-like family, member 3 1.18 235 Table A-1. Continued Unigene ID Description Mm.394313 Transcribed locus 1.17 Mm.131572 P21 (CDKN1A)-activated kinase 7 1.17 Mm.41681 Transmembrane protein 119 1.17 Mm.246674 DnaJ (Hsp40) homolog, subfamily C, member 1 1.13 Mm.177761 Chloride channel 2 1.12 Mm.427613 ATP-binding cassette, sub-family G (WHITE), member 1 1.11 Mm.42652 N-acetyltransferase 14 1.10 Mm.291864 Hypothetical LOC629059 1.10 Mm.41642 Regulator of G-protein signaling 4 1.09 Mm.314338 Activin receptor IIA 1.07 Mm.37199 Glutathione S-transferase, mu 1 1.07 Mm.738 Procollagen, type IV, alpha 1 1.07 Mm.330055 Aldehyde dehydrogenase 9, subfamily A1 1.06 Mm.338361 G protein-coupled receptor 75 1.06 Mm.311809 Protein tyrosine phosphatase, receptor type, M 1.06 Mm.23258 RAB, member of RAS oncogene family-like 2A 1.06 Mm.38607 N-acetylglucosamine-1-phosphotransferase, gamma subunit 1.05 Mm.100065 RIKEN cDNA 1300012616 gene 1.05 Mm.133607 Leucine rich repeat and fibronectin type III domain containing 2 1.04 Mm.223717 MAD homolog 1 (Drosophila) 1.03 Mm.205421 Fibronectin type III domain containing 3a 1.02 Mm.218844 Annexin A9 1.02 Mm.29253 Exosome component 8 1.01 Mm.268369 Leucine rich repeat containing 47 1.01 Mm.317293 Neuronal growth regulator 1 1.00 236 Figure A-Z: Genes with statistically higher expression in SN pc than ARC of saline treated controls. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed 8 h after MPTP or saline treatment. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each mouse were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that were significantly higher in SNpc than ARC in saline treated controls by at least 2 fold were idenfified. 237 Table A-2. Genes with higher expression in SNpc than ARC in saline treated controls Unigene ID Description Mm.14609 Aldehyde dehydrogenase family 1, subfamily A7 9.91 Mm.397323 Expressed sequence Al853839 7.99 Mm.276829 Solute carrier family 39 (zinc transporter), member 4 7.83 Mm.41970 Dopamine receptor 2 7.51 Mm.72070 Riken cDNA C130021l20 gene 7.33 Mm.84118 Predicted gene, E6639426 7.33 Mm.283137 Cholinergic receptor, nicotinic, alpha polypeptide 6 7.28 Mm.427936 Transcribed locus 7.27 Mm.222729 Guanylate cyclase 2c 6.89 Mm.4298 Transcribed locus 6.40 Mm.39825 Transcribed locus 6.25 Mm.57199 Ret proto-oncogene 6.16 Mm.110444 Cholinergic receptor, nicotinic, beta polypeptide 3 6.15 Mm.308735 Kelch—like 1 (Drosophila) 6.09 Mm.103778 Cholinergic receptor, nicotinic, alpha polypeptide 5 5.96 Mm.3507 Nuclear receptor subfamily 4, group A, member 2 5.74 Mm.23782 Glycosyltransferase 25 domain containing 2 5.45 Mm.268521 Insulin-like growth factor 1 5.16 ; Mm.5076 Tolloid-like 5.10 Mm.288474 Secreted phosphoprotein 1 5.06 Mm.412897 Transcribed locus 5.05 Mm.258708 Early B—cell factor 3 5.01 Mm.52392 RIKEN cDNA A930038C07 gene 5.00 . Mm.406232 Transcribed locus 4.93 ,Wmassssl Calmegin 4.81 Mm.405570 POU domain, class 3, transcription factor 2 4.79 Mm.239941 Pre B-cell leukemia transcription factor 3 4.77 I Mm.71924 Ankyrin repeat domain 38 4.75 Mm.74324 Solute carrier family 35, member D3 4.67 ‘ Mm.29221 Transcribed locus 4.66 Mm.295917 Gene model 540, (NCBI) 4.61 Mm.63569 Cholinergic receptor, nicotinic, alpha polypeptide 3 4.58 Mm.282800 Synuclein, gamma 4.55 Mm.393273 Transcribed locus 4.54 Mm.420796 Transcribed locus 4.53 Mm.37882 CDNA sequence BC065085 4.29 Mm.317854 Pregnancy-associated plasma protein A 4.19 Mm.113278 Potassium voltage-gated channel, delayed-rectifier, subfamily S, 4.17 member 3 Mm.42190 Unc4.1 homeobox (C. elegans) 4.05 238 Table A-2. Continued Unigene ID Description Mm.408043 Transcribed locus 3.99 Mm.217412 Multiple C2 domains, transmembrane 2 3.94 Mm.39599 6561-like 3.87 Mm.275374 Nuclear receptor interacting protein 3 3.86 Mm.292983 Vitrin 3.85 Mm.288805 DEP domain containing 7 3.83 Mm.292100 Fibrinogen-like protein 2 3.81 Mm.361919 Solute carrier family 18 (vesicular monoamine), member 2 3.68 Mm.282751 Anillin, actin binding protein (scraps homoloiDrosophila) 3.67 Mm.154541 RIKEN cDNA 3830431621 gene 3.67 Mm.129387 POU domain, class 3, transcription factor 2 3.66 Mm.10651 GTP cyclohydrolase 1 3.60 Mm.272976 Synaptic vesicle glycoprotein 2c 3.57 Mm.156558 Synaptotagmin XVII 3.46 Mm.30013 RIKEN cDNA 2010011l20 gene 3.45 Mm.83817 Villin-like 3.41 Mm.425860 Leucine rich repeat containingLilB 3.36 Mm.331690 RIKEN cDNA 5930437A14 gene 3.33 Mm.100125 SH3 domain binding glutamic acid-rich protein like 2 3.20 Mm.141275 RIKEN cDNA E130009112 gene 3.17 Mm.72799 Transmembrane protease, serine 5 (spinesin) 3.10 Mm.30172 Ras association (RalGDS/AF-6) domain family 6 3.08 Mm.41993 Solute carrier family 6 (neurotransmitter transporter, dopamine), 3.08 member 3 Mm.1333 Proprotein convertase subtilisin/kexin type 1 3.08 Mm.39151 Transcribed locus 3.04 Mm.422608 RIKEN cDNA 1190017012 gene 2.99 Mm.89682 Membrane bound O-acyltransferase domain containing 1 2.97 Mm.115970 A disintegrin-like and metallopetidase (reprolysin type) with 2.96 thrombospondin type 1 motif, 16 Mm.415763 Transcribed locus 2.95 Mm.220765 Cadherin 8 2.95 Mm.293363 Spindlin family, member 2 2.94 Mm.4860 Protein tyrosine phosphatase, receptor type, U 2.90 Mm.27789 FERM domain containing 43 2.90 Mm.246595 Hypocretin (orexin) receptor 1 2.86 Mm.159989 RIKEN cDNA 2810022L02 gene 2.86 Mm.102211 RIKEN cDNA 9330109K§gene 2.84 Mm.101836 RIKEN cDNA 9630033F20 gene 2.84 Mm.384353 Mab-21-like 1 (C. elegans) 2.82 Mm.391931 Membrane protein, palmitoylated 7 (MAGUK p55 subfamily 2.82 member 7) 239 Table A-2. Continued Unigene ID Description Mm.425526 Fibroblast growth factor 20 2.82 Mm.151332 Peroxin 2 2.81 Mm.39723 Kelch domain containing 8A 2.81 Mm.91728 Transcribed locus 2.81 Mm.397113 Transcribed locus 2.79 Mm.379143 Regulator of G-protein signaling 8 2.78 Mm.250866 Aldehyde dehydrogenase family 1, subfamily A1 2.77 Mm.384108 lmmunoglobulin superfamily, member 4 2.77 Mm.318710 RlKEN CDNA 3632451006 gene 2.71 Mm.186779 G protein-coupled receptor 151 2.70 Mm.7995 Fibroblast growth factor 13 2.69 Mm.125650 Apolipoprotein L, 2 2.68 Mm.360649 RIKEN cDNA A830006F12 gene 2.68 Mm.235938 Fragile histidine triad gene 2.67 Mm.102899 Transcribed locus 2.67 Mm.333264 Glutamate receptor interacting protein 2 2.66 Mm.280624 Testis expressed gene 15 2.64 Mm.211850 Arylsulfatase 6 2.60 Mm.133293 Membrane protein, palmitoylated 7 (MAGUK p55 subfamily 2.58 member 7) Mm.392745 10 days embryo whole body cDNA, RIKEN full-length enriched 2.57 library, clone22610306612 productzunclassifiable, full insert sequence Mm.133615 RIKEN cDNA 2810457I06 gene 2.56 Mm.253736 Palmdelphin 2.55 Mm.209715 Procollagen, type Xl, alpha 1 2.52 Mm.39649 RIKEN cDNA 3230216623 gene 2.50 Mm.30767 RIKEN cDNA 1300003313 gene 2.49 Mm.289643 Cysteine conjugate-beta lyase 2 2.47 Mm.3944 Kallikrein related-peptidase 6 2.46 Mm.130696 RIKEN cDNA 4930589M24 gene 2.45 Mm.434415 Acyl-CoA synthetase long-chain family member 4 2.45 Mm.155708 Ets variant gene 5 2.43 Mm.169261 Von Willebrand factor C and EGF domains 2.43 Mm.5195 Complexin 1 2.40 Mm.211477 Pleckstrin homology-like domain, family B, member 2 2.40 Mm.345834 0 day neonate cerebellum cDNA, RIKEN full-length enriched library, 2.38 clone2C230096|08 productzhypothetical protein, full insert sequence Mm.69013 RIKEN cDNA B430201A12 gene 2.38 Mm.30221 Insulin induced gene 1 2.37 Mm.209232 Contactin associated protein—like 4 2.33 Mm.432481 Forkhead box P2 2.32 240 Table A-2. Continued Unigene ID Description Mm.274308 N-ethylmaleimide sensitive fusion protein attachment protein beta 2.30 Mm.101707 SLIT and NTRK-Iike family, member 4 2.30 Mm.42012 Laminin, alpha 3 2.29 Mm.1292 Tyrosine hydroxylase 2.26 Mm.23462 Elastin microfibril interfacer 2 2.26 Mm.380993 PhosphatidylinositoI-specific phospholipase C, X domain containing 2.26 2 Mm.198414 Serine/threonine kinase 39, STE20/SP51 homolog (yeast) 2.25 Mm.400451 Musashi homolog 2 (Drosophila) 2.24 Mm.252369 Cholinergic receptor, nicotinic, alpha polypeptide 4 2.24 Mm.407309 Transcribed locus 2.23 Mm.394300 Transcribed locus 2.23 Mm.34235 Williams Beuren syndrome chromosome region 27 (human) 2.23 Mm.253 Regulator of chromosome condensation 2 2.22 Mm.285060 FCH and double SH3 domains 1 2.14 Mm.212855 Myosin regulatory light chain interacting protein 2.12 Mm.78923 Phosphatidylinositol-4—phosphate 5-kinase—like 1 2.11 Mm.268536 Myotubularin related protein 11 2.10 Mm.80318 Transmembrane protein 169 2.09 Mm.337023 Membrane-associated ring finger (C3HC4) 4 2.07 Mm.34175 RIKEN CDNA A530047Jll gene 2.06 Mm.37214 Transferrin 2.06 Mm.316210 FAT tumor suppressor homolog 4 (Drosophila) 2.04 Mm.291799 Retinol dehydrogenase 11 2.03 Mm.87676 Transmembrane protein 16K 2.03 Mm.51259 CDNA sequence BC035537 2.01 Mm.51434 Transcribed locus 1.99 Mm.43358 Pre B-cell leukemia transcription factor 1 1.99 Mm.11869 Expressed sequence AI427122 1.98 Mm.435523 Hypothetical protein LOC574403 1.96 Mm.426628 Expressed sequence C87490 1.96 Mm.428708 Special AT—rich sequence binding protein 1 1.95 Mm.226435 UDP-GalzbetaGlcNAc beta 1,3—galactosyltransferase, polypeptide 1 1.95 Mm.224306 Kelch-like 13 (Drosophila) 1.95 Mm.435789 Transcribed locus 1.94 Mm.268317 Pleckstrin homology domain containing, family H (with MyTH4 1.92 domain) member 1 Mm.255246 Cytochrome P450, family 26, subfamily b, polypeptide 1 1.89 Mm.133193 Keratin 222 1.88 Mm.435077 Transcribed locus 1.88 Mm.306954 Anterior pharynx defective 1a homolog (C. elegans) 1.86 Mm.158289 Myosin, heavy polypeptide 14 1.86 241 Table A-2. Continued Unigene ID Description Mm.56097 RIKEN cDNA 0330017J20 gene 1.86 Mm.53865 Ankyrin repeat domain 29 1.85 Mm.4744 Growth differentiation factor 5 1.84 Mm.398133 Transcribed locus 1.83 Mm.292168 Synuclein, alpha interacting protein (synphilin) 1.82 Mm.70371 RIKEN cDNA 2310005E10 gene 1.82 Mm.406348 Transcribed locus 1.82 Mm.41945 Tetratricopeptide repeat domain 22 1.82 Mm.45533 RIKEN cDNA 2310026E23 gene 1.82 Mm.153013 Regulator of G-protein signaling 6 1.81 Mm.335395 RAB6B, member RAS oncogene family 1.81 Mm.435503 AT motif binding factor 1 1.80 Mm.27469 Tetraspanin 2 1.80 Mm.338690 Solute carrier family 35, member F1 1.78 Mm.1892 Corticotropin releasing hormone receptor 1 1.77 Mm.306021 UDP galactosyltransferase 8A 1.76 Mm.32744 Opsin (encephalopsin) 1.75 Mm.240912 Ribulose-5-phosphate-3-epimerase 1.75 Mm.208125 A disintegrin-like and metallopeptidase (reprolysin type) with 1.74 thrombospondin type 1 motif, 6 Mm.434125 Transcribed locus 1.74 Mm.282257 Vav 3 oncogene 1.73 Mm.27005 Visinin-like 1 1.71 Mm.28893 RAP1, GTP-GDP dissociation stimulator 1 1.71 Mm.40068 Tubulin, beta 3 1.70 Mm.407234 Transcribed locus 1.69 Mm.213623 RIKEN cDNA 6430502M16 gene 1.69 Mm.426957 Transcribed locus 1.68 Mm.247457 Secernin 3 1.68 Mm.157069 Delta-like 1 homolog (Drosophila) 1.68 Mm.10214 Tuftelin 1 1.67 Mm.30119 Sterol-C4-methyl oxidase-like 1.67 Mm.431124 CDNA, clonezY260147N17, strandzunspecified 1.64 Mm.393375 Transcribed locus 1.63 Mm.316418 Arginine/serine-rich coiled-coil 1 1.62 Mm.393717 Transcribed locus 1.62 Mm.26479 Zinc fingerprotein 618 1.61 Mm.39330 Heat shock protein 4 like 1.60 Mm.87027 Cell cycle exit and neuronal differentiation 1 1.60 Mm.153566 Meteorin, glial cell differentiation regulator-like 1.59 Mm.248096 RUN and SH3 domain containing 2 1.58 Mm.20453 FK506 binding protein 1b 1.56 242 Table A-2. Continued Unigene ID Description Mm.2445 Acyl-Coenzyme A dehydrogenase, long-chain 1.56 Mm.133370 24-dehydrocholesterol reductase 1.55 Mm.275332 Autophagy-related 7 (yeast) 1.54 Mm.432562 Myelin-associated oligodendrocytic basic protein 1.54 Mm.3093 Serine (or cysteine) peptidase inhibitor, clade E, member 2 1.54 Mm.127058 StAR-related lipid transfer (START) domain containing 4 1.54 Mm.32550 DnaJ (Hsp40) homolog, subfamily C, member 12 1.54 Mm.140158 Cytochrome P450, family 51 1.53 Mm.3970 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 4 (Hu 1.52 antigen D) Mm.196701 Protocadherin beta 12 1.52 Mm.31263 RIKEN cDNA BZ30118H07 gene 1.52 Mm.275387 RIKEN cDNA 1810041L15 gene 1.49 Mm.154303 RIKEN cDNA 4930544621 gene 1.49 Mm.166647 G protein-coupled receptor 158 1.48 Mm.44721 Leucine-rich repeat LGI family, member 2 1.48 Mm.21995 Fatty acid desaturase domain family, member 6 1.47 Mm.125503 RAN binding protein 6 1.47 Mm.410111 Transcribed locus 1.46 Mm.434311 Cytochrome P450, family 2, subfamily u, polypeptide 1 1.46 Mm.102147 16 days neonate cerebellum cDNA, RIKEN full-length enriched 1.44 library, clone:9630041l08 productzunclassifiable, full insert sequence Mm.289915 Damage specific DNA binflg protein 1 1.43 Mm.29098 Galactose mutarotase 1.43 Mm.1517 Synaptobrevin like 1 1.42 Mm.29646 CNDP dipeptidase 2 (metallopeptidase M20 family) 1.42 Mm.377099 Melanoma antigen, family A, 7 1.42 Mm.264036 NAD(P)H dehydrogenase, quinone 2 1.42 Mm.124595 RIKEN cDNA A330008L17 gene 1.41 Mm.41523 RIKEN cDNA 6330416613 gene 1.41 Mm.380683 HIV-1 Rev binding protein 1.40 Mm.220817 Histidine acid phosphatase domain containing 1 1.40 Mm.432488 Male sterility domain containing 1 1.38 Mm.341423 Dedicator of cytokinesis 4 1.38 Mm.182574 Isochorismatase domain containing 1 1.38 Mm.18962 Catenin (cadherin associated protein), alpha 1 1.37 Mm.151962 Cache domain containing 1 1.37 Mm.26564 Oxysterol binding protein-like 11 1.37 Mm.72979 RIKEN cDNA 6430550H21 gene 1.36 Mm.397953 Transcribed locus 1.36 Mm.46782 La ribonucleoprotein domain family, member 2 1.35 243 Table A-2. Continued Unigene ID Description Mm.386792 Zinc finger protein 364 1.35 Mm.24183 Transgelin 3 1.34 Mm.314113 ELOVL family member 6, elongation of long chain fatty acids (yeast) 1.34 Mm.176695 Tripartite motif-containing 59 1.34 Mm.259191 O-linked N-acetylglucosamine (6lcNAc) transferase (UDP-N- 1.34 acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) Mm.93796 Mcf.2 transforming sequence 1.34 Mm.271620 Golgi—specific brefeldin A-resistance factor 1 1.34 Mm.192162 Calsyntenin 2 1.32 Mm.119646 Mitogen-activated protein kinase kinase kinase 7 interacting protein 1.32 3 Mm.194 FMS-like tyrosine kinase 3 1.32 Mm.158827 RIKEN cDNA 2410003P15 gene 1.31 Mm.138434 GRAM domain containing 13 1.31 Mm.40110 6 patch domain containing 3 1.30 Mm.196596 RIKEN cDNA 3110002H16 gene 1.30 Mm.193670 Eukaryotic translation initiation factor 5A2 1.29 Mm.54183 Olfactomedin 3 1.28 Mm.302755 Trinucleotide repeat containing 9 1.27 Mm.433866 Transcribed locus 1.26 Mm.291854 Pleckstrin homology domain containing, family 6 (with RhoGef 1.26 domain) member 3 Mm.425171 RIKEN cDNA 6430524H05 gene 1.25 Mm.341004 ATP-binding cassette, sub-family A (ABC1), member 8b 1.25 Mm.284654 WD repeat domain 37 1.25 Mm.371580 Sorbitol dehydrogenase 1.24 Mm.255026 Acyl-CoA synthetase short-chain family member 2 1.24 Mm.219675 6 elongation factor, mitochondrial 2 1.22 Mm.425766 Neurexin Ill 1.22 Mm.41728 Enoyl Coenzyme A hydratase domain containing 1 1.22 Mm.222685 Ceramide kinase 1.22 Mm.298283 Neurofilament, heavy polypeptide 1.21 Mm.275683 Islet cell autoantigen 1 1.21 Mm.130063 RIKEN cDNA 9330182L06 gene 1.20 Mm.258932 RIKEN cDNA 1810012P15 gene 1.20 Mm.425467 Solute carrier organic anion transporter family, member 331 1.19 Mm.247143 GDP-mannose 4, 6-dehydratase 1.19 Mm.298251 Leucine-rich repeat LGI family, member 1 1.19 Mm.195932 CDC42 effector protein (Rho GTPase binding) 2 1.19 Mm.170855 RIKEN cDNA B230380007 gene 1.18 Mm.945 Protein tyrosine phosphatase, receptor type, E 1.17 Mm.389061 PFTAIRE protein kinase 1 1.17 244 Table A-2. Continued Unigene ID Description Mm.302793 Synaptotagmin IX 1.16 Mm.290085 Cereblon 1.16 Mm.3676 Golgi transport 1 homolog B (S. cerevisiae) 1.16 Mm.5163 Ras-like without CAAX 2 1.15 Mm.33120 Histamine N-methyltransferase 1.15 Mm.275639 Glycine receptor, beta subunit 1.15 Mm.86472 RIKEN CDNA 1200014M14 gene 1.15 Mm.146332 Receptor accessory protein 1 1.14 Mm.433835 Transcribed locus 1.13 Mm.57225 Phosphoglucomutase 2-like 1 1.12 Mm.414514 Transcribed locus 1.11 Mm.291826 Adiponectin receptor 2 1.11 Mm.58836 Citrate synthase 1.11 Mm.251228 RNA terminal phosphate cyclase domain 1 1.10 Mm.34326 Zinc finger, DHHC domain containing 2 1.10 Mm.331630 A kinase (PRKA) anchor protein 2 1.10 Mm.38816 WD repeat domain 36 1.09 Mm.312233 TAF4B RNA polymerase II, TATA box binding protein (TBP)- 1.09 associated factor Mm.289441 Claudin 1 1.08 Mm.54201 Hypothetical protein C030014L02 1.08 Mm.33490 6A repeat binding protein, beta 2 1.07 Mm.432944 Chromatin modifying protein 2B 1.07 Mm.397106 Expressed sequence Al848218 1.07 Mm.6306 ATPase, Ca++ transporting, ubiquitous 1.07 Mm.30737 F—box protein 28 1.06 Mm.44068 RIKEN cDNA 3830406C13 gene 1.05 Mm.24431 TAF13 RNA polymerase II, TATA box binding protein (TBP)— 1.05 associated factor Mm.4065 RIKEN cDNA C230093N12 gene 1.04 Mm.219433 Zinc finger, SWIM domain containing 6 1.03 Mm.2942 Asparagine synthetase 1.03 Mm.347625 Phosphatidylinositol glycan anchor biosynthesis, class 2 1.02 Mm.419813 CUB and Sushi multiple domains 1 1.01 Mm.145488 Tyrosyl—tRNA synthetase 1.01 Mm.146984 Proteasome (prosome, macropain) inhibitor subunit 1 1.00 245 Figure A-3: Genes with statistically higher expression in ARC than SNpc of animals treated with MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/ kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed 8 h after MPTP or saline treatment. Amplified mRNA from 300-500 laser-captured TH immunofluorescent cells from ARC and SNpc of each mouse were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that were significantly higher in ARC than SNpc by at least 2 fold in MPTP treated animals were identified. 246 Table A-3. Genes with higher expression in ARC than SNpc 8h after MPTP treatment Unigene ID Description Log, FC Mm.277996 Pro-opiomelanocortin-alpha 9.74 Mm.140765 Endothelin converting enzyme-like 1 8.79 Mm.57138 Sine oculis-related homeobox 6 homolog (Drosophila) 7.74 Mm.154796 Neuropeptide Y 7.48 Mm.434306 Distal-less homeobox 1 7.13 Mm.336070 G protein-coupled receptor 101 7.02 Mm.75498 CART prepropeptide 6.94 Mm.2453 Somatostatin 6.86 Mm.42242 ISL1 transcription factor, LlM/homeodomain 6.60 Mm.393566 Estrogen receptor 1 (alpha) 6.29 Mm.34797 Cellular retinoic acid binding protein I 6.11 Mm.2509 Procollagen, type VI, alpha 1 6.09 Mm.414399 Transcribed locus 6.02 Mm.4655 Galanin 6.02 Mm.42096 6 substrate 5.69 Mm.429391 Transcribed locus 5.62 Mm.389327 Growth hormone releasing hormone 5.62 Mm.102196 RIKEN cDNA 2900001608 gene 5.48 Mm.200916 Glutathione peroxidase 3 5.43 Mm.208287 FXYD domain—containing ion transport regulator 6 5.40 Mm.150401 Serine/threonine kinase 32A 5.29 Mm.22650 Serine (or cysteine) peptidase inhibitor, clade A, member 3N 5.28 Mm.283194 Kelch domain containing 88 5.27 Mm.1528 Glypican 4 5.20 Mm.44305 RIKEN cDNA 1100001E04 gene 5.09 Mm.64201 Neurotensin 5.07 Mm.70980 Sortilin-related VP510 domain containing receptor 3 5.04 Mm.402016 Transcribed locus 5.00 Mm.2374 Tachykinin 2 4.94 Mm.277812 Villin 2 4.85 Mm.332425 Corin 4.83 Mm.6239 Prodynorphin 4.82 Mm.246573 RIKEN cDNA 9130014624 gene 4.74 Mm.335953 Diacylglycerol kinase kappa 4.72 Mm.397887 Unc-5 homolog D (C. elegans) 4.71 Mm.1440 Tachykinin 1 4.56 Mm.296806 SEC14-like 3 (S. cerevisiae) 4.46 Mm.5137 Double C2, beta 4.44 Mm.45054 Guanine deaminase 4.40 247 Table A-3. Continued Unigene ID Description Mm.45207 Melanoma antigen, family L, 2 4.35 Mm.290693 G protein-coupled receptor 6 4.31 Mm.262135 Protein kinase inhibitor beta, cAMP dependent, testis specific 4.31 Mm.159201 RIKEN cDNA 4932442L08 gene 4.28 Mm.3896 Distal-less homeobox 2 4.26 Mm.4672 G protein—coupled receptor 83 4.25 Mm.284102 Expressed sequence AI467606 4.23 Mm.154093 Procollagen, type XXIII, alpha 1 4.21 Mm.41395 Neuroglobin 4.12 Mm.34980 Transcribed locus 4.08 Mm.238343 Annexin A2 4.07 Mm.128861 Protocadherin 20 4.04 Mm.38674 Synaptotagmin-like 4 4.02 Mm.287187 Sphingomyelin phosphodiesterase, acid-like 3B 3.98 Mm.36006 Adenylate kinase 7 3.97 Mm.413640 Transcribed locus 3.95 Mm.41580 RABSB, member RAS oncogene family 3.89 Mm.126464 Transcribed locus 3.88 Mm.20108 Aldehyde oxidase 3 3.86 Mm.124728 AarF domain containing kinase 4 3.85 Mm.330975 G protein-coupled receptor 103 3.85 Mm.392524 G protein-coupled receptor 83 3.84 Mm.77895 Chondrolectin 3.80 Mm.391246 RIKEN cDNA 2610018603 gene 3.79 Mm.4489 Potassium voltage—gated channel, subfamily H (eag-related), 3.75 member 1 Mm.17993 RIKEN cDNA 1700027N10 gene 3.74 Mm.74610 CDNA sequence BC023892 3.72 Mm.273285 Tescalcin 3.71 Mm.35708 Leprecan-like 2 3.64 Mm.402920 Transcribed locus 3.63 Mm.273114 Gamma—aminobutyric acid (GABA-A) receptor, subunit alpha 5 3.58 Mm.396927 Transcribed locus 3.58 Mm.416176 Transcribed locus 3.57 Mm.171378 Uncoupling protein 2 (mitochondrial, proton carrier) 3.54 Mm.23297 Retrotransposon gag domain containing 4 3.50 Mm.405529 Transcribed locus 3.47 Mm.34901 GH regulated TBC protein 1 3.46 Mm.39617 Plexin domain containing 1 3.36 Mm.41854 Raftlin lipid raft linker 1 3.36 Mm.387697 Forkhead-associated (FHA) phosphopeptide binding domain 1 3.36 Mm.435441 Laminin, beta 3 3.34 248 :- - I I - o o Mm.23895 Tektin 2 3.31 Mm.101811 Dynein, axonemal, heavy chain 1 3.31 Mm.31056 Coiled—coil domain containing 1098 3.27 Mm.42257 Tektin 1 3.27 Mm.296819 RIKEN cDNA 3100002J23 gene 3.26 Mm.327654 LON peptidase N-terminal domain and ring finger 3 3.20 Mm.976 Flavin containing monooxygenase 1 3.20 Mm.1894 CDldl antigen 3.17 Mm.38450 Septin 9 3.15 Mm.384976 Expressed sequence AW492955 3.14 Mm.372587 RIKEN cDNA C730029A08 gene 3.14 Mm.28385 Transmembrane protein 1768 3.11 Mm.285934 Monooxygenase, DBH-like 1 3.10 Mm.126193 TRAF2 and NCK interacting kinase 3.10 Mm.42202 R-spondin homolog (Xenopus laevis) 3.08 Mm.423621 CD44 antigen 3.06 Mm.329622 CDC42 binding protein kinase gamma (DMPK-like) 3.05 Mm.324305 Gem-interacting protein 3.05 Mm.227117 Solute carrier family 30, member 10 3.05 Mm.39600 RIKEN cDNA C030019l05 gene 3.03 Mm.46662 Dapper homolog 1, antagonist of beta—catenin (xenopus) 3.02 Mm.32622 G protein-coupled receptor 179 3.02 Mm.336244 Nuclear RNA export factor 3 3.02 Mm.398480 RIKEN cDNA 3830409H07 gene 3.00 Mm.347711 Procollagen, type IV, alpha 4 3.00 Mm.78373 Coiled—coil domain containing 19 2.99 Mm.334207 RIKEN CDNA 4932409l22 gene 2.98 Mm.341593 Transmembrane protein 132E 2.96 Mm.281700 Huntingtin-associated protein 1 2.95 Mm.220358 Glucokinase 2.90 Mm.4876 Reticulocalbin 1 2.90 Mm.16347 Prepronociceptin 2.88 Mm.1239 Glial fibrillary acidic protein 2.88 Mm.187372 RIKEN cDNA 4632412N22 gene 2.86 Mm.287421 RIKEN cDNA 1700086L19 gene 2.85 Mm.19316 GRAM domain containing 1C 2.84 Mm.10099 Neuronal pentraxin 2 2.84 Mm.2899 Preproenkephalin 1 2.83 Mm.303386 Laminin, alpha 1 2.82 Mm.4352 Procollagen, type XVIII, alpha 1 2.80 Mm.113877 Glycine receptor, alpha 2 subunit 2.79 249 Table A-3. Continued Unigene ID Description Mm.266301 Double C2, alpha 2.77 Mm.1791 Dual specificity phosphatase 6 2.77 Mm.435516 Synaptic nuclear envelope 1 2.75 Mm.292568 Leucine rich repeat transmembrane neuronal 1 2.75 Mm.12743 Testis specific gene A2 2.75 Mm.20916 Wolfram syndrome 1 homolog (human) 2.73 Mm.329578 Neuropilin (NRP) and tolloid (TLL)-like 1 2.72 Mm.110505 lmmunoglobulin superfamily, member 1 2.72 Mm.39884 Receptor (calcitonin) activity modifying protein 3 2.71 Mm.315382 CDNA sequence BC040758 2.70 Mm.1433 Neuropeptide Y receptor Y2 2.70 Mm.360398 Transmembrane channel-like gene family 4 2.70 Mm.284863 BMP and activin membrane-bound inhibitor, homolog (Xenopus 2.68 laevis) _ Mm.51340 Ankyrin repeat and SOCS box-containing protein 4 2.61 Mm.159995 RIKEN cDNA A930021C24 gene 2.59 Mm.132535 Inositol 1,4,5-trisphosphate 3-kinase B 2.59 Mm.87155 Protein phosphatase, EF hand calcium-binding domain 2 2.58 Mm.356184 EF hand calcium binding protein 2 2.58 Mm.130952 Stam binding protein like 1 2.58 Mm.953 Sphingomyelin phosphodiesterase 2, neutral 2.56 Mm.335337 Similar to Protein C6orf117 2.55 Mm.425110 Hypothetical protein LOC620695 2.51 Mm.10299 Procollagen, type V, alpha 2 2.50 Mm.3475 Retinoid X receptor gamma 2.50 Mm.29891 Forkhead box 01 2.49 Mm.5304 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 2 2.48 Mm.275409 Transmembrane protein 46 2.46 Mm.308296 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- 2.44 acetylgalactosaminyltransferase-like 1 Mm.251774 Peroxidasin homolog (Drosophila) 2.43 Mm.256249 Paraneoplastic antigen MA3 2.41 Mm.4965 LIM homeobox protein 1 2.41 Mm.40322 Slit homolog 1 (Drosophila) 2.39 Mm.275751 Coiled-coil domain containing 57 2.39 Mm.272814 Paraneoplastic antigen MA1 2.38 Mm.132391 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 2.38 Mm.434650 Transcribed locus 2.37 Mm.40173 Potassium voltage-gated channel, subfamily F, member 1 2.35 Mm.4351 Arginine vasopressin receptor 1A 2.35 Mm.400754 Transcribed locus 2.35 Mm.275648 P02 and LIM domain 7 2.34 250 Table A-3. Continued Unigene ID Description Mm.433168 Transcribed locus 2.32 Mm.28244 CDNA sequence BC022623 2.31 Mm.3957 CDNA sequence BC034204 2.30 Mm.261591 Insulin receptor substrate 4 2.29 Mm.150294 Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4- 2.29 hydroxylase), alpha polypeptide III Mm.344095 Scavenger receptor class A, member 3 2.28 Mm.24005 Adhesion molecule with lg like domain 2 2.26 Mm.93193 RIKEN cDNA 4432405804 gene 2.26 Mm.268378 Calcium channel, voltage-dependent, T type, alpha 1H subunit 2.25 Mm.398518 Calcium channel, voltage-dependent, gamma subunit 4 2.24 Mm.281298 Growth arrest and DNA-damage-inducible 45 gamma 2.23 Mm.100167 Phosphodiesterase 8B 2.22 Mm.276747 Visual cortex cDNA, RIKEN full-length enriched library, 2.22 clonezK530032101 productzunclassifiable, full insert sequence Mm.225096 lntegrin alpha 6 2.22 Mm.433973 Protein tyrosine phosphatase, receptor type, K 2.20 Mm.20164 Glutathione peroxidase 7 2.18 Mm.41033 Transformation related protein 53 inducible protein 11 2.17 Mm.334569 RIKEN cDNA 4732435N03 gene 2.16 Mm.241063 Expressed sequence AI894139 2.16 Mm.394928 RIKEN cDNA 9430029N19 gene 2.16 Mm.158282 RIKEN cDNA 1700029J07 gene 2.15 Mm.96057 RIKEN cDNA A730017001figene 2.15 Mm.435564 Ephrin A5 2.14 Mm.291877 Bromodomain and PHD finger containing, 3 2.14 Mm.71498 Ligand dependent nuclear receptor corepressor-like 2.14 Mm.290207 E26 avian leukemia oncogene 2, 3' domain 2.13 Mm.33304 Potassium channel, subfamily K, member 2 2.11 Mm.33832 Solute carrier family 14 (urea transporter), member 1 2.11 Mm.84073 Bcl2-associated athanogene 3 2.10 Mm.7214 Annexin A3 2.10 Mm.205421 Fibronectin type III domain containing 3a 2.10 Mm.6388 Heat shock protein 1A 2.10 Mm.435439 Neuronal guanine nucleotide exchange factor 2.09 Mm.80685 Ciliary rootlet coiled-coil, rootletin 2.09 Mm.403263 Transcribed locus 2.08 Mm.276279 Zinc finger protein 667 2.06 Mm.275845 Synapsin II 2.04 Mm.44561 RAS protein-specific guanine nucleotide-releasing factor 1 2.02 Mm.436351 Transcribed locus 2.02 Mm.4541 SRY-box containing gene 2 2.01 251 Table A-3. Continued Unigene ID Description Mm.295397 DEP domain containing 6 2.01 Mm.32760 RIKEN cDNA 1700009P17 gene 2.00 Mm.36446 CDNA sequence BC007180 2.00 Mm.132155 RIKEN cDNA C030009122 gene 1.99 Mm.285 Kinase insert domain protein receptor 1.99 Mm.433558 Transcribed locus 1.98 Mm.283273 Inhibitor of DNA binding 4 1.96 Mm.390712 Potassium channel tetramerisation domain containing 4 1.95 Mm.57175 C083 antigen 1.95 Mm.121705 RIKEN cDNA A530088l07 gene 1.94 Mm.392156 Transcribed locus, weakly similar to NP_608540.1 [Drosophila 1.93 melanogaster] Mm.279932 RIKEN cDNA 8230342M21 gene 1.93 Mm.403692 Transcribed locus 1.92 Mm.18635 Regulator of G-protein signalling 10 1.91 Mm.252561 Heparan sulfate 6-O-sulfotransferase 2 1.91 Mm.403636 Transcribed locus 1.91 Mm.7821 Tumor protein DSZ-Iike 1 1.91 Mm.347452 Kinesin family member 17 1.89 Mm.157091 Adenylate cyclase 6 1.89 Mm.24262 Ankyrin repeat domain 54 1.89 Mm.11171 Dorso-medial telencephalon gene 2 1.88 Mm.329991 Scm-like with four mbt domains 2 1.88 Mm.250030 Ribosomal protein L12 1.88 Mm.181021 Procollagen, type IV, alpha 2 1.88 Mm.20355 Wingless-related MMTV integration site 4 1.88 Mm.208662 CDNA sequence BC029169 1.88 Mm.45888 RIKEN cDNA 2210417009 gene 1.88 Mm.89201 Zinc finger protein 179 1.86 Mm.391678 Odd Oz/ten-m homolog 4 (Drosophila) 1.86 Mm.393388 0 day neonate thymus cDNA, RIKEN full-length enriched library, 1.86 clone:A430010E21 productzunclassifiable, full insert sequence Mm.89976 Zinc finger, imprinted 1 1.85 Mm.183114 Transcribed locus 1.84 Mm.186678 CDNA sequence BC022687 1.83 Mm.316890 RIKEN cDNA 5330417C22 gene 1.83 Mm.139192 Guanine nucleotide binding protein, beta 4 1.83 Mm.379388 RIKEN cDNA A230106006 gene 1.83 Mm.4940 RIKEN cDNA 1110036003 gene 1.83 Mm.156919 Cathepsin Z 1.82 Mm.40588 Ring finger protein 182 1.81 Mm.201493 Kruppel-like factor 12 1.79 252 Table A-3. Continued Unigene ID Description Mm.136238 Zinc finger and BTB domain containing 20 1.79 Mm.154284 Solute carrier family 22 (organic cation transporter), member 21 1.78 Mm.326526 Zinc finger protein 516 1.77 Mm.29293 SEC24 related gene family, member 0 (S. cerevisiae) 1.76 Mm.332684 Kinetochore associated 1 1.75 Mm.262113 RIKEN CDNA 5730593F17 gene 1.75 Mm.38832 Fibronectin type III domain containing 38 1.74 Mm.394729 RIKEN cDNA 4632427E13 gene 1.74 Mm.186943 Transmembrane protein 131 1.73 Mm.297949 Phytanoyl-CoA dioxygenase domain containing 1 1.73 Mm.10685 Neuropeptide Y receptor Y5 1.73 Mm.252862 D-aspartate oxidase 1.72 Mm.20325 N—acetyl galactosaminidase, alpha 1.71 Mm.25181 Zinc finger, CCHC domain containing 11 1.71 Mm.158143 Nuclear receptor subfamily 2, group F, member 2 1.70 Mm.248337 Vasorin 1.70 Mm.402837 Transcribed locus 1.70 Mm.360537 Predicted gene, E6619719 1.70 Mm.39691 15 days embryo head cDNA, RIKEN full-length enriched library, 1.68 clone20930025C07 productzunclassifiable, full insert sequence Mm.396273 STlPl homology and U-Box containing protein 1 1.67 Mm.87051 Thymocyte selection-associated HMG box gene 1.67 Mm.24684 Fos-like antigen 2 1.67 Mm.155887 RIKEN cDNA 1600021P15 gene 1.67 Mm.381165 Polycomb group ring finger 5 1.67 Mm.337647 Zinc finger, BED domain containing 4 1.67 Mm.333327 Histamine receptor H 1 1.66 Mm.434173 Transcribed locus 1.65 Mm.18889 Park2 co-regulated 1.64 Mm.29253 Exosome component 8 1.63 Mm.404309 Transcribed locus 1.63 Mm.320691 Potassium voltage—gated channel, Shel-related family, member 2 1.63 Mm.196189 Angiopoietin-Iike 4 1.62 Mm.420886 Transcribed locus 1.61 Mm.279872 PRP3 pre-mRNA processing factor 3 homolog (yeast) 1.60 Mm.38548 Regulator of G-protein signaling 9 1.59 Mm.76237 Transcribed locus 1.58 Mm.407113 Transcribed locus 1.58 Mm.331842 Major facilitator superfamily domain containing 2 1.58 Mm.40038 Apoptosis-inducing factor, mitochondrion-associated 3 1.58 Mm.184971 RIKEN cDNA A930009L07 gene 1.57 Mm.292567 Cysteine-rich with EGF—like domains 2 1.57 253 Table A-3. Continued Unigene ID Description Mm.17649 RIKEN cDNA 1700026008 gene 1.57 Mm.8149 Male germ cell—associated kinase 1.57 Mm.183650 Zinc finger protein 647 1.56 Mm.422738 WT1-interacting protein 1.56 Mm.159842 Calcium channel, voltage-dependent, alphaZ/delta subunit 1 1.55 Mm.408606 Transcribed locus 1.55 Mm.89564 Pregnancy upregulated non—ubiquitously expressed CaM kinase 1.53 Mm.738 Procollagen, type IV, alpha 1 1.53 Mm.434093 Transcribed locus 1.53 Mm.248445 Tripartite motif protein 25 1.53 Mm.204834 Solute carrier family 1 (glial high affinity glutamate transporter), 1.52 member 3 Mm.397123 RIKEN cDNA 6330404F12 gene 1.51 Mm.24204 Alanine and arginine rich domain containing protein 1.51 Mm.392175 CREB regulated transcription coactivator 3 1.49 Mm.314338 Activin receptor IIA 1.49 Mm.17964 KH domain containing, RNA binding, signal transduction associated 1.49 3 Mm.91794 Transcribed locus 1.48 Mm.33171 Ankyrin repeat and SOCs box—containing protein 5 1.48 Mm.435847 Expressed sequence AV025504 1.46 Mm.87663 Calcium channel, voltage-dependent, gamma subunit 5 1.46 Mm.272517 RWD domain containing 2 1.45 Mm.41364 RIKEN cDNA 0330038006 gene 1.45 Mm.359762 Peptidylprolyl isomerase (cyclophilin)-Iike 6 1.45 Mm.28534 Leucine rich repeat containing 1 1.45 Mm.275071 Jun oncogene 1.44 Mm.394224 Androgen receptor 1.44 Mm.272120 Glutamic acid decarboxylase 1 1.44 Mm.193212 Hydroxypyruvate isomerase homolog (E. coli) 1.43 Mm.24626 RIKEN cDNA 4921526F01 gene 1.43 Mm.434145 Transcribed locus 1.43 Mm.392845 Androgen receptor 1.42 Mm.331573 Reversion-inducing-cysteine-rich protein with kazal motifs 1.42 Mm.21926 DNA segment, Chr 5, ERATO Doi 40, expressed 1.42 Mm.31203 Glutathione S-transferase, mu 4 1.40 Mm.258746 RIKEN cDNA 1190002N15 gene 1.40 Mm.103477 Natriuretic peptide receptor 2 1.39 Mm.128580 DNA methyltransferase (cytosine-5) 1 1.39 Mm.295686 Zinc finger CCCH type containing 12C 1.39 Mm.32582 Early B-cell factor 4 1.38 Mm.329416 Coiled-coil domain containing 112 1.37 254 Table A-3. Continued Unigene ID Description Mm.287163 Btg3 associated nuclear protein 1.37 Mm.292016 Splicing factor, arginine/serine-rich 7 1.36 Mm.133607 Leucine rich repeat and fibronectin type III domain containing 2 1.36 Mm.223717 MAD homolog 1 (Drosophila) 1.36 Mm.70979 G protein-coupled receptor 176 1.35 Mm.383297 Inositol 1,4,5-trisphosphate 3-kinase B 1.35 Mm.22418 Peroxisome biogenesis factor 5 1.35 Mm.390409 Proline-rich polypeptide 6 1.34 Mm.597 Platelet-activating factor acetylhydrolase, isoform 1b, alphal 1.34 subunn Mm.136736 Endothelial differentiation, sphingolipid G-protein-coupled 1.34 receptor, 3 Mm.155573 W0 repeat domain 31 1.34 Mm.153643 Protocadherin 17 1.33 Mm.393348 RIKEN cDNA 4930562019 gene 1.32 Mm.395887 SET domain containing 6 1.32 Mm.2930 Peter pan homolog (Drosophila) 1.31 Mm.10812 Phosphodiesterase 9A 1.31 Mm.196464 Guanine nucleotide binding protein, alpha inhibiting 2 1.31 Mm.379020 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1 1.31 Mm.240435 Oxysterol binding protein-like 6 1.30 Mm.35324 Somatostatin receptor 4 1.30 Mm.246674 DnaJ (Hsp40) homolog, subfamily C, member 1 1.29 Mm.6988 Aminolevulinate, delta-, dehydratase 1.29 Mm.171304 Integrator complex subunit 2 1.27 Mm.424974 MARCKS-like 1 1.27 Mm.250731 Microtubule associated serine/threonine kinase 3 1.27 Mm.42191 Odd Oz/ten-m homolog 3 (Drosophila) 1.27 Mm.281800 Solute carrier family 35 (UDP-glucuronic acid/UDP-N- 1.26 acetylgalactosamine dual transporter), member 01 Mm.247537 Ankyrin repeat domain 39 1.26 Mm.182434 Follistatin-like 1 1.25 Mm.317293 Neuronal growth regulator 1 1.24 Mm.1786 Adenine phosphoribosyl transferase 1.24 Mm.3255 Proprotein convertase subtilisin/kexin type 7 1.24 Mm.400798 Transcribed locus 1.23 Mm.389552 F-box protein 10 1.23 Mm.389966 RAB30, member RAS oncogene family 1.23 Mm.6442 Polycystic kidney disease 2 1.23 Mm.22119 Fc receptor, lgG, low affinity III 1.23 Mm.425327 Glutamate receptor, ionotropic, delta 2 1.22 Mm.226704 Gamma-aminobutyric acid (GABA-A) receptor, subunit beta 1 1.22 255 Table A-3. Continued Unigene ID Description Mm.200203 RUN and TBC1 domain containing 2 1.21 Mm.38607 N—acetylglucosamine—l-phosphotransferase, gamma subunit 1.21 Mm.29678 Polypyrimidine tract binding protein 1 1.21 Mm.41702 SRY-box containing gene 11 1.20 Mm.347 RIKEN CDNA 1810007M14 gene 1.20 Mm.33421 RIKEN cDNA 1110012J17 gene 1.19 Mm.116769 Quiescin QS-like 1 1.19 Mm.40329 Calcium/calmodulin-dependent protein kinase I gamma 1.19 Mm.305152 Apolipoprotein E 1.19 Mm.209711 RIKEN CDNA A730017C20 gene 1.19 Mm.46675 Kelch repeat and BTB (POZ) domain containing 11 1.18 Mm.38172 Mitogen activated protein kinase 7 1.18 Mm.26237 Cryptochrome 1 (photolyase—like) 1.18 Mm.24028 Rap guanine nucleotide exchange factor (GEF) 3 1.17 Mm.390108 CKLF-like MARVEL transmembrane domain containing 3 1.17 Mm.279427 Zinc finger, CCHC domain containing 8 1.17 Mm.286892 Procollagen, type IV, alpha 5 1.17 Mm.396682 RIKEN cDNA C030009012 gene 1.16 Mm.3390 Myosin IB 1.15 Mm.242072 Tial cytotoxic granule-associated RNA binding protein—like 1 1.15 Mm.19119 LysM, putative peptidoglycan-binding, domain containing 2 1.14 Mm.136586 Spermine oxidase 1.14 Mm.3468 Suppressor of cytokine signaling 3 1.13 Mm.28853 Pituitary tumor-transforming 1 interacting protein 1.13 Mm.378951 Kinesin family member C3 1.12 Mm.363427 HIRA interacting protein 3 1.12 Mm.208955 CDNA sequence BC031353 1.12 Mm.232525 Anthrax toxin receptor 1 1.10 Mm.384704 Transmembrane protein 108 1.09 Mm.234641 RAR-related orphan receptor beta 1.09 Mm.42652 N-acetyltransferase 14 1.09 Mm.102305 Trinucleotide repeat containing 6a 1.08 Mm.249966 V-rel reticuloendotheliosis viral oncogene homolog A (avian) 1.07 Mm.275864 RAB34, member of RAS oncogene family 1.07 Mm.393354 CUG triplet repeat, RNA binding protein 1 1.07 Mm.291831 6lycosylphosphatidylinositol specific phospholipase 01 1.06 Mm.32959 Androgen-induced proliferation inhibitor 1.06 Mm.28765 Translocating chain-associating membrane protein 1 1.04 Mm.351459 Sirtuin 1 ((silent mating type information regulation 2, homolog) 1 1.04 (S. cerevisiae) Mm.274784 Heterogeneous nuclear ribonucleoprotein H3 1.03 Mm.416039 Transcribed locus 1.03 256 Mm.426078 Mm.54460 Mm.391777 Mm.155896 Mm.332838 Mm.433262 Mm.275304 Mm.3l324 Gene model 967 related Max dimerization nuclear Glutamate kainate 2 Transcribed locus member RAS 257 Figure A-4: Genes with statistically higher expression in SNpc than ARC of animals treated with MPTP. Male mice (n=8/group) were treated with a single injection of MPTP (20 mg/kg; s.c.) or saline (10 ml/kg; s.c.). All animals were killed 8 h after MPTP or saline treatment. Amplified mRNA from 300-500 laser—captured TH immunofluorescent cells from ARC and SN of each mouse were hybridized to an Affymetrix mouse 430 2.0 chip. Data was normalized and genes that were significantly higher in SNpc than ARC by at least 2 fold in MPTP treated animals were identified. 258 Table A-4. Genes with Higher Expression in SNpc than ARC 8h after MPTP treatment Unigene ID Description Mm.14609 Aldehyde dehydrogenase family 1, subfamily A7 9.49 Mm.397323 Expressed sequence AI853839 7.97 Mm.276829 Solute carrier family 39 (zinc transporter), member 4 7.79 Mm.427936 Transcribed locus 7.51 Mm.72070 Riken cDNA C130021l2%ene 7.40 Mm.283137 Cholinergic receptor, nicotinic, alpha polypeptide 6 7.25 Mm.41970 Dopamine receptor 2 7.24 Mm.84118 Predicted gene, E6639426 6.49 Mm.222729 Guanylate cyclase 2c 6.49 Mm.4298 Transcribed locus 6.35 Mm.39825 Transcribed locus 6.24 Mm.110444 Cholinergic receptor, nicotinic, beta polypeptide 3 6.19 Mm.103778 Cholinergic receptor, nicotinic, alpha polypeptide 5 6.07 Mm.393273 Transcribed locus 6.06 Mm.3507 Nuclear receptor subfamily 4, group A, member 2 5.95 Mm.57199 Ret proto-oncogene 5.90 Mm.308735 Kelch-like 1 (Drosophila) 5.75 Mm.52392 RIKEN cDNA A930038C07 gene 5.60 Mm.414106 Transcribed locus 5.46 Mm.23782 Glycosyltransferase 25 domain containing 2 5.42 Mm.406232 Transcribed locus 5.41 Mm.268521 Insulin-like growth factor 1 5.17 Mm.5076 Tolloid-Iike 5.10 Mm.9431 Protease, serine, 12 neurotrypsin (motopsin) 5.09 Mm.297371 POU domain, class 3, transcription factor 1 5.09 l Mm.239941 Pre B—cell leukemia transcription factor 3 4.97 Mm.74324 Solute carrier family 35, member 03 4.97 I Mm.420796 Transcribed locus 4.89 Mm.412897 Transcribed locus 4.88 Mm.405570 POU domain, class 3, transcription factor 2 4.83 Mm.30013 RIKEN cDNA 2010011I20 gene 4.79 Mm.358581 Calmegin 4.58 Mm.63569 Cholinergic receptor, nicotinic, alpha polypeptide 3 4.57 Mm.258708 Early B-cell factor 3 4.52 Mm.295917 Gene model 540, (NCBI) 4.49 Mm.39599 GSGl-like 4.44 Mm.137991 Eph receptor A5 4.38 Mm.29221 Transcribed locus 4.27 Mm.113278 Potassium voltage-gated channel, delayed-rectifier, subfamily S, 4.24 259 Table A-4. Continued Unigene ID Description member 3 Mm.392745 10 days embryo whole body cDNA, RIKEN full-length enriched 4.23 library, clone:2610306612 product:unclassifiable, full insert sequence Mm.141275 RIKEN cDNA E130009112 gene 4.16 Mm.42190 Unc4.1 homeobox (C. elegans) 4.05 Mm.332576 Meningioma 1 4.03 Mm.379143 Regulator of G-protein signaling 8 4.02 Mm.408043 Transcribed locus 4.00 Mm.129387 POU domain, class 3, transcription factor 2 4.00 Mm.384108 lmmunoglobulin superfamily, member 4 3.84 Mm.425860 Leucine rich repeat containing 38 3.83 Mm.388885 POU domain, class 3, transcription factor 1 3.80 Mm.288805 DEP domain containing 7 3.78 Mm.71924 Ankyrin repeat domain 38 3.76 Mm.125650 Apolipoprotein L, 2 3.71 Mm.40875 0 day neonate cerebellum cDNA, RIKEN full-length enriched library, 3.65 clone:C230022P04 product:unclassifiable, full insert sequence Mm.30172 Ras association (RalGDS/AF-6) domain family 6 3.65 Mm.275374 Nuclear receptor interacting protein 3 3.64 Mm.412261 Transcribed locus, strongly similar to XP_001055606.1 similar to 3.57 Oxysterol-binding protein-related protein 3 (OSBP-related protein 3) (ORP-3) [Rattus norvegicus] Mm.154541 RIKEN cDNA 3830431621 gene 3.51 Mm.100125 SH3 domain binding glutamic acid-rich protein like 2 3.47 Mm.83817 Villin-like 3.47 Mm.39723 Kelch domain containing 8A 3.46 Mm.220765 Cadherin 8 3.43 Mm.193274 R-spondin 2 homolog (Xenopus laevis) 3.42 Mm.101836 RIKEN cDNA 9630033F20 gene 3.41 Mm.37882 CDNA sequence BC065085 3.40 Mm.246595 Hypocretin (orexin) receptor 1 3.39 Mm.333264 Glutamate receptor interacting protein 2 3.32 Mm.102899 Transcribed locus 3.31 Mm.6255 Paired-like homeodomain transcription factor 3 3.27 Mm.102211 RIKEN cDNA 9330109K16 gene 3.25 Mm.115970 A disintegrin-like and metallopetidase (reprolysin type) with 3.21 thrombospondin type 1 motif, 16 Mm.334011 Malic enzyme 3, NADP(+)-dependent, mitochondrial 3.20 Mm.5195 Complexin 1 3.20 Mm.394300 Transcribed locus 3.16 Mm.380993 Phosphatidylinositol-specific phospholipase C, X domain containing 3.16 260 Table A-4. Continued Unigene ID Description 2 Mm.26751 Synaptotagmin-like 2 3.14 Mm.156558 Synaptotagmin XVII 3.13 Mm.317854 Pregnancy-associated plasma protein A 3.11 Mm.361919 Solute carrier family 18 (vesicular monoamine), member 2 3.11 Mm.14099 Zinc finger protein 385 3.11 Mm.293363 Spindlin family, member 2 3.11 Mm.415763 Transcribed locus 3.02 Mm.39090 Glutamate receptor, ionotropic, NMDAZC (epsilon 3) 3.01 Mm.72799 Transmembrane protease, serine 5 (spinesin) 3.01 Mm.141936 Insulin-like growth factor binding protein 2 3.01 Mm.17484 Synuclein, alpha 3.01 Mm.4860 Protein tyrosine phosphatase, receptor type, U 3.00 Mm.331690 RIKEN cDNA 5930437A14 gene 2.99 Mm.276248 Sphingosine-l—phosphate phosphotase 2 2.98 Mm.89682 Membrane bound O-acyltransferase domain containing 1 2.97 Mm.298731 Immunoglobin superfamily, member 21 2.97 Mm.159989 RIKEN cDNA 2810022L02 gene 2.95 Mm.360649 RIKEN cDNA A830006F12 gene 2.92 Mm.345834 0 day neonate cerebellum cDNA, RIKEN full-length enriched library, 2.90 clone:C230096l08 productzhypothetical protein, full insert sequence Mm.82308 Protocadherin beta 3 2.89 Mm.428708 Special AT-rich sequence binding protein 1 2.89 Mm.7995 Fibroblast growth factor 13 2.87 Mm.151332 Peroxin 2 2.85 Mm.292983 Vitrin 2.81 Mm.403758 Transcribed locus 2.81 Mm.384353 Mab-21-like 1 (C. elegans) 2.81 Mm.268536 Myotubularin related protein 11 2.79 Mm.169261 Von Willebrand factor C and EGF domains 2.77 Mm.255246 Cytochrome P450, family 26, subfamily b, polypeptide 1 2.75 Mm.211477 Pleckstrin homology-like domain, family 8, member 2 2.75 Mm.425526 Fibroblast growth factor 20 2.74 Mm.292100 Fibrinogen-Iike protein 2 2.74 Mm.103351 ATP-binding cassette, sub-family A (ABC1), member 7 2.73 Mm.133615 RIKEN cDNA 2810457|06 gene 2.72 Mm.282751 Anillin, actin binding protein (scraps homolog, Drosophila) 2.71 Mm.35141 Mannoside acetylglucosaminyltransferase 5, isoenzyme B 2.69 Mm.27789 FERM domain containing 48 2.68 Mm.318710 RIKEN cDNA 3632451006 gene 2.68 Mm.435467 Testis-specific kinase 2 2.68 261 Table A-4. Continued Unigene ID Description Mm.41993 Solute carrier family 6 (neurotransmitter transporter, dopamine), 2.65 member 3 Mm.17629 Caspase recruitment domain family, member 10 2.65 Mm.155708 Ets variant gene 5 2.62 Mm.253736 Palmdelphin 2.62 Mm.46782 La ribonucleoprotein domain family, member 2 2.61 Mm.422608 RIKEN CDNA 1190017012 gene 2.61 Mm.210857 Myelin oligodendrocyte glycoprotein 2.60 Mm.273082 Synaptic vesicle glycoprotein 2 b 2.59 Mm.53865 Ankyrin repeat domain 29 2.58 Mm.235938 Fragile histidine triad gene 2.57 Mm.10651 GTP cyclohydrolase 1 2.56 Mm.46797 Adrenergic receptor, beta 1 2.55 Mm.42012 Laminin, alpha 3 2.55 Mm.376140 Sushi domain containing 5 2.55 Mm.39151 Transcribed locus 2.51 Mm.30221 Insulin induced gene 1 2.50 Mm.240912 Ribulose—S-phosphate-3—epimerase 2.47 Mm.34608 Citrate lyase beta like 2.45 Mm.316210 FAT tumor suppressor homolog 4 (Drosophila) 2.44 Mm.397113 Transcribed locus 2.44 Mm.40915 5T6 (alpha—N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N- 2.43 acetylgalactosaminide alpha-2,6-sialyltransferase 5 Mm.236287 RIKEN cDNA 1700019003 gene 2.42 Mm.426321 LEM domain containing 1 2.42 Mm.40550 Transcribed locus 2.42 Mm.250866 Aldehyde dehydrogenase family 1, subfamily A1 2.41 Mm.78923 Phosphatidylinositol-4-phosphate 5-kinase-like 1 2.39 Mm.34344 Pseudouridylate synthase 7 homolog (S. cerevisiae)—like 2.38 Mm.39649 RIKEN cDNA 8230216623 gene 2.38 Mm.80318 Transmembrane protein 169 2.37 Mm.198414 Serine/threonine kinase 39, STE20/SPSl homolog (yeast) 2.37 Mm.20453 FK506 binding protein 1b 2.37 Mm.285060 FCH and double SH3 domains 1 2.36 Mm.406348 Transcribed locus 2.35 Mm.192699 Phospholipase C, gamma 2 2.34 Mm.407111 Transcribed locus 2.34 Mm.434385 RIKEN cDNA C330011K17 gene 2.32 Mm.289643 Cysteine conjugate-beta lyase 2 2.31 Mm.209715 Procollagen, type XI, alpha 1 2.31 Mm.34235 Williams Beuren syndrome chromosome region 27 (human) 2.28 Mm.435077 Transcribed locus 2.26 262 Table A-4. Continued Unigene ID Description Mm.212855 Myosin regulatory light chain interacting protein 2.26 Mm.3944 Kallikrein related-peptidase 6 2.25 Mm.28893 RAP1, GTP-GDP dissociation stimulator 1 2.24 Mm.23462 Elastin microfibril interfacer 2 2.23 Mm.252369 Cholinergic receptor, nicotinic, alpha polypeptide 4 2.22 Mm.130696 RIKEN cDNA 4930589M24 gene 2.20 Mm.30767 RIKEN cDNA 1300003813 gene 2.20 Mm.432481 Forkhead box P2 2.19 Mm.435523 Hypothetical protein LOC574403 2.18 Mm.407309 Transcribed locus 2.17 Mm.11869 Expressed sequence AI427122 2.15 Mm.138434 GRAM domain containing 1B 2.14 Mm.133193 Keratin 222 2.13 Mm.65316 Nitric oxide synthase 1 (neuronal) adaptor protein 2.12 Mm.153013 Regulator of G-protein signaling 6 2.12 Mm.341377 Aryl-hydrocarbon receptor 2.12 Mm.27005 Visinin—Iike 1 2.10 Mm.125874 Hormonally upregulated Neu-associated kinase 2.10 Mm.432488 Male sterility domain containing 1 2.08 Mm.291824 Transmembrane protein 32 2.08 , Mm.426628 Expressed sequence C87490 2.07 ‘ Mm.64962 Matrix-remodelling associated 7 2.06 Mm.45533 RIKEN cDNA 2310026E23 gene 2.06 Mm.274308 N-ethylmaleimide sensitive fusion protein attachment protein beta 2.06 Mm.435789 Transcribed locus 2.05 Mm.391931 Membrane protein, palmitoylated 7 (MAGUK p55 subfamily 2.04 member 7) Mm.69013 RIKEN cDNA B430201A12 gene 2.03 Mm.158289 Myosin, heavy polypeptide 14 2.03 Mm.43358 Pre B-cell leukemia transcription factor 1 2.00 Mm.381 Adipose differentiation related protein 2.00 Mm.392499 Transcribed locus 1.99 Mm.395863 Transcribed locus 1.97 Mm.426957 Transcribed locus 1.97 Mm.335395 RAB6B, member RAS oncogene family 1.95 Mm.87676 Transmembrane protein 16K 1.95 Mm.136116 Leucine rich repeat containing 20 1.94 Mm.40068 Tubulin, beta 3 1.94 Mm.5162 Serine dehydratase-like 1.93 Mm.87027 Cell cycle exit and neuronal differentiation 1 1.92 Mm.209232 Contactin associated protein-like 4 1.92 Mm.12239 Geminin 1.91 263 Table A-4. Continued Unigene ID Description Mm.400451 Musashi homolog 2 (Drosophila) 1.91 Mm.46764 CDP-diacylglycerol synthase 1 1.89 Mm.396875 Regulator of G—protein signalling 7 binding protein 1.88 Mm.226435 UDP-GalzbetaGlcNAc beta 1,3-galactosyltransferase, polypeptide 1 1.88 Mm.23639 RIKEN cDNA 6330530A05 gene 1.88 Mm.1292 Tyrosine hydroxylase 1.87 Mm.70371 RIKEN CDNA 2310005E10 gene 1.86 Mm.49665 T-cell lymphoma breakpoint associated target 1 1.86 Mm.86823 NAT9 1.83 Mm.41290 GTPase activating RANGAP domain-like 3 1.82 Mm.292168 Synuclein, alpha interacting protein (synphilin) 1.82 Mm.4744 Growth differentiation factor 5 1.81 Mm.432562 Myelin-associated oligodendrocytic basic protein 1.81 Mm.29646 CNDP dipeptidase 2 (metallopeptidase M20 family) 1.80 Mm.176695 Tripartite motif-containing 59 1.80 Mm.435503 AT motif binding factor 1 1.80 Mm.282257 Vav 3 oncogene 1.79 Mm.100348 Regulator of G-protein signalling 7 binding protein 1.78 Mm.338690 Solute carrier family 35, member F1 1.78 Mm.60061 Regulating synaptic membrane exocytosis 1 1.78 Mm.4654 Protein tyrosine phosphatase, non-receptor type 5 1.78 Mm.51434 Transcribed locus 1.78 Mm.27969 UDP-N-acetylglucosamine pyrophosphorylase 1 1.78 Mm.153566 Meteorin, glial cell differentiation regulator-like 1.78 Mm.247457 Secernin 3 1.78 Mm.433275 Transcribed locus 1.77 Mm.306021 UDP galactosyltransferase 8A 1.77 Mm.27469 Tetraspanin 2 1.76 Mm.41945 Tetratricopeptide repeat domain 22 1.76 Mm.44075 Fibronectin type III domain containing 5 1.76 Mm.390803 Pannexin 2 1.75 Mm.337023 Membrane-associated ring finger (C3HC4) 4 1.75 Mm.412289 Transcribed locus 1.75 Mm.298251 Leucine-rich repeat LGI family, member 1 1.74 Mm.32744 Opsin (encephalopsin) 1.74 Mm.275332 Autophagy—related 7 (yeast) 1.73 Mm.314113 ELOVL family member 6, elongation of long chain fatty acids (yeast) 1.73 Mm.44721 Leucine-rich repeat LGI family, member 2 1.73 Mm.393375 Transcribed locus 1.72 Mm.56946 POU domain, class 3, transcription factor 4 1.72 Mm.124595 RIKEN cDNA A330008L17 gene 1.72 264 .I“‘ - - - I I - . o - Mm.57225 Phosphoglucomutase 2-like 1 1.70 Mm.316418 Arginine/serine-rich coiled-coil 1 1.70 Mm.403011 Transcribed locus 1.69 Mm.55143 Dickkopf homolog 3 (Xenopus laevis) 1.69 Mm.224306 Kelch-like 13 (Drosophila) 1.68 Mm.1892 Corticotropin releasing hormone receptor 1 1.68 Mm.3970 ELAV (embryonic lethal, abnormal vision, Drosophila)-|ike 4 (Hu 1.67 antigen 0) Mm.407200 Transcribed locus 1.67 Mm.264036 NAD(P)H dehydrogenase, quinone 2 1.65 Mm.275387 RIKEN CDNA 1810041L15 gene 1.65 Mm.18962 Catenin (cadherin associated protein), alpha 1 1.65 Mm.334029 Predicted gene, E6622320 1.64 Mm.27687 RUN and SH3 domain containing 1 1.63 Mm.41203 Solute carrier family 2 (facilitated glucose transporter), member 6 1.62 Mm.391904 Glutamate receptor, metabotropic 1 1.62 Mm.10214 Tuftelin 1 1.62 Mm.37214 Transferrin 1.61 Mm.29098 Galactose mutarotase 1.61 Mm.139607 CDNA sequence BC003266 1.60 Mm.317049 RIKEN cDNA 5730508809 gene 1.60 Mm.127058 StAR-related lipid transfer (START) domain containing 4 1.60 Mm.259470 Oxysterol binding protein-like 1A 1.59 Mm.9870 Centromere protein Q 1.59 Mm.205422 Sterile alpha motif domain containing 10 1.57 Mm.34175 RIKEN cDNA A530047J11 gene 1.56 Mm.291799 Retinol dehydrogenase 11 1.56 Mm.229342 Acetyl-Coenzyme A acetyltransferase 2 1.55 Mm.41728 Enoyl Coenzyme A hydratase domain containing 1 1.55 Mm.101707 SLIT and NTRK—like family, member 4 1.54 Mm.146332 Receptor accessory protein 1 1.53 Mm.386754 Leucine—rich repeats and transmembrane domains 1 1.53 Mm.392637 Solute carrier family 36 (proton/amino acid symporter), member 1 1.53 Mm.298283 Neurofilament, heavy polypeptide 1.53 Mm.212927 Transmembrane protein 38a 1.53 Mm.27589 Lactate dehydrogenase D 1.52 Mm.89646 Lactation elevated 1 1.52 Mm.289702 Synaptotagmin I 1.52 Mm.407234 Transcribed locus 1.52 Mm.172720 Leucine rich repeat containing 59 1.50 Mm.19133 Amyloid beta (A4) precursor-like protein 2 1.50 Mm.945 Protein tyrosine phosphatase, receptor type, E 1.50 265 Table A-4. Continued Unigene ID Description Mm.423496 Hypothetical protein LOC636791 1.49 Mm.392829 Hypothetical protein C130006E23 1.49 Mm.398492 Transcribed locus 1.49 Mm.1517 Synaptobrevin like 1 1.48 Mm.389247 Nebulin 1.48 Mm.39330 Heat shock protein 4 like 1.48 Mm.388924 Potassium voltage-gated channel, shaker-related subfamily, beta 1.47 member 2 Mm.41523 RIKEN cDNA 6330416613 gene 1.47 Mm.12057 Hypothetical protein C130086A10 1.46 Mm.3093 Serine (or cysteine) peptidase inhibitor, clade E, member 2 1.46 Mm.268317 Pleckstrin homology domain containing, family H (with MyTH4 1.45 domain) member 1 Mm.289915 Damage specific DNA binding protein 1 1.45 Mm.433866 Transcribed locus 1.45 Mm.194 FMS-like tyrosine kinase 3 1.45 Mm.390807 Protein tyrosine phosphatase 4a3 1.44 Mm.173826 Dymeclin 1.43 Mm.336158 CDNA sequence AK129302 1.43 Mm.40110 6 patch domain containing 3 1.43 Mm.434311 Cytochrome P450, family 2, subfamily u, polypeptide 1 1.43 Mm.336329 RALBP1 associated Eps domain containing protein 2 1.42 Mm.140158 Cytochrome P450, family 51 1.42 Mm.158827 RIKEN cDNA 2410003P15 gene 1.42 Mm.5915 DPH5 homolog (S. cerevisiae) 1.41 Mm.103560 Jun dimerization protein 2 1.41 Mm.2942 Asparagine synthetase 1.40 Mm.26479 Zinc fingerprotein 618 1.40 Mm.21108 Monoamine oxidase A 1.40 Mm.109840 Regulator of G-protein signaling 11 1.40 Mm.259191 O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N- 1.39 acetylglucosamine:polypeptide-N—acetylglucosaminyl transferase) Mm.210447 EH domain binding protein 1-like 1 1.38 Mm.425467 Solute carrier organic anion transporter family, member 331 1.38 Mm.132583 Kelch-like 4 (Drosophila) 1.38 Mm.153111 Required for meiotic nuclear division 1 homolog (S. cerevisiae) 1.38 Mm.280254 Abhydrolase domain containing 5 1.37 Mm.133370 24-dehydrocholesterol reductase 1.37 Mm.290802 Guanosine monophosphate reductase 1.37 Mm.193670 Eukaryotic translation initiation factor 5A2 1.36 Mm.157119 Sortilin 1 1.36 Mm.27844 ATP synthase, H+ transporting, mitochondrial F0 complex, subunits 1.36 266 Table A-4. Continued Unigene ID Description Mm.33490 GA repeat binding protein, beta 2 1.36 Mm.392446 Mus musculus, clone IMAGE11512359, mRNA 1.36 Mm.302793 Synaptotagmin IX 1.35 Mm.268902 Complexin 2 1.34 Mm.192162 Calsyntenin 2 _ 1.34 Mm.245297 Cytochrome P450, family 4, subfamily v, polypeptide 3 1.34 Mm.386792 Zinc finger protein 364 1.34 Mm.31263 RIKEN cDNA B230118H07jgene 1.33 Mm.18036 E2F transcription factor 1 1.32 Mm.341423 Dedicator of cytokinesis 4 1.32 Mm.38037 MON1 homolog A (yeast) 1.32 Mm.96867 Ring finger protein 141 1.31 Mm.347625 Phosphatidylinositol glycan anchor biosynthesis, class Z 1.31 Mm.3645 Myosin Va 1.31 Mm.125503 RAN binding protein 6 1.31 Mm.377099 Melanoma antigen, family A, 7 1.31 Mm.24641 RIKEN cDNA 2810405J04 gene 1.31 Mm.290534 SH3 binding domain protein 5 like 1.30 Mm.435680 Transcribed locus 1.29 Mm.252316 Glutathione synthetase 1.28 Mm.26134 Protein phosphatase 2 (formerly 2A), regulatory subunit 8 (PR 52), 1.28 beta isoform Mm.21995 Fatty acid desaturase domain family, member 6 1.27 Mm.247143 GDP-mannose 4, 6-dehydratase 1.27 Mm.209503 Tetratricopeptide repeat domain 27 1.27 Mm.30737 F-box protein 28 1.27 Mm.33120 Histamine N-methyltransferase 1.27 Mm.240434 Apoptosis-inducing factor, mitochondrion-associated 1 1.25 ‘ Mm.190774 Basonuclin 2 1.25 Mm.397953 Transcribed locus 1.24 Mm.414514 Transcribed locus 1.24 Mm.121789 POU domain, class 3, transcription factor 3 1.24 Mm.21109 Gelsolin 1.24 Mm.392075 Calumenin 1.24 Mm.257067 Ly6/neurotoxin 1 1.23 Mm.312233 TAF4B RNA polymerase II, TATA box binding protein (TBP)- 1.23 associated factor Mm.219675 G elongation factor, mitochondrial 2 1.23 Mm.170855 RIKEN cDNA 8230380007 gene 1.23 Mm.341747 RIKEN cDNA 6330442E10 gene 1.23 Mm.331630 A kinase (PRKA) anchor protein 2 1.22 Mm.222685 Ceramide kinase 1.22 267 Table A-4. Continued Unigene ID Description Mm.40477 0 day neonate lung cDNA, RIKEN full-length enriched library, 1.22 clone:E030046E07 product:unclassifiable, full insert sequence Mm.29580 Stathmin-like 2 1.21 Mm.2445 Acyl—Coenzyme A dehydrogenase, long-chain 1.21 Mm.38433 Tryptophanyl-tRNA synthetase 1.21 Mm.258932 RIKEN cDNA 1810012P15 gene 1.20 Mm.157069 Delta-like 1 homolog (Drosophila) 1.19 Mm.275683 Islet cell autoantigen 1 1.19 Mm.141054 RIKEN CDNA 1110011C06 gene 1.17 Mm.389061 PFTAIRE protein kinase 1 1.17 Mm.41022 RNA binding motif protein 19 1.15 Mm.425766 Neurexin Ill 1.15 Mm.406721 Transcribed locus 1.14 Mm.54183 Olfactomedin 3 1.14 Mm.6306 ATPase, Ca++ transporting, ubiquitous 1.13 Mm.38816 WD repeat domain 36 1.12 Mm.3676 Golgi transport 1 homolog B (S. cerevisiae) 1.12 Mm.386776 Estrogen related receptor, alpha 1.11 Mm.255026 Acyl-CoA synthetase short-chain family member 2 1.11 Mm.219433 Zinc finger, SWIM domain containing 6 1.10 Mm.419813 CUB and Sushi multiple domains 1 1.10 Mm.397106 Expressed sequence Al848218 1.09 Mm.433835 Transcribed locus 1.09 Mm.389855 RIKEN cDNA C230052I12 gene 1.08 Mm.434709 Transcribed locus 1.08 Mm.26564 Oxysterol binding protein-like 11 1.08 Mm.29150 Dynein light chain Tctex-type 3 1.08 Mm.27925 Dysbindin (dystrobrevin binding protein 1) domain containing 1 1.07 Mm.24431 TAF13 RNA polymerase II, TATA box binding protein (TBP)- 1.07 associated factor Mm.409762 Transcribed locus 1.06 Mm.432777 Transcribed locus, strongly similar to XP_225688.3 similar to zinc 1.05 finger protein [Rattus norvegicus] Mm.380683 HIV-1 Rev binding protein 1.05 Mm.74208 Periphilin 1 1.05 Mm.197441 Protocadherin beta 16 1.05 Mm.277242 RIKEN CDNA A530089|17 gene 1.05 Mm.46722 PDZ domain containing 1 1.04 Mm.211429 Ectonucleotide pyrophosphatase/phosphodiesterase 6 1.04 Mm.58836 Citrate synthase 1.04 Mm.251228 RNA terminal phosphate cyclase domain 1 1.04 Mm.182912 Growth hormone inducible transmembrane protein 1.04 268 Mm.340211 Mm.289441 Mm.216217 Mm.405108 Mm.28309 Mm.36507 Mm.210095 Mm.432944 Mm.290085 Mm.290704 Mm.160172 Mm.41761 COX10 homolog, cytochrome c oxidase assembly protein, heme A: fa nsferase Claudin 1 RIKEN cDNA 2900011008 ne Transcribed locus RIKEN cDNA 0610033I05 Nudix nucleoside di linked motif 12 ATPase, Ca++ tra rti membrane 3 Chromatin mod in 28 Cereblon RIKEN cDNA C330007P06 Kv channel interacti n 4 5',3'-nucleotidase, mitochondrial 269 References 270 Abeliovich A, Schmitz Y, Farinas I, Choi-Lundberg 0, Ho WH, Castillo PE, Shinsky N, Verdugo JM, Armanini M, Ryan A, Hynes M, Phillips H, Sulzer D, Rosenthal A (2000) Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. 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