CONTAINING DIFFERENTIALLY SUSCEPTIBLE DOPAMINERGIC NEURONS  By   Joseph Robert Patterson                      A DISSERTATION  Submitted to Michigan State University in partial fulfillment of the requirements  for the degree of  Genetics-Doctor of Philosophy  2016   ABSTRACT  CONTAINING DIFFERENTIALLY SUSCEPTIBLE DOPAMINERGIC NEURONS  By Joseph Robert Patterson The hallmark pathologies of Parkinson disease (PD) are the formation of Lewy bodies and the progressive loss of nigrostriatal dopamine (NSDA) neurons. In mice, the NSDA neurons are preferentially damaged through exposure to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Another population of DA neurons that are initially damaged by MPTP, but are able to recover are the tuberoinfundibular DA (TIDA) neurons. Parkin is a product of the PARK2 gene, which is linked to autosomal recessive or juvenile PD. Parkin has multiple functions in neurons and is predicted to protect against the neurotoxic effects of MPTP. Potential transcription factors of parkin were identified using TFSEARCH, PROMO, and Patch 1.0, and refined to 11 based the transcription factor being identified in all three programs, being known to be found in the brain, and known to respond to a type of stress that MPTP could cause. The candidate transcription factors were examined at 6 h after MPTP in regions containing the cell bodies of TIDA and NSDA neurons. From these candidates, only FosB  Further examination of the temporal expression and cellular localization of    Regions containing the cell bodies of the TIDA (arcuate nucleus; ARC) and NSDA (substantia nigra; SN) neurons were dissected and processed for correlates with parkin, increasing in the ARC and not in the SN. Furthermore, total FosB protein was localized to nuclei of NSDA and TIDA neurons, and fractions derived from the ARC and SN. Though the number of DA neurons expressing total FosB does not change at 6 h post-increase in the nuclear fraction from the ARC.  AAV-NSDA and TIDA neurons, in both cases, parkin increased about 2-fold. The predominantly dimerizes with the FosBs and inhibits their ability to act as transcription factors was injected into the ARC. The AAV-the increase of parkin after MPTP in the TIDA neurons. Taken together, the tion factors of parkin, since they are predicted to bind the Park2 promoter, their expression correlates with the differential expression of parkin, increases prior to parkin, expreto MPTP. iv  ACKNOWLEDGEMENTS  for very long. We keep moving forward, opening new doors, and doing new things, because we're curious and curiosity keeps leading us down new paths". I feel this quote best sums up not only how to approach graduate school and science, but life. It is important to recognize what has happened and learn from it, but unless you "keep moving forward", you will never get anywhere. In the completion of this dissertation, there are many people that have played some role that need to be acknowledged. First off, I have to thank my advisers, Dr. Keith Lookingland and Dr. John Goudreau. Keith, I want to thank you for the opportunity to grow as a scientist and the independence to develop and explore my own ideas. John, I would like to thank you for always suggesting new avenues to explore, as well as introducing me to researchers in the field.  I want to thank my current and former labmates Dr. Matthew Benskey, Dr. Chelsea Tiernan, Dr. Tyrell Simkins, Dr. Hae-young Hawong, Teri Lansdell, and Brittany Winner. You have all made the time in the lab interesting. I also want to thank the rotating graduate students I have worked with, especially Laura Harding, for her contribution to the Nrf2 aspects of this dissertation. I must also thank all of the undergraduate  v  students that I have worked with, especially Elizabeth Kim, who aided in quantification of data, as well as solution making, and animal care.   I want to thank Dr. AJ Robison and his lab for the use of their viral vectors and their invaluable knowledge concerning FosB and FosB. I also want to thank my committee members, Dr. Christina Chan and Dr. Monique Floer for their insight on the project. I also want to acknowledge the Genetics Program and its director Dr. Cathy Ernst. They have helped fund my studies through teaching assistantships. Special thanks to Dr. Jennifer Schisa, my undergraduate research and MS research advisor, work in your lab is what started me down this path.      Most of all I want to thank my family, as they are the ones that tolerated me throughout it all. To my wife Cassie, I want to thank you for putting up with the long hours, late nights, and my ramblings throughout the last few years. Your love and support is what got me through. To my son Jordan, you brighten my day and inspire me to be better. I want to thank my parents, Robert and Nancy, for their support throughout school. I also want to thank my in-laws for keeping Cassie and Jordan company while I was busy, as well as Tuesday night dinners. Finally I want to acknowledge my dog Echo, who kept me company while I was writing my dissertation.       vi  TABLE OF CONTENTS  LIST OF TABLES .................................................................................. ix  LIST OF FIGURES ................................................................................. x  KEY TO ABBREVIATIONS ..................................................................... xx  Chapter 1: General Introduction ............................................................. 1 Parkinson Disease .............................................................................. 1 NSDA Neurons and PD ........................................................................ 2 Etiology of PD .................................................................................... 9 MPTP .............................................................................................. 13 Parkin ............................................................................................. 18 Parkin and MPTP ............................................................................... 22 Summary ........................................................................................ 23 Central Hypothesis ........................................................................... 24 Specific Aim 1. Idenitfication of candidate transcription factors of       parkin ............................................................................................. 24 Specific Aim 2. Chacacterization of candidate transcription factors of  parkin ............................................................................................. 24 Specific Aim 3. Manipulation of expression of trancription factors and their subsequent effects on parkin expression ............................................. 25  Chapter 2: Methods ............................................................................ 26 Animals .......................................................................................... 26 Drug Treatment ............................................................................... 26 Brain Removal and Median Eminence Dissection .................................. 26 Tissue Preparation for Conventional Western Blots ............................... 29 BCA Assay for Western Blots ............................................................. 32 Western Blot Analyses ...................................................................... 32 Nuclear and Cytoplasmic Fraction Isolation .......................................... 36 Immunofluorescent Staining and Quantification ................................... 39 BCA Protein Assay for Neurochemistry ................................................ 40 Neurochemical Analysis of DA and DOPAC in ST and ME........................ 41 Stereotaxic Surgeries ....................................................................... 43 Cell Culture ..................................................................................... 49 Cell Harvesting and Processing .......................................................... 49 Cell Viability Assay ........................................................................... 50 DCF-DA Assay ................................................................................. 50 Statistical Analysis ........................................................................... 51   vii  Chapter 3: Examination of Potential Regulators of Parkin......................... 52 Introduction .................................................................................... 52 DNA Methylation .............................................................................. 57 Results ......................................................................................... 57 Discussion .................................................................................... 62 Histones and Histone Modifications ..................................................... 63 Results ......................................................................................... 63 Discussion .................................................................................... 75 Transcription Factors ........................................................................ 76 Results ......................................................................................... 76 UCSC Genome Browser and ENCODE ............................................. 76 PATCH 1.0 .................................................................................. 82 TFSearch .................................................................................... 85 PROMO ...................................................................................... 87 Discussion .................................................................................... 92  Chapter 4: Assessment of Putative Transcription Factors of Parkin .......... 102 Introduction .................................................................................. 102  ........................................................... 102 Nrf1 and ER Stress ...................................................................... 104 Nrf2 and Oxidative Stress ............................................................. 105 AP-1 Transcription Factors ............................................................ 106 NRF1, Parkin and Mitochondrial Biogenesis ..................................... 107 SRY and the TH Promoter ............................................................. 107 Transcription Factor Regulation of the Parkin Promoter .................... 108 Acute MPTP-induced Parkin Expression in the ARC ........................... 108 ATF4 ............................................................................................ 109 Results ....................................................................................... 109 Discussion .................................................................................. 119 Nrf1 and Nrf2 ................................................................................ 120 Results ....................................................................................... 120 Discussion .................................................................................. 128 NRF1 ............................................................................................ 130 Results ....................................................................................... 130 Discussion .................................................................................. 142 SRY .............................................................................................. 143 Results ....................................................................................... 143 Discussion .................................................................................. 146 AP-1 ............................................................................................. 147 Results ....................................................................................... 147 Discussion .................................................................................. 162 Overall Conclusions ........................................................................ 164   viii  Chapter 5: Examination of FosB/FosB Expression and Localization in the ARC and SN ..................................................................................... 165 Introduction .................................................................................. 165 Temporal Expression of AP-1 Transcription Factors ............................. 166 Results ....................................................................................... 166 Discussion .................................................................................. 188 Sub- ...................................... 189 Results ....................................................................................... 189 Discussion .................................................................................. 196 Localization of Total FosB in DA Neurons, Total Neurons, and Glial Cells 197 Results ....................................................................................... 197 Discussion .................................................................................. 204 Long- .................. 205 Results ....................................................................................... 205 Discussion .................................................................................. 227 Overall Conclusions ........................................................................ 229  Chapter 6: Effects of Experimental Manipulation of FosB on Parkin Expression in Cell Culture and in the ARC and SN ................................. 230 Introduction .................................................................................. 230 Transcriptional Activators of the FosB Gene ....................................... 233 Results ....................................................................................... 233 Discussion .................................................................................. 240  ............................... 241 Results ....................................................................................... 241 Discussion .................................................................................. 255 AAV- ......................................... 257 Results ....................................................................................... 257 Discussion .................................................................................. 267 AAV- ................. 269 Results ....................................................................................... 269 Discussion .................................................................................. 277 Overall Conclusions ........................................................................ 278  Chapter 7: General Discussion ........................................................... 280 Differences Between TIDA and NSDA Neurons ................................... 280 Potential Pathway Involved in Parkin Regulation ................................ 283 FosBs Beyond Parkin ...................................................................... 286 Potential Future Directions .............................................................. 289 Concluding Remarks ....................................................................... 291  REFERENCES ................................................................................... 293   ix  LIST OF TABLES  Table 2.1 List of primary antibodies used ............................................... 34  Table 2.2 List of secondary antibodies used ........................................... 35  Table 3.1 Putative transcription factors of the mouse parkin promoter identified by PATCH 1.0 ....................................................................... 83  Table 3.2 Putative transcription factors of the mouse parkin promoter identified by TFSearch ......................................................................... 86  Table 3.3 Putative transcription factors of the mouse parkin promoter identified by PROMO ........................................................................... 89  Table 5.1 Mean percentage of TH-immunoreactive neurons, total neurons, and glial cells containing total FosB ..................................................... 203  Table 5.2 Pearson and Spearman correlation statistics in the comparison of  .................................................................. 224             x  LIST OF FIGURES  Figure 1.1 Location of NSDA and TIDA neuronal pathways ......................... 6  Figure 1.2 Coronal sections containing ST and SN ..................................... 7  Figure 1.3 Schematic diagram of a NSDA axon terminal ............................ 8  Figure 1.4 Mechanism of action of MPTP ................................................ 15  Figure 1.5 Coronal sections containing ME and ARC  ............................... 17  Figure 1.6 Schematic depicting the structure of the parkin protein ............ 21  Figure 2.1 Removal of the median eminence (ME) .................................. 28  Figure 2.2 Microdissection of the ST, ARC, and SN .................................. 31  Figure 2.3 Fresh brain sectioning for nuclear and cytoplasmic fraction isolation ............................................................................................ 38  Figure 2.4 Example HPLC traces for neurochemistry ............................... 42  Figure 2.5 Optimization of stereotaxic AAV delivery into the SN ................ 45  Figure 2.6 Representative images showing localization and spread of the AAV-GFP viral vector as compared to a saline sham control ..................... 46  Figure 2.7 Optimization of AAV delivery to the ARC ................................. 47  Figure 2.8 Representative images showing localization and spread of the AAV-GFP viral vector as compared to a saline sham control ..................... 48  Figure 3.1 Coding strand sequences of the mouse bi-directional promoter of Park2 (green) and Pacrg (black) ........................................................... 55  Figure 3.2 Coding strand sequences of the human Park2 (green) and mouse Park2 (black) promoters ...................................................................... 56  Figure 3.3 GC percentage and CpG islands of the mouse Park2 promoter .. 59  Figure 3.4 DNaseI hypersensitivity from the PSU group ........................... 60  xi   Figure 3.5 DNaseI hypersensitivity from the UW group ............................ 61  Figure 3.6 Histone modification marks from the Caltech group ................. 66  Figure 3.7 Histone modification marks from the SYDH group .................... 67  Figure 3.8 Histone modification marks from the LICR group in the cerebellum  ........................................................................................................ 68  Figure 3.9 Histone modification marks from the LICR group in the heart ... 69  Figure 3.10 Histone modification marks from the LICR group in the liver ... 70  Figure 3.11 Histone modification marks from the LICR group in MEL cells .. 71  Figure 3.12 Histone modification marks from the PSU group in CH12 cells       ........................................................................................................ 72  Figure 3.13 Histone modification marks from the PSU group in erythroblasts   ........................................................................................................ 73  Figure 3.14 Histone modification marks from the PSU group in megakaryocytes ................................................................................. 74  Figure 3.15 Known transcription factor binding via ChIP-seq data from the Caltech group .................................................................................... 78  Figure 3.16 Known transcription factor binding via ChIP-seq data from the LICR group ........................................................................................ 79  Figure 3.17 Known transcription factor binding via ChIP-seq data from the PSU group ......................................................................................... 80  Figure 3.18 Known transcription factor binding via ChIP-seq data from the SYDH group ....................................................................................... 81  Figure 3.19 Putative transcription factors of parkin identified by one or more program ............................................................................................ 99  Figure 3.20 Predicted transcription binding sites on the mouse and human Park2 promoters ............................................................................... 100   xii  Figure 4.1 Comparison of the effects of MPTP on ATF4 in the ARC of mice       ...................................................................................................... 111  Figure 4.2 Comparison of the effects of MPTP on ATF4 in the SN of mice . 112  Figure 4.3 Comparison of the effects of MPTP on phosphorARC of mice ..................................................................................... 113  Figure 4.4 Comparison of the effects of MPTP on phosphoSN of mice ....................................................................................... 114  Figure 4.5 Comparison of the effects of MPP+ on ATF4 in MN9D dopaminergic neuronal cell culture ......................................................................... 115  Figure 4.6 Comparison of the effects of MPP+ on parkin in MN9D dopaminergic neuronal cell culture ...................................................... 116  Figure 4.7 Comparison of the effects of tunicamycin on ATF4 in MN9D dopaminergic neuronal cell culture ...................................................... 117  Figure 4.8 Comparison of the effects of tunicamycin on parkin in MN9D dopaminergic neuronal cell culture ...................................................... 118  Figure 4.9 Comparison of the effects of MPTP on Nrf1 (120 and 95 kDa) in the ARC of mice ............................................................................... 122  Figure 4.10 Comparison of the effects of MPTP on Nrf1 (120 and 95 kDa) in the SN of mice ................................................................................. 123  Figure 4.11 Comparison of the effects of MPTP on Nrf2 in the ARC of mice       ...................................................................................................... 124  Figure 4.12 Comparison of the effects of MPTP on Nrf2 in the SN of mice 125  Figure 4.13 Parkin expression in the ARC of WT and Nrf2KO mice ........... 126  Figure 4.14 Parkin expression in the SN of WT and Nrf2KO mice ............ 127  Figure 4.15 Comparison of the effects of MPTP on NRF1 in the ARC of mice     ...................................................................................................... 132  Figure 4.16 Comparison of the effects of MPTP on NRF1 in the SN of mice       ...................................................................................................... 133   xiii  Figure 4.17 Comparison of the effects of MPTP on ROS formation and accumulation in the ME of mice .......................................................... 134  Figure 4.18 Comparison of the effects of MPTP on ROS formation and accumulation in the ARC of mice ........................................................ 135  Figure 4.19 Comparison of the effects of MPTP on ROS formation and accumulation in the ST of mice ........................................................... 136  Figure 4.20 Comparison of the effects of MPTP on ROS formation and accumulation in the SN of mice .......................................................... 137  Figure 4.21 Comparison of ROS presence in brain regions containing TIDA neurons ........................................................................................... 138  Figure 4.22 Comparison of ROS presence in brain regions containing NSDA neurons ........................................................................................... 139  Figure 4.23 Comparison of ROS presence in brain regions containing axon terminals of the TIDA or NSDA neurons ............................................... 140  Figure 4.24 Comparison of ROS presence in brain regions containing cell bodies of the TIDA or NSDA neurons ................................................... 141  Figure 4.25 Comparison of the effects of MPTP on SRY in the ARC of mice       ...................................................................................................... 144  Figure 4.26 Comparison of the effects of MPTP on SRY in the SN of mice . 145  Figure 4.27 Comparison of the effects of MPTP on c-Fos in the ARC of mice      ...................................................................................................... 149  Figure 4.28 Comparison of the effects of MPTP on c-Fos in the SN of mice       ...................................................................................................... 150  Figure 4.29 Comparison of the effects of MPTP on c-Jun in the ARC of mice     ...................................................................................................... 151  Figure 4.30 Comparison of the effects of MPTP on c-Jun in the SN of mice       ...................................................................................................... 152  Figure 4.31 Comparison of the effects of MPTP on FosB in the ARC of mice      ...................................................................................................... 153   xiv  Figure 4.32 Comparison of the effects of MPTP on FosB in the SN of mice        ...................................................................................................... 154  Figure 4.33     ................................................................................................... 155  Figure 4.34 Comparison of the effects of  ...................................................................................................... 156  Figure 4.35 Comparison of the effects of MPTP on JunD in the ARC of mice      ...................................................................................................... 157  Figure 4.36 Comparison of the effects of MPTP on JunD in the SN of mice        ...................................................................................................... 158       ................................................................................................... 159  Figure 4.38 Comparison of the effects of  ...................................................................................................... 160  Figure 4.39 Compar/JunD in the ARC and SN of mice .................................................................... 161  Figure 5.1 Time course effects of MPTP on FosB expression in the ARC and SN .................................................................................................. 168  sB expression in the ARC and SN .................................................................................................. 169  Figure  4, 6, 8, 12, or 24 h post-MPTP ............................................................... 170   4, 6, 8, 12, or 24 h post-MPTP ............................................................... 171  Figure 5.5 Time course effects of MPTP on c-Fos expression in the ARC and SN .................................................................................................. 172  Figure 5.6 Representative Western blots for c-Fos in the ARC at 4, 6, 8, 12, or 24 h post-MPTP ............................................................................ 173  Figure 5.7 Representative Western blots for c-Fos in the SN at 4, 6, 8, 12, or 24 h post-MPTP ................................................................................ 174  xv   Figure 5.8 Time course effects of MPTP on c-Jun expression in the ARC and SN .................................................................................................. 175  Figure 5.9 Representative Western blots for c-Jun in the ARC at 4, 6, 8, 12, or 24 h post-MPTP ............................................................................ 176  Figure 5.10 Representative Western blots for c-Jun in the SN at 4, 6, 8, 12, or 24 h post-MPTP ............................................................................ 177  Figure 5.11 Time course effects of MPTP on JunD expression in the ARC and SN .................................................................................................. 178  nD expression in the ARC and SN ............................................................................................ 179   4, 6, 8, 12, or 24 h post-MPTP ........................................................... 180   4, 6, 8, 12, or 24 h post-MPTP ............................................................... 181  io expression in the ARC and SN ............................................................................... 182  Figure 5.16 Early time course effects of MPTP on FosB expression in the ARC and SN ............................................................................................ 184  sB expression in the ARC and SN ..................................................................................... 185  e ARC at 1, 2, or 4 h post-MPTP ...................................................................... 186  he SN at 1, 2, or 4 h post-MPTP .......................................................................... 187   fractions derived from the ARC ........................................................................ 190  Figure 5.21 Effects of MPTP on FosB  fractions derived from the ARC ................................................................................... 191   xvi  and nuclear fractions derived from the ARC at 6 h post-MPTP ................ 192  ic fractions derived from the SN ......................................................................... 193  r fractions derived from the SN ..................................................................................... 194  and nuclear fractions derived from the SN at 6 h post-MPTP .................. 195  -immunoreactive neurons in the ARC and SN ........................................ 199  f all neurons in the ARC and SN ................................................................ 201  Figure 5.28 Total Fon glial cells in the ARC and SN ............................................................................................ 202  Figure 5.29 Extended time course effects of MPTP on DA concentration in the ST .................................................................................................. 207  Figure 5.30 Extended time course effects of MPTP on DA concentration in the ME .................................................................................................. 208  Figure 5.31 Extended time course effects of MPTP on TH expression in the ST .................................................................................................. 209  Figure 5.32 Representative Western blots for TH in the ST at 1, 3, or 7 d post-MPTP ....................................................................................... 210  Figure 5.33 Extended time course effects of MPTP on DAT expression in the ST .................................................................................................. 211  Figure 5.34 Extended time course effects of MPTP on VMAT expression in the ST .................................................................................................. 212  Figure 5.35 Representative Western blots for DAT and VMAT in the ST at 1, 3, or 7 d post-MPTP .......................................................................... 213  Figure 5.36 Extended time course effects of MPTP on weight change....... 215   xvii  Figure 5.37 Extended time course effects of MPTP on FosB expression in the ARC ................................................................................................ 216  Figure 5.38 Extended time course effects of MPTP the ARC ........................................................................................... 217  e ARC at 1, 3, or 7 d post-MPTP ...................................................................... 218  Figure 5.40 Extended time course effects of MPTP on parkin expression in the ARC ........................................................................................... 219  Figure 5.41 Representative Western blots for parkin in the ARC at 1, 3, or 7 d post-MPTP..................................................................................... 220  Figure 5.42 Extended time course effects of MPTP on parkin expression in the SN ............................................................................................ 221  Figure 5.43 Representative Western blots for parkin in the SN at 1, 3, or 7 d post-MPTP ....................................................................................... 222  Figure 5.44 Linear regressions plotted for FosB as compared to parkin at 1, 3, and 7 days post-MPTP ....................................................................... 225  compared to parkin at 1, 3, and 7 days post-MPTP ................................................................... 226  Figure 6.1 Early time course effects of MPTP on phosphorylated CREB (Ser133) in the ARC and SN .............................................................. 234  Figure 6.2 Early time course effects of MPTP on total CREB in the ARC and SN .................................................................................................. 235  Figure 6.3 Early time course effects of MPTP on phosphorylated SRF (Ser103) in the ARC and SN .............................................................. 236  Figure 6.4 Early time course effects of MPTP on total SRF in the ARC and SN    ................................................................................................... 237  Figure 6.5 Representative Western blots for phosphorylated CREB (Ser133) and phosphorylated SRF (Ser103) in the ARC and SN at 1, 2, or 4 h post-MPTP .............................................................................................. 238   xviii  Figure 6.6 Representative Western blots for total CREB and SRF in the ARC and SN at 1, 2, or 4 h post-MPTP........................................................ 239  Figure 6.7 Effects of MPP+ on stored DA in MN9D cells .......................... 243  Figure 6.8 Cell viability of MN9D cells at 24 h post MPP+ ........................ 244  Figure 6.9 FosB expression in MN9D cells at 6 and 12 h post MPP+ ......... 245  +  ..... 246  D cells at 6 and 12 h post-MPP+  ................................................................... 247  Figure 6.12 Parkin expression in MN9D cells at 6 and 12 h post MPP+ ..... 248  Figure 6.13 Representative Western blot for parkin in MN9D cells at 6 and 12 h post-MPP+ ..................................................................................... 249  Figure 6.14 FosB expression in MN9D cells at 6 and 12 h post PMA ......... 250  9D cells at 6 and 12 h post PMA ....... 251  9D cells at 6 and 12 h post-PMA ..................................................................... 252  Figure 6.17 Parkin expression in MN9D cells at 6 and 12 h post PMA ....... 253  Figure 6.18 Representative Western blot for parkin in MN9D cells at 6 and 12 h post-PMA ...................................................................................... 254  Figure 6.19 Parkin in the ARC after AAV-- mediated overexpression       ...................................................................................................... 259  the ARC of mice injected with either AAV-GFP or AAV- ....................... 260  Figure 6.21 Parkin in the SN after AAV--mediated overexpression .. 261  SN of mice injected with either AAV-GFP or AAV-sB ......................... 262  Figure 6.23 DA concentration in the ST after AAV- ....................... 263   xix  Figure 6.24 DOPAC concentration in the ST after AAV- ................. 264  Figure 6.25 DOPAC/DA ratio in the ST after AAV- ......................... 265  Figure 6.26 DA concentrations in the ME after AAV- ..................... 266  Figure 6.27 FosB in the ARC after AAV-D expression and 24 h post-MPTP .............................................................................................. 271  -D expression and 24 h post-MPTP .............................................................................................. 272  mice injected with either AAV-GFP or AAV- ................... 273  Figure 6.30 Parkin in the ARC after AAV-D expression and 24 h post-MPTP .............................................................................................. 274  ARC of mice injected with either AAV-GFP or AAV- ......... 275  Figure 6.32 DA concentrations in the ME after AAV--MPTP .............................................................................................. 276  Figure 7.1 Predicted parkin regulatory pathway involved in the response to acute MPTP exposure ........................................................................ 285                      xx  KEY TO ABBREVIATIONS  3V Third ventricle AAV Adeno-associated virus AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA Analysis of Variance AP-1 Activator protein 1 ARC Arcuate nucleus ARE Antioxidant response element ATF4 Activating transcription factor 4 BCA Bicinchoninic acid BDNF Brain derived neurotrophic factor BSA Bovine serum albumin °C Degrees Celsius Ca2+ Calcium Caltech California Institute of Technology CaMKII Calmodulin kinase II CamKIV Calmodulin kinase IV cAMP Cyclic adenosine monophosphate  CCCP Carbonyl cyanide 3-chlorophenyl hydrazone ChIP Chromatin immunoprecipitation ChIP-Seq Chromatin immunoprecipitation sequencing  xxi  CREB cAMP response element binding protein CTCF CCCTC-binding factor Cul3 Cullin3 Cy3 Cyanine 3 d Day(s) D1 Dopamine receptor D1 D2 Dopamine receptor D2 DA Dopamine DAT Dopamine transporter DCF-DA 2', 7'-dichlorofluorescine diacetate DDC Dopa decarboxylase ddH2O Double-distilled dihydrogen monoxide (water) DMEM Dulbecco's modified eagle medium DNA Deoxyribonucleic acid DOPA 3, 4-Dihydroxyphenylalanine DOPAC 3, 4-Dihydroxyphenylacetic acid DOPAL 3, 4-Dihydroxyphenylacetaldehyde EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol--aminoethyl ether)-N,N,N',N'- tetraacetic acid    ENCODE Encyclopedia of DNA elements ER Endoplasmic reticulum  xxii  GADD43 Growth arrest and DNA damage inducible protein 34 GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFAP Glial fibrillary acidic protein GTP Guanosine triphosphate h Hour(s) H3 Histone protein 3 HAT Histone acetyltransferase HPLC High pressure liquid chromatography i.p. Intraperitoneal IBR In-between RING kb Kilobase KCl Potassium chloride kDa Kilodalton Keap1 Kelch-like ECH-associate protein 1 kg Kilogram KO Knock-out LAT L-amino acid transporter L-DOPA Levodopa LICR Ludwig Institute for Cancer Research LRRK2 Leucine-rich repeat kinase 2 LyF-1 Lymphoid transcription factor 1 MAO-B Monoamine oxidase B  xxiii  Mb Megabase ME Median eminance MEL cells Murine erthroleukemia cells MeOH Methanol mg Miligram MgCl2 Magnesium chloride min Minute(s) miRNA Micro ribonucleic acid ml Milliliter mM Millimolar mm Millimeter MN9D Murine mesencephalon-derived dopaminergic neuronal cell  line 9D  MPP+ 1-methyl-4-phenylpyridinium MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mRNA Messenger ribonucleic acid MSN Medium spiny neuron MyoD Myogeneic differentiation NAc Nucleus accumbens NaCl Sodium chloride NAD+/NADH Nicotinamide adenine dinucleotide NaOH Sodium hydroxide NeuN Neuronal nuclei  xxiv  ng Nanogram NGS Normal goat serum NMDA N-methyl-D-aspartate  Nrf1 Nuclear factor (erythroid-derived 2)-like 1  NRF1 Nuclear respiratory factor 1 Nrf2 Nuclear factor (erythroid-derived 2)-like 2 NSDA Nigrostriatal dopamine Pacrg Park2 co-regulated gene PARIS Parkin interacting substrate PBS Phosphate buffered saline PD Parkinson disease PERK PKR- pg Picogram PGC- Peroxisome proliferator-activated receptor gamma    PINK1 PTEN-induced putative kinase 1 PKA Protein kinase A PMA Phorbol 12-myristate 13-acetate PSU Pennsylvania State University REP Repressor element of parkin  RING Really interesting new gene RIPA buffer Radioimmunoprecipitation assay buffer ROS Reactive oxygen species  xxv  RTPCR Real-time polymerase chain reaction s Second(s) s.c. Subcutaneous SDS Sodium dodecyl sulfate SEM Standard error of the mean Ser Serine SIRT2 Sirtin 2 SN Substantia nigra SNCA -synuclein  SOS Sodium octyl sulfate SRF Serum response factor SRY Sex determining region on chromosome Y ST Striatum  SYDH Stanford/Yale TCF11 Transcription factor 11 TDF Testis determining factor  TGX Tris-glycine extended TH Tyrosine hydroxylase TIDA Tuberoinfundibular dopamine TPA 12-O-tetradecanoylphorbol-12-acetate TRE TPA-response element Tris-HCl Tris-hydrochloric acid  xxvi  tRNA Transfer ribonuclei acid Ubl Ubiquitin-like UCSC University of California Santa Cruz  Microgram  Micromolar uORF Upstream open reading frame UPR Unfolded protein response UPS Ubiquitin proteasome system USF Upstream stimulating factor V Volts VMAT Vesicular monoamine transporter VTA Ventral tegmental area WT Wild-type  1  Chapter 1: General Introduction  Parkinson Disease Parkinson disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the degeneration of the nigrostriatial (NS) dopamine (DA) neurons and the formation of Lewy bodies or intraneuronal protein aggregates (Nuytemans et al., 2010). The loss of the NSDA neurons leads to the classic motor symptoms of PD, which are resting tremor, bradykinesia, rigidity, and postural instability (Jankovic, 2008; Fahn, 2003). Additionally, other there are non-motor symptoms associated with PD, such as depression, hyposmia, anhedonia, gastrointestinal issues, sleep disorder, cognitive impairment and neuropsychiatric symptoms (Sveinbjorndottir, 2016).  The first clinical description of PD was in Dr. James Parkinson's 1817 "An Essay on the Shaking Palsy". Dr. Parkinson described the symptoms as "involuntary tremulous motion, with lessened muscular power, in parts not in action even when supported; with propensity to bend the trunk forward, and to pass from a walking to a running pace". This description from Dr. Parkinson best describes the resting tremor and postural instability associated with PD. Though Parkinson was the first to clinically describe the disease, mention of its symptoms can be found throughout history. Symptoms of PD, mostly the tremors, were alluded to in two books of the   2  Old Testament and once in the New Testament, described by Leonardo da Vinci, referred to as "the palsy" by William Shakespeare, and mentioned in ancient medical texts, such as Charaka Samhita from India, the Akkadian Diagnostic Handbook from Mesopotamia, and De Tremore, Palpitatione, Convulsione et Rigore by Galen of Pergamon (Raudino, 2012).   NSDA Neurons and PD Cell bodies of the NSDA neurons are located in the substantia nigra pars compacta (SNpc) and axons project to and terminate in the striatum (ST) (Figures 1.1 and 1.2). DA synthesis within DA neurons starts with the transport of dietary tyrosine into the neurons via the large neutral amino acid transporter (LAT) (Fernstrom and Fernstrom, 2007). Tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis, adds a hydroxyl group to tyrosine, converting it to 3,4-dihydroxyphenylalanine (DOPA) (Levitt et al., 1965). A carboxyl group is then removed by DOPA decarboxylase (DDC), producing DA, which is packaged into synaptic vesicles via the vesicular monoamine transporter (VMAT) (Weihe et al., 1994; Lovenberg et al., 1962) (Figure 1.3). An action potential causes synaptic vesicles within the pre-synaptic axon terminal to release DA into the synaptic cleft.  The NSDA neurons synapse on the medium spiny neurons (post-synaptic), which possess D1 or D2-like DA receptors. Both D1 and D2 receptors are G-  3  protein coupled receptors. DA binding to the receptor leads to the exchange -subunit acts on adenylate cyclase, either stimulatory or inhibitory based on s i subunit respectively. Inhibition of adenylate cyclase results in a decrease in cyclic adenosine monophosphate (cAMP), where stimulation increases cAMP (Kebabian and Greengard, 1971). This regulation of cAMP modifies the activity of protein kinase A, of which cAMP is a co-factor. Protein kinase A can phosphorylate TH, increasing its activity and DA synthesis (Kim et al., 1993). In addition to post-synaptic DA receptors, NSDA neurons have pre-synaptic D2 autoreceptors, which released DA can act on and repress DA synthesis in a negative feedback loop. DA not interacting with DA receptors can be scavenged using the DA transporter (DAT), which is used to re-uptake DA into the terminals of the pre-synaptic neurons (Shimada et al., 1991). The recaptured DA, as with newly synthesized DA, can be either packaged into synaptic vesicles or metabolized. Monoamine oxidase B (MAO-B) found bound to the outer membrane of mitochondria will deaminate DA, producing 3, 4-dihydroxyphenylacetaldehyde (DOPAL) (Hafer et al., 1987; O'Carroll et al., 1983). The aldehyde on DOPAL is oxidized by aldehyde dehydrogenase, forming 3, 4-dihydroxyphenylacetic acid (DOPAC), which can exit the axons via simple diffusion (Marchitti et al., 2007) (Figure 1.3).                 4  The role of the NSDA neurons terminating in the ST is in the regulation of the basal ganglia and motor control. The basal ganglia are comprised of interconnected nuclei that connect to the motor cortex and function to coordinate movement. The circuitry of the direct pathway involves D1 receptor expressing medium spiny neurons, which send inhibitory signals to the globus pallidus interna and substantia nigra pars reticulata. The globus pallidus interna and substantia nigra pars reticulata send inhibitory signals to the thalamus, which sends excitatory signals to the motor cortex. The motor cortex, in turn, sends inhibitory signals to the brain stem, spinal cord and subthalamic nucleus. The inhibitory signals from the motor cortex to the subthalamic nucleus sends excitatory signals to the globus pallidus interna and subtantia nigra pars reticulata. The circuitry of the indirect pathway involves the D2 receptor expressing medium spiny neurons, which send inhibitory signals to the globus pallidus externa, which in turn sends inhibitory signals to the subthalamic nucleus, globus pallidus interna and substantia nigra pars reticulata.  Increased DA release from the NSDA neurons terminating near medium spiny neurons interacts with D1 and D2 receptors. Medium spiny neurons with D1 receptors are activated by DA, which inhibits neurons in the globus pallidus interna and substantia nigra pars reticulata that normally inhibit thalamic neurons. This allows neurons in the thalamus to send excitatory signals to the motor cortex. Medium spiny neurons with D2 receptors are   5  inhibited by DA, which blocks the inhibition of neurons in the globus pallidus externa, allowing the globus pallidus externa neurons to inhibit neurons in the subthalamic nucleus, globus pallidus interna and subtantia nigra pars reticulata. This allows neurons in the thalamus to send excitatory signals to the motor cortex. The loss of the NSDA neurons leads to the decrease of DA acting on both types of medium spiny neurons, and thus has the inverse effect as DA release, decreasing the signal sent to the motor cortex, causing the motor symptoms associated with PD.          6   Figure 1.1 Location of NSDA and TIDA neuronal pathways. The NSDA neurons (red) have cell bodies in the SN and project rostral to the ST. The TIDA (green) neurons are housed entirely in the MBH, with cell bodies in the ARC and axon terminals in the ME. Also shown as references, are the locations of the lateral ventricle, olfactory bulb, cerebral cortex, hippocampus, and cerebellum.     7   Figure 1.2 Coronal sections containing ST and SN. The ST (blue) is located in a section rostral to the SN (red). Coronal sections are modified from the mouse brain library (www.mbl.org).     8   Figure 1.3 Schematic diagram of a NSDA axon terminal. TH adds a hydroxyl group to tyrosine, converting it to DOPA. DOPA has a carboxyl group removed by DDC, converting it to DA. DA is packaged into synaptic vesicles by VMAT. DA can then be released into the synapse. Once in the synapse, DA can contact D1 or D2 receptors on the post-synaptic neurons (post-synaptic neurons possess either D1 or D2, not both as depicted in the simplified figure), or the D2 receptor on the pre-synaptic axon terminal. DA binding to the D2 receptor leads to the inhibition of TH. DA in the synapse can also re-enter the axon through DAT. DA scavenged by DAT or not packaged into vesicles, can be broken down into DOPAC by MAO-B.          9  Etiology of PD  Though the exact cause of PD is unknown, there are both genetic and environmental factors shown to be associated with the disease. There are 19 loci that have been identified based on family histories and genome wide association studies as Parkinson disease related or Park loci. The most commonly studied are parkin (Park2-synuclein (SNCA, or Park1, and Park4), leucine-rich repeat kinase 2 (LRRK2 or Park8), PTEN-induced putative kinase 1 (PINK1 or Park6) and DJ-1 (Park6) (Nuytemans et al., 2010). Parkin is an E3 ubiquitin ligase that tags misfolded proteins for degradation, but this protein has other functions than just within the ubiquitin proteasome system (UPS) as discussed below (see Parkin). Individuals with parkin mutations have been found to have a loss of NSDA neurons, noradrenergic neurons in the locus coeruleus, and variable Lewy -synuclein protein has been suggested to normally function -synuclein are a key component of Lewy body aggregates associated with PD (Cookson, 2012; Cookson, 2009). The mechanism by which LRRK2 leads to PD is not known, however, some mutations in the gene affect kinase activity (MacLeod et al., 2006; Klein and Westenberger, 2012). PINK1 on defective or damaged mitochondria can phosphorylate parkin, leading to the ubiquitylation of mitochondrial proteins such as mitofusin and voltage-dependent anion channel 1. This process labels mitochondria for degradation through mitophagy (Poole et al., 2008,   10  2010; Geisler et al., 2010). DJ-1 is predicted to function as an oxidative stress sensor via conserved cysteine residues (Wilson, 2011; Waak et al., 2009). Along with parkin, these genes account for six of the nineteen Park loci (Klein and Westenberger, 2012; Hardy, 2010; Nuytemans et al., 2010).  Less is known about the link between PD and the other Park loci. Park3, Park10, Park12, and Park16 do not have exact gene names and Park10 is recognized as a risk locus, rather than having a Mendelian inheritance pattern (Hernandez et al., 2016; Mitsui and Tsuji, 2014, Klein and Westenberger, 2012; Hardy, 2010). The loci with confirmed genes can be grouped into categories. UCH-L1 (Park5) and FOXO7 (Park15) are involved in the UPS, PLA2G6 (Park14) is involved in lipid metabolism, ATP13A2 (Park9) and VPS35 (Park7) with lysosome function, HTRA2 (Park13) is associated with mitochondria, GIGYF2 (Park11) is predicted to function in the regulation of tyrosine kinase receptor signaling, EIF4G1 (Park18) with translation, and DNAJC16 (Park19) encodes a heatshock protein (Hernandez et al., 2016; Mitsui and Tsuji, 2014, Klein and Westenberger, 2012; Hardy, 2010). With the exception of Park10 and Park16 which do not follow a known inheritance pattern, and Park12 which follows an X-linked pattern, the other Park loci follow either autosomal dominant (Park1, Park3, Park4, Park5, Park8, Park11, Park13, Park17, and Park18) or autosomal recessive (Park2, Park6, Park7, Park9, Park14, Park15, and Park19) Mendelian inheritance patterns (Hernandez et al., 2016). These monogenic forms of PD   11  are responsible for approximately 30% of familial and less than 6% of sporadic PD (Kumar et al., 2011). In addition to monogenic forms of parkinsonism, there are risk loci that can factor into polygenic parkinsonism. Examples of risk loci identified via GWAS are MAPT which is associated with microtubules, GBA which is associated with the lysosome, ATXN3 which is associated with the UPS, ATXN2 which does not have a known function, NUCKS1 which is involved in mitosis, SIPA1L2 which is involved in the Ras signaling pathway, TMEM163 which is predicted to function in the recruitment of cations to vesicles, STK39 which is a serine/threonine kinase involved in stress response, MCCC1 which is a subunit of 3-methylcrotonyl-CoA carboxylase, TMEM175 which is a component of potassium channels in lysosomes, SCARB2 which is involved in membrane transport to lysosomes, HLA-DQB1 which is a component of a histocompatibility complex, GPNMB which is a transmembrane glycoprotein, FGF20 which is a fibroblast growth factor, INPP5F which is a specific phosphatase, MIR4697 which encodes a miRNA (microRNA), CCDC62 which is a coactivator for nuclear receptors, GCH1 which is an enzyme in tetrahydrobiopterin synthesis, VPS13C which plays a role in vesicle trafficking, STX1B which is predicted to play a role in synaptic vesicle exocytosis, SREBF which is a transcription factor involved in sterol syntheis, RIT2 which is involved in the mitogen activated protein kinase pathway, and DDRGK1 which plays a role in endoplasmic reticulum stress   12  (Hernandez et al., 2016; Mitsui and Tsuji, 2014; Hardy, 2010). Some short nucleotide polymorphisms in SNCA and LRRK2 are also associated with risk in the context of polygenic PD, in addition to those involved in monogenic PD (Hernandez et al., 2016). Both monogenic and polygenic factors have a role in the risk of developing PD, as well as the age of onset. Rare mutations in monogenic genes that are highly penetrant, such as those in Park2 are more likely to cause PD at an early age (Escott-Price et al., 2015). This compares to polygenic causes of PD, where common variants of multiple risk loci cause the disease. In this case there tends to be a lower occurrence of the disease and a later age of onset. The caveat however is that with increased common variants of risk loci, the prevalence of the disease can increase as well as the potential for an earlier age of onset (Escott-Price et al., 2015).  In addition to genetic risk factors, there are environmental risk factors that mostly revolve around rural living and farming. In addition to rural living itself, other aspects such as drinking well water and consequential exposure to manganese, herbicides such as paraquat and pesticides such as rotenone have all been suspected (Bellou et al., 2016; Pezzoli et al., 2013; Mortimer et al., 2012; Noyce et al., 2012; Van der Mark et al., 2012; Brown et al., 2006). In the early 1980s heroin addicts in San Francisco who used a synthetic opiate desmethylprodine exhibited Parkinsonian symptoms, more specifically the inability to initiate movement. These symptoms were found   13  to be caused by a by-product produced in the production of desmethylprodine, called 1-methyl-4-phenyl-1,2,3,6-tetrahydropyradine (MPTP). Exposure to MPTP in primates results in the selective degeneration of the NSDA neurons, but with no Lewy body pathology (Dauer and Przedborski, 2003; Langston et al., 1999; Ballard et al., 1985).  MPTP The selectivity of MPTP for DA neurons is based on the mechanism of action of the drug. MPTP is highly lipophilic and readily crosses the blood brain barrier. MPTP is metabolized in glial cells by monoamine oxidase-B into 1-methyl-4-phenylpyridinium (MPP+) (Markley et al., 1984; Dauer and Przedborski, 2003). This toxic metabolite is selectively taken up into the DA neurons through DAT, which normally functions in the re-uptake of released DA from the synapse (Chan et al., 1991; Dauer and Przedborski, 2003) (Figure 1.4). MPP+ has many adverse effects on the neurons including the uptake via the VMAT and displacement of stored DA from the synaptic vesicles (potentially leading to accumulation of toxic DA metabolites), inhibition of mitochondrial Complex I, and interactions with cytosolic enzymes, each of which can cause oxidative stress (Chan et al., 1991; Liu et al., 1992; Dauer and Przedborski, 2003). In some MPTP dosing paradigms, indicators of oxidative stress are present, such as lipid peroxidation and increased superoxide dismutase (Hung and Lee, 1998). Oxidative stress   14  could also be related to other effects of MPTP, such as microtubule destabilization (Cartelli et al., 2013; Cappelletti et al., 2005). In vitro, MPP+ has also been shown to act as a proteasome inhibitor (Lansdell, et al., submitted).       15   Figure 1.4 Mechanism of action of MPTP. MPTP readily crosses the blood-brain barrier and is converted to MPP+ by mitochondrial MAO-B in glial cells. MPP+ released from glial cells enter DA neurons through DAT. In DA neurons, MPP+ can enter into synaptic vesicles via VMAT and displace stored DA, resulting in generation of toxic DA metabolites. MPP+ in vitro can directly inhibit the proteasome, destabilize microtubules and inhibit mitochondrial Complex I.    16  In mice, acute MPTP (single subcutaneous injection of 20 mg/kg) decreases stored DA in the axon terminal regions of the NSDA, MLDA, and TIDA neurons. DA in the NSDA neurons does not recover, whereas DA in the TIDA neurons fully recovers by 24 h after MPTP (Behrouz et al., 2007). The cell bodies of the TIDA neurons are located in the arcuate nucleus (ARC) of the mediobasal hypothalamus (Figure 1.5). The axons of TIDA neurons are short relative to those of NSDA neurons and do not extend out of the mediobasal hypothalamus, instead the axons project basally and terminate outside the blood-brain barrier in the median eminence (ME) (Figure 1.5). TIDA neurons are also unaffected in PD (Matzuk et al., 1985). Recovery of the TIDA neurons is not only dependent on de novo protein synthesis, but also increased expression of parkin, which occurs in the TIDA but not the NSDA neurons (Benskey et al., 2012; 2015).          17   Figure 1.5 Coronal sections containing ME and ARC. Within the MBH the ME (blue) is located directly below the third ventricle and the ARC (red) is flanking the third ventricle. Coronal sections are modified from the mouse brain library (www.mbl.org).       18  Parkin Parkin is an enzyme containing 465 amino acids in humans and 464 amino acids in mice (Kitada et al., 1998; Li and Gehring, 2015). Both human and mouse parkin have a ubiquitin like domain (Ubl), linker region, three really interesting new gene (RING0, RING1, and RING2) domains, a repressor element of parkin (REP) and an in-between RING domain (IBR) (Wenzel et al., 2011; Hristova et al., 2009; Zhang et al., 2000; Li and Gehring, 2015) (Figure 1.6). These domains allow parkin to function as an E3 ubiquitin ligase (Zhang et al., 2000; Li and Gehring, 2015). In the UPS, an E1 activating enzyme binds ubiquitin in an ATP dependent reaction and in turn, transfers the ubiquitin to the E2 conjugating enzyme. A misfolded protein substrate bound to an E3 ubiquitin ligase interacts with the E2 conjugating enzyme, which transfers the ubiquitin to the substrate. This process is repeated, forming a polyubiquitin chain. The ubiquitinated substrate can then be transferred to the proteasome to be degraded (Pickart, 2001; Li and Gehring, 2015). In the context of parkin, each domain aids in the function of parkin as an E3 ubiquitin ligase.     The Ubl domain interacts with Rpn10, a regulatory subunit of the 26S proteasome, and is predicted to aid in the binding of parkin to the proteasome (Sakata et al., 2003; Wilkinson et al., 2000). In the context of the UPS, the RING1 domain of parkin interacts with the E2 conjugating enzyme and RING2 contains a catalytic cysteine involved in ubiquitin   19  transfer to the substrate (Zhang et al., 2000). RING0 can interact with 14-3-3 chaperone proteins, which leads to the inhibition of the ubiquitin ligase activity of parkin (Hristova et al., 2009; Sato et al., 2006). REP located between the IBR and RING2 domains can interact with RING1 to block the E2 conjugating enzyme from binding (Trempe et al., 2013). In addition to its role as an E3 ubiquitin ligase, parkin has other functions. The RING1, linker, and RING2 domains can bind to microtubules and aid in their assembly and stability (Yang et al., 2005; Ren et al., 2003,2009). Parkin can affect proteasome activity, where in the ST of mice deficient in parkin, there is decreased proteasome activity as compared to wild-type (WT) mice (Lansdell, et al., submitted).  Parkin can regulate mitochondrial dynamics, including degradation, fusion, fission, and biogenesis. PINK1 is constitutively expressed and subsequently degraded in the mitochondrial matrix. When mitochondria are damaged, depolarization of the membrane leads to PINK1 localization to the outer mitochondrial membrane. PINK1 recruits parkin which ubiquitinates proteins of the mitochondrial membrane and leads to the formation of a mitophagosome that fuses with a lysosome to degrade the mitochondria (Clark et al., 2006; Park et al., 2006, 2009; Poole et al., 2008; Vives-Bauza et al., 2010; Pickrell et al., 2015; Li and Gehring, 2015; Lutz et al., 2009; Narendra et al., 2008). In fission and fusion, key proteins involved in each process, such as dynamin-related protein 1, mitofusin 2, and optic atrophy 1,   20  are substrates of parkin. However, parkin functions more prominently in fission, where overexpression of parkin leads to elongated mitochondria and the loss of parkin leads to increased fusion (Poole et al., 2008; Buhlman et al., 2014; Chen et al., 2005; Ziviani et al., 2010; Li and Gehring, 2015).  The regulation of mitochondrial biogenesis by parkin is via peroxisome proliferator-activated receptor---regulator of mitochondrial biogenesis that is transcriptionally repressed by parkin-interacting substrate (PARIS) (Shin et al., 2011). The E3 ubiquitin ligase activity of parkin degrades PARIS, resulting in increased expression of PGC-together stimulate mitochondrial biogenesis (Russell et al., 2004; Wu et al., 1999). Finally, parkin has been shown to be a transcription factor of presenilin-1 and presenilin-2, two proteins involved in Alzheimer disease (Duplan et al., 2013). Parkin as a transcription factor uses the RING1-IBR-RING2 domains and has a consensus sequence or GCCGGAG (Duplan et al., 2013).        21   Figure 1.6 Schematic depicting the structure of the parkin protein. Shown is the human parkin protein with 465 amino acids. The location of the Ubl, linker region, RING0, RING1, IBR, REP, and RING2 domains are shown, with length in amino acids of each domain indicated by the numbers below.                      22  Parkin and MPTP Recovery of DA stores in axon terminals of the TIDA neurons is dependent on parkin and over-expression of parkin in the NSDA neurons rescues the loss of TH after acute MPTP exposure (Benskey et al., 2015). Comparing regions containing TIDA and NSDA neuronal axon terminals, at 4 h post-MPTP, there are high levels of MPP+ in both the ST and ME, but by 24 h MPP+ is cleared from these tissues (Benskey et al., 2012). After a single 20 mg/kg subcutaneous injection of MPTP, parkin mRNA in TIDA neuronal cell bodies in the ARC increases almost 3-fold 8 h post-injection, and parkin protein almost 2.5 fold within 36 h (Benskey et al., 2012). In contrast, in NSDA neuronal cell bodies in the SN parkin mRNA expression remains unchanged 8 h post-MPTP and parkin protein decreases by 36 h post exposure (Benskey et al., 2012). In the context of chronic MPTP (mice treated every 3.5 days for 35 days with MPTP were allowed to recover 21 days), parkin in the ARC remains elevated over the 21-day recovery period (Benskey et al., 2013). These results indicate that differential susceptibility of central DA neurons to acute neurotoxicant exposure is correlated with de novo synthesis of parkin. In comparing the effects of MPTP and known functions of parkin, it is evident that parkin has the potential to counteract the effects of MPTP. MPTP can cause depletion of DA vesicular storage and impaired expression of TH, both of which parkin has been shown to counteract (Benskey et al., 2015).   23  MPTP causes the destabilization of microtubules, which parkin can counteract through stabilization and assembly of microtubules (Cartelli et al., 2013; Yang et al., 2005; Ren et al., 2003, 2009). MPTP inhibits mitochondrial Complex I and damages mitochondria, whereas parkin has a significant role in mitochondrial fission, fusion, degradation, and biogenesis (Li and Gehring, 2015; Shin et al., 2011). It is apparent then that parkin is a neuroprotective protein that is central to the differential susceptibility between the TIDA and NSDA neurons to acute neurotoxic insult. Through the identification of regulators of parkin, such as transcription factors, the underlying cause of the differential expression of parkin may be discovered, as well as the pathways involved, thereby providing potential avenues for the development of therapeutic neuroprotective strategies for the treatment of PD.     Summary Regional differences in parkin expression after MPTP correspond with the recovery of DA neurons. i.e. parkin increases in the ARC and the TIDA neurons recover, whereas parkin does not increase in the SN and the NSDA neurons do not recover (Benskey et al., 2012). These changes in parkin expression occur within the context of acute MPTP exposure, but chronic neurotoxicant exposure has similar regional effects demonstrating a prolonged increase in parkin expression up to 3 weeks after cessation of MPTP treatment (Benskey et al., 2013).    24  Differential regulation of parkin expression could potentially be due to differences in transcription factor regulation of the parkin promoter between TIDA and NSDA neurons. By finding candidate transcription factors, followed by the regional, temporal, and sub-cellular expression after MPTP, parkin transcription factors can identified. Expression of these transcription factors can then be altered via adeno-associated virus (AAV) expression vectors, where expression of transcriptional activators are expected to increase parkin expression and transcriptional repressors decrease parkin expression.  Central Hypothesis: Differential regulation of parkin expression in response to neurotoxic insult is due to unique expression patterns of transcription factors in the DA neurons as predicted by their susceptibility to acute MPTP administration.   Specific Aim 1. Identification of candidate transcription factors of parkin. Hypothesis: Through predictive transcription factor binding software, candidate transcription factors of parkin can be identified.  Specific Aim 2. Characterization of candidate transcription factors of parkin.  Hypothesis: Characterization of regional differences in temporal expression patterns and sub-cellular localization of candidate   25  transcription factors in the ARC and SN will be able to narrow down the list of candidate transcription factors.    Specific Aim 3. Manipulation of expression of transcription factors and their subsequent effects on parkin expression.  Hypothesis: Through the overexpression of proteins consistent with the expectations of a parkin promoter transcriptional activator, parkin expression is predicted to increase. Additionally, by preventing expression or function of parkin promoter transcriptional activators, subsequent increased parkin expression in the ARC after MPTP should be blocked.               26  Chapter 2: Methods  Animals C57BL/6J male WT mice were obtained from The Jackson Laboratory, Bar Harbor, ME. Mice were housed 3-5 to a cage in a room with a 12 h light/dark cycle (lights on 0700 h) and provided food and water ad libitum. All animal use was performed with approval from the Michigan State University Institutional Animal Care and Use Committee (AUF 10/14-183-00).  Drug Treatment MPTP (Sigma Aldrich #M0896 or Santa Cruz Biotechnology Inc. sc-206178) was dissolved in 0.9% saline and diluted to 2.0 mg/ml based on the free base of MPTP. Animals were randomly assigned to treatment groups and were injected with MPTP (20 mg/kg; s.c.) or vehicle (0.9% saline, 10 ml/kg; s.c.).   Brain Removal and Median Eminence Dissection At designated times after MPTP injection, mice were killed by decapitation with a guillotine. Scissors were used cut the skin midline along the top of the head, starting at the base of the skull and ending rostral to the eyes. One blade of the scissors were inserted into the spinal column and the other blade used to make two bilateral cuts. The frontal bone of the skull was cut   27  between the eyes. The skull was then cut along the sagittal suture, starting at the neck and proceeding rostral to the frontal bone cut. Forceps were used to peel open the skull, then the brain was carefully lifted back to expose and the optic nerves were cut. The brain was then teased out onto a wet piece of filter paper on a chilled Petri dish.  The brain was positioned to expose the ventral side and a water bottle was used to gently clean the brain as well as to better expose the third ventricle. One blade of the iridectomy scissors was inserted into the third ventricle and two cuts were made lateral of the median eminence (ME) (Figure 2.1, Panels B-C). The boundaries of the median eminence were defined by the noticeable vasculature present in the tissue. The ME was lifted with forceps and a third cut was made near the optic chiasm to detach the tissue (Figure 2.1, Panels D-E). The ME was then placed into buffer, differing based on the analytical endpoint of the experiment. A water bottle was used to again clean the brain and open the third ventricle, before the brain could be further processed, differing based on the analytical endpoint of the experiment as described below.   28   Figure 2.1 Removal of the median eminence (ME). (A) The brain before dissection, arrow points to the ME. Typically vascularization of the ME can be visualized and is used to demarcate the ME. (B) Scissors were inserted into the third ventricle (3V) and the right side of the ME was cut. (C) Scissors were reinserted into the third ventricle and the left side of the ME was cut. (D) Forceps were used to grasp and lift the ME, further exposing the third ventricle. (E) While still holding the ME, the scissors were used to cut the ME near the optic chiasm. The ME was then removed and placed in buffer. (F) The ventral surface of the brain showing the remaining mediobasal hypothalamus and 3V after removal of the ME.    29  Tissue Preparation for Conventional Western Blots Brains were frozen on dry ice, wrapped in aluminum foil and stored at -80 °C until further processed. A cryostat set at approximately -10 °C was Coronal sections containing the striatum (ST), arcuate nucleus (ARC) and substantia nigra (SN) were collected on glass slides (Figure 2.2). Three sections were taken through the ST, starting just prior to where the corpus callosum joins the right and left hemispheres and ending prior to the anterior commissure connecting the hemispheres. Two sections were taken through the ARC, starting caudal of the optic chiasm where the third ventricle is present and the optic tracts begin to ascend into the brain. One section was taken through the SN, the section was taken when the hippocampus had descended halfway down the brain and the SN was just starting to be visible. Between collecting these sections, the brain was  Within the coronal sections, ST, ARC, and SN brain regions were individually microdissected using micropunching tools. For the ST, a modified 18 gauge needle punching tool (1 mm inner diameter) was used to bilaterally dissect the ST just below and lateral to the corpus callosum (Figure 2.2, Top Panel). For the ARC, an 18 gauge needle punching tool was used to take a single punch from each section containing the ARC (Figure 2.2, Middle Panel). The punch was centered along the third ventricle and   30  was taken half to a third of the way up the third ventricle dorsal to the ventral surface of the hypothalamus. For the SN, a 21 gauge oval needle from the darker area of the midbrain representative of the SN (Figure 2.2, Bottom Panel). These punches were dorsal relative to the darker areas and contained some of the area above.  Tissue punches were transferred into RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium hatase inhibitor). Samples were lysed by sonication and centrifuged at 10,000 x g for 10 min. Supernatant fractions containing soluble proteins were collected and kept at -80 °C until used for Western blots. Aliquots of each sample were used for BCA assays to load equal protein in each well for Western blots.     31   Figure 2.2 Microdissection of the ST, ARC, and SN. Top panels show a 500 Left panels show the sections before brain regions were removed and right panels show the sections after brain regions were removed.      32  BCA Assay for Western Blots BCA reagents were mixed at a 50:1 (bicinchoninic acid:copper sulfate) 2O). A serial dilution of BSA (250, 125, 62.5, 31.25, 15.62, 7.81 and 0 ng) were used for the standards. To each tube, 0.5 ml of mixed BCA reagents were added. Tubes were incubated for 15 min in a 60 °C water bath and scanned with a Tecan Infinite M1000 Pro Microplate Reader to determine protein concentration.  Western Blot Analyses Protein from each sample was run on a 4-20% TGX gel (Bio-Rad), and transferred to a PVDF membrane. Membranes were activated in methanol, blocked in either 5% BSA or milk, and incubated with primary antibodies overnight at 4 °C (Table 2.1). Membranes were washed and incubated at RT with the appropriate secondary antibody (Table 2.2). Membranes were rmo Scientific #34077) substrate kit with the Li-Cor Fc Odyssey Infrared Imaging System. The -actin, which was used to normalize each protein of interest. Band densitometry was performed using Li-Cor Image Studio Lite (version 5.0).    33  Bands for the protein of interest and loading control had a box drawn around them, and the signal measured. Sample signals were normalized by membrane using the formula: Signal X1 = (X1/Y1n/Yn) where X1 is the signal from the protein of interest, Y1 is the loading control signal, and n/Yn) is the sum of each protein of interest to loading control ratio on the membrane. Using the above formula multiple membranes were compared. After statistics were performed, fold change was calculated by dividing the treatment over the vehicle (for the treatment group) and vehicle divided by vehicle (for the control group). Error was propagated using the formula: 1/MEAN1)2 + (SEM2/MEAN2)2)].              34  Table 2.1 List of primary antibodies used. Antibody, the blocking buffer it was diluted with, animal it was raised in, company it was purchased from, and working dilution are shown.  Primary Antibody Blocking Buffer Source Company Dilution ATF4 4% BSA in PBST Rabbit Cell Signaling #11815 1:500 c-Fos 4% BSA in PBST Rabbit Cell Signaling #4384 1:500 c-Jun 4% BSA in PBST Rabbit Millipore #09-754 1:500 CREB 4% BSA in PBST Rabbit Cell Signaling #9197 1:500 CREB-P (ser133) 4% BSA in PBST Rabbit Cell Signaling #9198 1:500 DAT 4% BSA in PBST Rat Millipore #MAB369  1:500  4% BSA in PBST Rabbit Cell Signaling #5324 1:500 -P (ser51) 4% BSA in PBST Rabbit Cell Signaling #3597 1:500 FosB 4% BSA in PBST Rabbit Cell Signaling #2251 1:500 GAPDH 4% Milk in PBST Mouse Sigma Aldrich #G8795 1:500 Histone H3 4% BSA in PBST Rabbit Cell Signaling #4499 1:500 JunD 4% BSA in PBST Rabbit Cell Signaling #5000 1:500 NRF1 4% BSA in PBST Rabbit Cell Signaling #12381 1:500 NRF1/TCF11 4% BSA in PBST Rabbit Cell Signaling #8052 1:500 NRF2 4% BSA in PBST Rabbit Abcam #AB137550 1:500 Parkin 4% Milk in PBST Mouse Cell Signaling #4221 1:500 Parkin 4% BSA in PBST Rabbit Cell Signaling #2132 1:500 SRF 4% BSA in PBST Rabbit Cell Signaling #5147 1:500 SRF-P (ser103) 4% BSA in PBST Rabbit Cell Signaling #4261 1:500 SRY 4% Milk in PBST Mouse Abcam #AB22166 1:500 TH 4% BSA in PBST Rabbit Millipore #AB152  1:500 TH-P (ser40) 4% BSA in PBST Rabbit Cell Signaling #2791 1:500 VMAT2 4% BSA in PBST Rabbit Millipore #AB1767  1:500 -actin 4% Milk in PBST Mouse Cell Signaling #3700 1:500    35  Table 2.2 List of secondary antibodies used. Antibody, the blocking buffer it was diluted with, animal it was raised in, company it was purchased from, and working dilution are shown.  Secondary Antibody Blocking Buffer Source Company Dilution Goat anti-rabbit 4% BSA in PBST Goat Cell Signaling #7074 1:2000 Goat anti-rat 4% BSA in PBST Goat Cell Signaling #7077 1:2000 Horse anti-mouse 4% Milk in PBST Horse Cell Signaling #7076 1:2000                     36  Nuclear and Cytoplasmic Fraction Isolation Nuclear extractions were performed based on methods reported by Karunakaran and Ravindranath (2009) and Korner et al. (1989). After the ME was removed, fresh brains were placed in cold PBS, and sectioned with a razor blade using a Zivic brain matrix (Figure 2.3). Sections containing the ST (1 mm), ARC (2 mm) and SN (1 mm) were collected and the regions were individually microdissected from fresh tissue, similar to in Figure 2.2. The ST tissue was placed into tissue buffer for later neurochemical analysis and the ARC and SN were placed into PBS (pH 7.4) in a 0.5 ml microcentrifuge tube. Tubes were briefly centrifuged (2,000g for 30 s), PBS MgCl2phosphatase inhibitor cocktails) added to each tube. Four gentle strokes with to homogenize the tissue. Samples were incubated on ice in weak lysis buffer for 5 min and centrifuged at 10,000 g for 10 min at 4 °C. Supernatant HEPES (pH 7.9) 0.84 M NaCl, 1.5 mM MgCl2, 0.4 mM EDTA, 0.5 mM  inhibitor cocktails) added to the pellet. Pellet was re-suspended, incubated on ice for 30 min (vortexing every 10 min), and centrifuged at 10,000 g for 10 min at 4°C. Supernatant was collected (nuclear fraction), a BCA assay used to   37  measure protein concentration in both the cytoplasmic and nuclear fractions, and equal amounts of protein used for Western blotting.              38   Figure 2.3 Fresh brain sectioning for nuclear and cytoplasmic fraction isolation. Brains were positioned ventral side up in a Zivic brain matrix. The first razor blade was placed in a caudal portion of the brain as a backstop, followed by the most rostal blade. Two large bumps can be seen, one on each side of the optic nerves, the first slot in the matrix that intersects these was selected as the position of the most rostral blade. The third blade placed was just caudal of the optic chiasm, followed by the fourth blade four slots caudal, and fifth blade six slots caudal. The final blade was placed two slots caudal of the most rostral blade. All but the most caudal razor were removed and the three indicated sections (ST, ARC, and SN) were collected and brain regions dissected the same as with frozen sections.            39  Immunofluorescent Staining and Quantification Mice were anesthetized with a lethal dose of ketamine:xylazine (24.4 mg/kg:3.6 mg/kg; i.p.) and were considered unresponsive via pedal reflex/toe pinch test. Animals were suspended on a mesh grate at an approximately 30° angle, and a superficial incision made across the belly (enough to cut the skin but not damage internal organs). Two lateral incisions were made through the diaphragm and ribs. A hemostat was attached to the xiphoid cartilage at the base of the ribs and used to retract the ribs and expose the heart. A needle connected to a peristaltic pump was inserted into the left ventricle and the right atrium was cut. Mice were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). Perfused brains were removed and transferred to vials of 4% parafomaldehyde for 24 h at 4 °C.  Fixed brains were placed in 20% sucrose-phosphate buffer until fully infiltrated SN were cut on a cryostat (-apart in 0.05 M phosphate buffer (pH 7.4). Brain sections were washed in phosphate buffer with 0.1% Triton X100, then blocked in 5% NGS or 5% BSA. Sections were transferred into 1:1000 rabbit anti-FosB (SCBT #sc-48), 1:500 sheep anti-TH (Millipore #AB1542), 1:500 goat anti-GFAP (SCBT #sc-6170) or 1:500 rabbit anti-NeuN Cy3 conjugate (Millipore #ABN78C3) (Table 2.1). Sections were washed, incubated with 1:1000 F(ab')2 anti-rabbit Alexa   40  Fluor® 488 conjugate (Cell Signaling #4412), 1:1000 donkey anti-sheep Alexa Fluor® 594 conjugate (Thermo Scientific #A11016), or 1:1000 donkey anti-goat Alexa Fluor® 594 conjugate (Thermo Scientific #A11058), and mounted.  Staining order was crucial for double labeling. For FosB and TH double labeling, the order was block with BSA, sheep anti-TH, donkey anti-sheep, reblock with NGS, rabbit anti-FosB, and goat anti-rabbit. For FosB and GFAP double labeling, the order was block with BSA, goat anti-GFAP, donkey anti-goat, reblock with NGS, rabbit anti-FosB, and goat anti-rabbit. For FosB and NeuN double labeling, the order was block with NGS, rabbit anti-FosB, goat anti-rabbit, and then rabbit anti-NeuN. Slides were imaged with a Nikon TE2000-U inverted fluorescent microscope. ImageJ was used to quantify cells co-apart (Abramoff et al., 2004). The percentage of the cells containing total FosB were calculated and averaged across animals within each treatment group. Final image plates were made in Adobe Photoshop CS2.  BCA Protein Assay for Neurochemistry  The pellets from the neurochemistry sample preparation were re-suspended in vortexed, and centrifuged for 10 min at 10,000g. BCA reagents were mixed   41  n was added). A serial dilution of BSA (250, 125, 62.5, 31.25, 15.62 and 0 ng) were used for the standards. Tubes were incubated for 15 min in a 60 °C water bath and scanned with a Tecan Infinite M1000 Pro Microplate Reader to determine protein concentration.  Neurochemical Analysis of DA and DOPAC in ST and ME animal was dissected as described above and then transferred immediately  centrifuged (2,000g for 30 s), sonicated with three 1 s bursts. The proteins were pelleted by centrifugation for 1 min at 12,000g. The supernatant were transferred to a fresh tube and brought to a final volume using a beveled Hamilton syringe. The final volumes were based on the samples, a bilateral The samples were injected into the HPLC using a mobile phase of 0.03% SOS, 10% MeOH, pH 2.3 and detected using electrochemical detector with an oxidation potential of +0.4 V. Dopamine (DA) and 3,4-dihydroxyphenylacidic acid (DOPAC) content was quantified by comparison of peak heights of the standards to each sample, and then normalized to protein content (Figure 2.4).     42   Figure 2.4 Example HPLC traces for neurochemistry. A standard containing different catacholamines of a known concentration was run at the beginning and end of each set of samples. Peaks for DA and DOPAC were hand measured for samples and compared to standards               43  Stereotaxic Surgeries Mice were anesthetized with ketamine:xylazine (12.2 mg/kg:1.8 mg/kg; i.p.) and were considered unresponsive and ready for surgery via pedal reflex/toe pinch test. Animals were placed into the stereotaxic frame. The incisor bar was set at a height of 9.5 mm and both ear bars set at 15.0 mm, thereby placing the head at an approximately 15° angle. The scalp was swabbed three times with 70% ethanol followed by three times with Betadine to clean the surgical site. A sterile scalpel was used to make an incision along the rostrocaudal axis of the scalp, cutting the tissue from between the eyes to just rostral of the ears. The skin was retracted to expose the skull and the skull swabbed with a 1.5% hydrogen peroxide solution to remove the periosteum and highlight Bregma, or the intersection between the coronal and sagittal sutures.  A 30 gauge blunt Hamilton syringe was attached to a siliconized pulled glass micropipette (60- AAV-GFP, AAV--vector. A Dremel drill was used to bore a small hole in the skull at the following needle insertion points. Unilateral SN injections of 500 nl were injected at 3.3 mm caudal, 1.6 mm and -1.6 mm lateral relative to Bregma and -4.6 mm ventral to the surface of the skull (Figures 2.5 and 2.6). Bilateral ARC injections of 250 nl were performed at a 10° angle at 2.4 mm caudal, 1.3 mm and -1.3 mm lateral relative to Bregma and 6.3 ventral to   44  the surface of the skull (Figures 2.7 and 2.8). AAV Injections were performed at a flow rate of 125 nl/min using an automated micropump (World Precision Instruments).  Following the injection, the needle was left in place for 5 min to prevent backflow. After the needle was removed, the hole in the skull was filled with sterile bone wax. A generic antibiotic ointment (mixture of polymyxin B sulfate, bacitracin and neomycin sulfate) was swabbed onto the tissue around the opening of the skull and the tissue was closed with surgical staples. Mice were injected with ketoprofen (50 mg/kg; s.c.) and transferred to cages on heating pads until completely awake. Mice were moved to a recovery room and their health monitored daily for four weeks, at which time they were used for experiments.           45   Figure 2.5 Optimization of stereotaxic AAV delivery into the SN. Bilateral injections of 500 nl was made at coordinates -3.3 mm caudal, 1.6 mm and -1.6 mm lateral relative to Bregma, and -4.6 mm ventral to the surface of the skull. (Top Panel) Needle positions are depicted as red lines on the coronal section above. (Bottom Panel) To optimize coordinates, green dye was injected unilaterally, the brain frozen, sectioned, and imaged. Coordinates were readjusted until green dye was delivered to the correct location. Two examples of sections with green dye in the correct location are shown.   46   Figure 2.6 Representative images showing localization and spread of the AAV-GFP viral vector as compared to a saline sham control. DA neurons (TH-IR neurons, red) from a single brain are shown overlapping (yellow) with infected cells (green) localized to the SN, and not spreading into the ventral tegmental area. Scale bar is 10     47   Figure 2.7 Optimization of AAV delivery to the ARC. Bilateral ARC injections of 250 nl were performed at a 10° angle at 2.4 mm caudal, 1.3 mm and -1.3 mm lateral relative to Bregma, and 6.3 mm ventral to the surface of the skull. (Top Panel) Needle positions are depicted as red lines on the coronal section above. (Bottom Panel) To optimize coordinates, green dye was injected unilaterally, the brain frozen, sectioned, and imaged. Coordinates were readjusted until green dye was delivered to the correct location. Two examples of sections with green dye in the correct location are shown.   48   Figure 2.8 Representative images showing localization and spread of the AAV-GFP viral vector as compared to a saline sham control. DA neurons (TH-IR neurons, red) from a single brain are shown overlapping (yellow) with infected cells (green) Images are from the same brain section, with some virus crossing over to the saline side. Scale bar is 10                   49  Cell Culture MN9D cells (Choi et al., 1992) were cultured on poly-D-coated plates in DMEM media (Sigma D5648) supplemented with 0.37% sodium bicarbonate (Sigma S5761) 50 U/ml Penicillin/Streptomycin (Gibco 15070-063) and 10% Fetal Clone 3 (Hyclone SH30109.03). MN9D cells were differentiated through the addition of 1mM n-butyrate added to the media for 5-7 days (media and n-butyrate changed on day 3 and 5). Cells were treated with either MPP+ which were added to the media.  Cell Harvesting and Processing Media was removed and PBS (pH 7.4) was used to gently wash the cells. Fresh PBS was added, a cell scraper was used to detach the cells and the cells suspended in 1 ml of PBS. Cells in PBS were collected in a  Western blots. For neurochemistry and Western blot samples, cells were pelleted by centrifugation (10 min at as tissue, described above.    50  Cell Viability Assay The aliquot of the cell suspension were taken from the 0 and 24 h (MPP+, aliquot and gently mixed. Aliquots were pipetted onto a hemocytometer and examined using a microscope with a 10X phase-contrast objective. White and blue cells in four quadrants of the hemocytometer were counted for each sample. White cells that did not absorb the trypan blue were counted as live cells, whereas blue cells that absorbed the dye were counted as dead cells. The number of white (living) cells was divided by the total of both the white and blue (total) cells for each sample.   DCF-DA Assay Brains were removed, ME dissected, frozen and sectioned as described asection for 2', 7'-dichlorofluorescin diacetate (DCF-DA) assay and second section taken for neurochemistry) were collected on glass slides and regions dissected as previously described. These brain regions were transferred immediately into PBS (pH 7.4) and kept at -80 °C until the DCF-DA assay. On the day of the DCF-DA assay, samples were briefly centrifuged (2,000g for 30 -DA. Samples were incubated for 1 h at 37 °C, DCF-  51  of lysis buffer (0.1% SDS in tris-HCl; pH 7.5). Samples were sonicated until the tissue punch was completely broken up. Samples were centrifuged at 6,000 g at 4 °C for 20 min. and the supernatant plated in a black well plate. Samples were plated in quadruplicate randomly in a 384 well plate. Relative fluorescent units at an excitation of 485 nm and emission of 520 nm were collected for each well.   Statistical Analysis One-way analysis of variance (ANOVA) tests were used to detect differences between three or more groups, and two-sample t-tests or paired t-tests were used to detect differences between two groups. Sample sizes had a power greater than or equal to 0.80. A p value less than or equal to 0.05 was considered a significant difference. In experiments where an ANOVA was used and a significant difference was found, post hoc Tukey tests were used for comparisons between groups. Investigators conducting endpoint data collection were blinded to treatment group assignment.         52  Chapter 3: Examination of Potential Regulators of Parkin  Introduction The differential regulation of parkin between brain regions in response to MPTP is likely due to transcriptional regulation of the Park2 gene. The Park2 gene is controlled by a bi-directional promoter, which also regulates expression of the parkin co-regulated gene (Pacrg) gene (Asakawa et al., 2001; West et al., 2004) (Figure 3.1). Park2 and Pacrg are arranged head to head with approximately 200 bp between transcriptional start sites, 204 bp in humans and 203 bp in mice (Asakawa et al., 2001; West et al., 2003) (Figure 3.2). Hypermethylation of the region between the Park2 and Pacrg transcriptional start sites causes a decrease in the expression of both parkin and PACRG in tumor cell lines, further supporting the importance of the bi-directional promoter for both genes (Agirre et al., 2006). Though hypermethylation has been shown in tumor cell lines to alter parkin expression, methylation of the Park2 promoter does not contribute to the development of PD (Cai et al., 2011). In addition, inhibition of DNA methyltransferase has no effect on parkin transcription (Wang et al., 2013). Since parkin is expressed in both the TIDA and NSDA neurons, and there is no evidence to suggest methylation of the promoter has clinical implications, it is unlikely that methylation of the promoter is responsible for the differential expression of parkin, especially the increased expression in the   53  TIDA neurons following acute neurotoxicant exposure. Alternatively, the differential regulation of parkin could be due to histone modifications and/or the expression of transcription factors within the neurons.  Similar to other bi-directional promoters, the Park2 promoter in both humans and mice does not have a TATA or CAAT box, and contains multiple CpG islands (Asakawa et al., 2001; Orekhova and Rubtsoz, 2011; UCSC Genome Browser). Previous work on the Park2 promoter has identified key regions within the promoter sequence important for transcription factor binding. The region 0 to -72 bp upstream of the parkin transcriptional start site is sufficient to drive transcription of parkin, with the region -38 to -72 bp most important for the binding of transcription factors (West et al., 2003). This region contains a putative N-myc binding site, which has been shown to be a transcriptional repressor of parkin (West et al., 2004). The studies performed by West and others were based on a luciferase reporter in a neuroblastoma cell line and did not examine the promoter beyond -151 bp relative to the parkin transcriptional start site. In addition to the 34 bp region identified by West and others, another highly conserved region of the parkin promoter was identified. The region -155 to -178 bp upstream of the transcriptional start site of parkin is conserved among humans, mice, horses, and cattle (Bouman et al., 2011). This region contains a putative CREB/ATF/AP-1 binding site, and some cell culture models have indicated that ATF4 is a transcriptional activator of parkin (Bouman et al., 2011; Sun   54  et al., 2012). Taken together, this not only shows transcriptional regulation of parkin via transcription factors, but also suggests that the regions -38 to 72 bp and -155 to -178 bp may be crucial to the regulation of parkin. In order to predict which transcription factors may play a role in the differential regulation of parkin between brain regions, data concerning the Park2 promoter was collected from ENCODE, PATCH, PROMO, and TFSearch.        55   Figure 3.1 Coding strand sequences of the mouse bi-directional promoter of Park2 (green) and Pacrg (black). Park2 and Pacrg are oriented head to head, with both genes transcribed from different strands. Sequences were aligned and show the transcriptional start site and direction of transcription (arrows) as well as the first five transcribed bases of each gene (capital letters). Rows are 40 bases long.   56   Figure 3.2 Coding strand sequences of the human Park2 (green) and mouse Park2 (black) promoters. Sequences were aligned, bases identical in each are indicated by lines, gaps in the sequence when aligned are indicated by dashes. Rows are 40 bases long.       57  DNA Methylation  Results Data from ENCODE concerning CpG islands, DNaseI hypersensitivity, ChIP-seq data for histone modifications and transcription factors within the Park2 promoter was collected from the Penn State University (PSU), California Institute of Technology (Caltech), University of Washington (UW), Ludwig Institute for Cancer Research (LICR), and Stanford/Yale (SYDH) groups and examined (Rosenbloom et al., 2015, 2013; Kent et al., 2002). The region between Park2 and Pacrg is marked as having CpG islands, which begin in the transcribed region of Pacrg and continue well into the Park2 region (Figure 3.3). The GC percent shown in ENCODE indicates areas within the promoter that range from high to low GC content, with the promoter overall having high GC content, with a few valleys towards the middle with low GC content (Rosenbloom et al., 2015, 2013; Kent et al., 2002). As a whole, the GC content of the mouse promoter is 64.0% and in humans is 72.5% (Rosenbloom et al., 2015, 2013; Kent et al., 2002). The high GC content and presence of CpG islands suggests methylation of the promoter may play a role in the regulation of parkin.  The possible role of nucleosome positioning and histone modification in the regulation of parkin was examined. DNaseI hypersensitivity from ENCODE collected from the PSU group shows the parkin promoter is highly DNaseI hypersensitive in GATA-1 erythroid progenitor cells (Figure 3.4).   58  From the UW group, the parkin promoter was shown to be highly DNaseI hypersensitive in murine erythroleukemia cells, ES-CJ7 cells, CD19 positive B-cells, mouse cerebrum, cerebellum, and whole brain (8 week and embryonic day 14.5) (Figure 3.5) (Rosenbloom et al., 2015, 2013; Kent et al., 2002). DNaseI cuts naked DNA, where histones interacting with DNA (also known as nucleosomes) protect DNA from DNaseI. Hypersensitivity to DNaseI suggests that nucleosomes are most likely not present in the promoter region.   59   Figure 3.3 GC percentage and CpG islands of the mouse Park2 promoter. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of CpG island rich regions within the sequence are denoted by the green bar. Percentage of guanine and cytosine in the sequence in clusters of five nucleotides are shown in black (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    60      Figure 3.4 DNaseI hypersensitivity from the PSU group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location DNaseI hypersensitive regions within the sequence are shown in GATA-1 erythroid progenitor cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    61    Figure 3.5 DNaseI hypersensitivity from the UW group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location DNaseI hypersensitive regions within the sequence are shown in whole brain, cerebrum, CD19 positive B-cells, 3131 cells, ES-CJ7 cells, and MEL cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).          62  Discussion The bidirectional nature of the promoter, which controls both Park2 and Pacrg suggests the promoter is key to the regulation of parkin expression. After exposure to MPTP, mRNA for both parkin and PACRG increase only in the ARC, further supporting the hypothesis that the common promoter regulates expression of both genes (Behrouz dissertation). Through the examination of the promoter, especially the potential epigenetic factors involved, what regulates parkin may be deduced. Along with transcription factors, the promoter has the potential to be regulated by methylation of the DNA, positioning and modification of the histones.  In terms of DNA methylation, the GC content of the promoter becomes important. Traditionally, the cytosines within DNA can be modified with the addition of a methyl group to the fifth carbon. As the guanine on one strand will pair with a cytosine on the other strand, the guanine is also representative of a potentially methylatable cytosine. Within the mouse promoter the GC content is 64.0% and in the human promoter it is 72.5%. The relatively high GC content of the promoter suggests the presence of CpG sites that have the potential to be methylated. Within the mouse promoter, ENCODE shows the entire promoter located within a CpG island that continues into the Park2 gene (Rosenbloom et al., 2015, 2013; Kent et al., 2002). This suggests that DNA methylation may play a inhibitory role in the   63  regulation of parkin since DNA methylation is usually associated with low expression or silencing of a gene,.  Assuming this is true for parkin, a decrease in DNA methylation associated with increased parkin promoter activity would be expected in TIDA neurons, and maintained basal levels or possibly an increase would be expected in NSDA neurons. Specifically in the SN, MPTP has been show to alter the methylation of a few genes in a genome-wide study (Hu et al., 2015). In their study the found decreased methylation at 39 loci and increased methylation at 5 loci, including the start of the PD related gene Uchl1 (Hu et al., 2015). Among all their hits, there was no mention of change in methylation of Park2, or any other PD related genes. This suggests another mechanism is probably responsible for the differential expression of parkin after MPTP. The basal methylation status of the promoter would also be predicted to be low in both TIDA and NSDA neurons, since parkin is normally expressed in both the ARC and SN.  Histones and Histone Modifications Results Concerning nucleosomes, modification of the histones can also have a role in gene regulation. Overall, ChIP-seq data did not show histone modifications present directly in the promoter, but rather at the beginning of the Pacrg and Park2 genes. The Caltech group looked at H3K4me2 and   64  H3K4me3 in C2 cells. H3K4me2 is found near the beginning of the Pacrg gene, and H3K4me3 is found near the start of the Park2 gene (Figure 3.6). From the SYDH group, H3K4me and H3K4me3 markings were examined in MEL cells, and only H3K4me3 appeared to be present at both the starts of the Park2 and Pacrg genes, but not in the center of the promoter (Figure 3.7). The LICR group looked at H3K4me, H3K4me3, H3K27a, and H3K27me3 in the cerebellum; H3K4me (8 week and embryonic day 14.5), H3K4me3 (8 week and embryonic day 14.5), H3K9a (8 week), H3K27a (8 week and embryonic day 14.5), H3K27me3 (8 week), H3K36me3 (8 week), and H3K79me2 (8 week) in the liver; H3K4me (8 week and embryonic day 14.5), H3K4me3 (8 week and embryonic day 14.5), H3K9a (8 week), H3K27a (8 week and embryonic day 14.5), H3K27me3 (8 week), H3K36me3 (8 week), and H3K79me2 (8 week) in the heart; and H3K4me, H3K4me3, H3K9a, H3K27a, H3K27me3, H3K36me3, and H3K79me2 in MEL cells (Figures 3.8-11) (Rosenbloom et al., 2015, 2013; Kent et al., 2002). All of the histone modification markers were located more towards the Park2 gene and less within the promoter. The histone marks present were H3K4me3 and H3K27a in the cerebellum; H3K4me3, H3K9a and H3K27a in the heart (both 8 weeks and embryonic day 14.5); H3K4me3, H3K9a and H3K27a in the liver (both 8 weeks and embryonic day 14.5); and H3K4me3 and H3K9a in MEL cells (Figures 3.8-11). The PSU group looked at H3K4me3, H3K9me3, H3K27me3, and H3K36me3 in CH12 cells (Figure 3.12), H3K4me3,   65  H3K9me3, H3K27me3, and H3K36me3 in erythroblasts (Figure 3.13), and H3K4me, H3K4me3, H3K9me3, H3K27me3, and H3K36me3 in megakaryocytes (Figure 3.14). In all three cell types, only H3K4me3 appeared to be present, and more towards the Park2 gene than the promoter (Figures 3.12-14) (Rosenbloom et al., 2015, 2013; Kent et al., 2002).               66    Figure 3.6 Histone modification marks from the Caltech group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in C2 cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    67    Figure 3.7 Histone modification marks from the SYDH group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in CH12 and MEL cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).   68    Figure 3.8 Histone modification marks from the LICR group in the cerebellum. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in cerebellum tissue (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    69    Figure 3.9 Histone modification marks from the LICR group in the heart. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in heart tissue (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    70    Figure 3.10 Histone modification marks from the LICR group in the liver. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in liver tissue (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    71    Figure 3.11 Histone modification marks from the LICR group in MEL cells. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in MEL cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    72    Figure 3.12 Histone modification marks from the PSU group in CH12 cells. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in CH12 cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    73    Figure 3.13 Histone modification marks from the PSU group in erythroblasts. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in erythroblasts (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    74    Figure 3.14 Histone modification marks from the PSU group in megakaryocytes. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Location of histone marks within the region are shown in megakaryocytes (Rosenbloom et al., 2015, 2013; Kent et al., 2002).              75  Discussion Similar to DNA methylation, modification and localization of histones could play a role in the regulation of parkin. Data from ENCODE shows the parkin promoter is DNaseI hypersensitive in different cell lines, cerebrum, cerebellum, and whole brain. DNaseI hypersensitivity suggests the absence or presence of few nucleosomes within the promoter, as nucleosomes would be expected to protect the DNA. Without nucleosomes blocking access to the parkin promoter, it should be free to be influenced by transcription factors. Examining the reported position of nucleosomes directly, ENCODE shows H3K4 tri-methylation and H3K9 acetylation in cell lines, cerebellum, heart and liver (Rosenbloom et al., 2015, 2013; Kent et al., 2002). These histone marks are shown to be present within the promoter and/or the start of Park2. Reported histone marks shown to be absent were H3K4 mono and di-methylation, H3K9 tri-methylation, H3K27 acetylation, H3K27 tri-methylation, H3K36 tri-methylation, and H3K79 di-methylation. The presence of H3K4 tri-methylation and H3K9 acetylation associated with Park2 and its promoter, are both indicative of a promoter of an active or potentially active gene (Karmodiya et al., 2012; Bannister and Kouzarides, 2011; Shahbazian and Grunstein 2007; Martin and Zhang 2005).  Taken together, epigenetic factors may have some influence on the promoter, but probably not a determining factor in the differential regulation after neurotoxicant exposure. Since parkin is normally expressed in both the   76  ARC and SN, it is unlikely a repressive mechanism such as DNA methylation is being removed to increase parkin expression in the ARC in response to toxicant exposure. The presence of already permissive histone modifications also suggests that there is no hindrance to parkin expression, so an increase in another factor would be expected to cause toxicant-induced differential expression of parkin.  Transcription Factors Results UCSC Genome Browser and ENCODE  Examination of the available transcription factor binding data from ENCODE, suggests a few transcription factors that may regulate parkin. From the Caltech group, myogenin and MyoD are shown to interact with the promoter region (Figure 3.15). From data from the LICR group, RNA polymerase 2 and CTCF was shown to bind to the promoter in the cerebellum, heart, liver, and MEL cells; and p300 was shown to only bind in tissue from the heart (Figure 3.16). From data from the PSU group, GATA-1, Tal1, and FlI1 were not shown to interact with the promoter in erythroblasts, GATA-1 erythroid progenitor cells, or megakaryocytes (Figure 3.17). From data from the SYDH group, CTCF, GATA-1, c-Jun, and p300 did not appear to strongly bind in CH12 or MEL cells, where RNA polymerase 2 bound in   77  both CH12 and MEL cells (Figure 3.18) (Rosenbloom et al., 2015, 2013; Kent et al., 2002).                      78    Figure 3.15 Known transcription factor binding via ChIP-seq data from the Caltech group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Locations of transcription factor binding within the region are shown for C2 cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).      79    Figure 3.16 Known transcription factor binding via ChIP-seq data from the LICR group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Locations of transcription factor binding within the region are shown for MEL cells, cerebellum, heart, and liver tissue (Rosenbloom et al., 2015, 2013; Kent et al., 2002).    80   Figure 3.17 Known transcription factor binding via ChIP-seq data from the PSU group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Locations of transcription factor binding within the region are shown for erythroblasts, GATA-1 erythroid progenitor cells, and megakaryocytes (Rosenbloom et al., 2015, 2013; Kent et al., 2002).   81    Figure 3.18 Known transcription factor binding via ChIP-seq data from the SYDH group. Image is modified from ENCODE data from chromosome 17. The region between Pacrg (dark blue) and the Park2 (light blue) genes is considered the promoter region. Locations of transcription factor binding within the region are shown for CH12 and MEL cells (Rosenbloom et al., 2015, 2013; Kent et al., 2002).                      82  PATCH1.0 Data from the program PATCH 1.0, predicting the binding of transcription factors to the promoter region, shows 98 potential transcription factors varying in predicted binding scores, from the mouse promoter region (Matys et al., 2003) (Table 3.1). PATCH 1.0 is a pattern-based program for predicting transcription factor binding sites based on similarity to a consensus binding site. Transcription factors identified were constrained to those found in vertebrates only and the program default settings were used, with a mismatch penalty of 100, maximum number of mismatches of 2, and the lower score boundary set at 85.0. Transcription factors identified by Patch 1.0 were AhR, AP-2, AP-2alphaA, AP-2alphaB, AP-4, AR, ARIX, Arnt, C/EBP, C/EBPbeta, CACCC binding factor, c-Ets-2, c-Fos, c-Jun, c-Myb, CNBP, CREB, CREBbeta, CRE-BP1, CTCF, E12, E2F, E2F-1, E2F-4, E47, EBP-80, ETF, f(alpha)-f(epsilon), FXR, GammaCAC1, GammaCAC2, GATA-1, GR, Gsh-1, HEB, HIF-1, hnRNP K, HrpF, IL-6, INSAF, IPF1, IRE-ABP, ISGF-3, Isl-1, LBP-1, LUN-1, LXR-alpha, LXR-beta, LyF-1, MAZ, MAZi, MTF-1, MyoD, Myogenin, NF-1, NFAT-1, NF-ATc, NF-ATp, NF-ATx, NF-E, NF-Y, NRF-1, Pax-2, Pax-5, Pax-8, PPAR-alpha, PPAR-gamma, PR B, Pu box, RE-BP, RXR-alpha, RXR-beta, SMAD-3, SMAD-4, Sp1, Sp3, Sp4, SRF, SRY, STAT1, STAT3, STAT5A, STAT5B, STAT6, T3R-alpha, T3R-alpha1, T3R-beta1, Tal-1, USF, USF-1, USF-2, v-Jun, WT1, XF1, XF2, XPF-1, XrpFl, and ZAP.    83  Table 3.1 Putative transcription factors of the mouse parkin promoter identified by PATCH 1.0. Transcription factors along with their respective highest binding score are shown.    Transcription Factor Highest Score Transcription Factor Highest Score AhR 100.0 MAZ 85.7 AP-2 85.7 MAZi 85.7 AP-2alphaA 85.7 MTF-1 100.0 AP-2alphaB 85.7 MyoD 100.0 AP-4 100.0 Myogenin 87.5 AR 100.0 NF-1 85.7 ARIX 100.0 NFAT-1 100.0 Arnt 100.0 NF-ATc 100.0 C/EBP 100.0 NF-ATp 100.0 C/EBPbeta 100.0 NF-ATx 100.0 CACCC-BF 100.0 NF-E 100.0 c-Ets-2 100.0 NF-Y 100.0 c-Fos 100.0 NRF-1 100.0 c-Jun 100.0 Pax-2 100.0 c-Myb 100.0 Pax-5 100.0 CNBP 85.7 Pax-8 100.0 CREB 100.0 PPAR-alpha 100.0 CREBbeta 100.0 PPAR-gamma 100.0 CRE-BP1 100.0 PR B 85.7 CTCF 100.0 Pu box 100.0 E12 100.0 RE-BP 100.0 E2F 85.7 RXR-alpha 100.0 E2F-1 85.7 RXR-beta 100.0 E2F-4 85.7 SMAD-3 100.0 E47 87.5 SMAD-4 100.0 EBP-80 100.0 Sp1 100.0 ETF 85.7 Sp3 100.0 f(alpha)-f(epsilon) 87.5 SP4 100.0 FXR 100.0 SRF 100.0 GammaCAC1 100.0 SRY 85.7 GammaCAC2 100.0 STAT1 100.0 GATA-1 100.0 STAT3 87.5 GR 85.7 STAT5A 100.0 Gsh-1 87.5 STAT5B 100.0 HEB 100.0 STAT6 100.0    84  Table 3.1 Cont'd. Transcription Factor Highest Score Transcription Factor Highest Score HIF-1 85.7 T3R-alpha 100.0 hnRNP K 85.7 T3R-alpha1 100.0 HrpF 100.0 T3R-beta1 100.0 IL-6 100.0 Tal-1 87.5 INSAF 87.5 USF 100.0 IPF1 100.0 USF-1 100.0 IRE-ABP 85.7 USF-2 100.0 ISGF-3 100.0 v-Jun 85.7 Isl-1 100.0 WT1 87.5 LBP-1 100.0 XF1 100.0 LUN-1 100.0 XF2 100.0 LXR-alpha 100.0 XPF-1 100.0 LXR-beta 100.0 XrpFI 100.0 LyF-1 100.0 ZAP 100.0                85  TFSearch Data from the program TFSearch, predicting the binding of transcription factors to the promoter region, shows 22 potential transcription factors predicted to bind the mouse promoter region (Table 3.3). As with Patch 1.0, TFSearch is a pattern based program for predicting transcription factor binding sites based on similarity to a consensus binding site. Transcription factors were identified using program default settings, with a lower limit of an 85.0% binding prediction score. Transcription factors identified by TFSearch were AP-1, c-Ets, c-Myc, CRE-BP, E2F, Evi-1, GATA-1, GATA-2, GATA-3, HSF2, IK-2, Lyf-1, MyoD, MZF1, NF-E2, Nkx-2, p300, Sox-5, Sp1, SRY, STATx, and USF.                     86  Table 3.2 Putative transcription factors of the mouse parkin promoter identified by TFSearch. Transcription factors along with their respective highest binding score are shown.  Transcription Factor Highest Score AP-1 88.7 c-Ets 87.3 c-Myc 89.1 CRE-BP 87.5 E2F 86.2 Evi-1 90.7 GATA-1 91.8 GATA-2 91.7 GATA-3 92.5 HSF2 85.9 Ik-2 91.7 Lyf-1 85.7 MyoD 88.4 MZF1 91.1 NF-E2 86.4 Nkx-2 97.7 p300 85.1 Sox-5 85.6 Sp1 87.7 SRY 100.0 STATx 90.4 USF 88.8           87  PROMO Data from the program PROMO, predicting the binding of transcription factors to the promoter region, shows 193 potential transcription factors predicted to bind the mouse promoter region (Messeguer et al., 2002; Farre et al., 2003) (Table 3.4). PROMO is a matrix based program for predicting transcription factor binding sites. Unlike Patch 1.0 and TFSearch, PROMO assigns a dissimilarity score based on a position weight matrix for each transcription factor. A position weight matrix is based around the consensus binding sequence and accounts for common variations at different bases. Transcription factors were identified using program default settings, with an upper limit of 15.0% dissimilarity to the consensus transcription factor binding sequence. Transcription factors identified by PROMO were ABI4, Adf-1, AhR, AhR:Arnt, AIRE, ALF1B, Alfin1, ANT, Antp, AP-2, AP2-alphaA, AP-4, Arnt, ATF3, BR-C Z2, BR-C Z3, BTEB3, BTEB4, C/EBP, C/EBPalpha, C/EBPbeta, C/EBPbeta(p20), C/EBPdelta, CAC-binding protein, Cdx-1, c-Ets-1, c-Ets-1 54, c-Fos, c-Jun, c-Myb, COE1, COE2, CP2, c-Rel, CREMtau, CREMtau1, CREMtau2, Crx, Cutl1, D1, DBP, DEF:GLO, DEF:GLOLSQUA, DP-1, DRF1.1, DRF1.3, DSXF, DSXM, E12, E2F, E2F-1, E2F-1:DP-1, E2F-5, E47, E4F1, E74A, Egr-1, EllaE-A, Elf-1, Elk-1, ENKTF-1, ETF, Eve, f(alpha)-f(epsilon), FACB, FOXN2, FOXO3a, GAL4, GCF, GKLF, GR-alpha, GT-1, HELIOS, HES-1, HMG 1(Y), HNF-1A, HNF-1B, HNF-3, HNF-3beta, HOXA3, HSF1 (long), HSF1 (short), IA-1, IPF1, IRF-1, IRF-3, Isl-1, JunB, JunD, Kr,   88  Lf-A1, LVc, LyF-1, MATalpha2, Max-1, MAZ, MCM1, MF3, MNB1a, MYB2, MYBAS1, Myf-3, Myf-5, MyoD, myogenin, MZF-1, Ncx, NF-1, NF-1/L, NF-AT1, NF-AT2, NF-AT3, NF-ATI, NF-E4, Nkx2-1, NRF-1, Nrf2:MafK, p300, p53, Pax-2, Pax2a, Pax-5, Pax-6, Pax-9a, Pax-9b, PBF, POU1F1a, PR A, PR B, Prd, PUR alpha, PUR beta, R2, RC2, RelA, RFX1, Sp1, Sp3, Spz1, STAT1beta, STAT3, STAT4, STAT5A, StuAp, SUT1, T11, TAF, T-Ag, TCF-2, TRMI, Ubx, unc-86, USF, USF1, USF-1, USF2b, VDR, Vpr, VSF-1, WTI I, YY1, ZF5, Zic1, Zic2, and Zic3. From the 193 potential transcription factors, 164 are not repeated in the list, where AhR (2 times), C/EBPalpha (4 times), C/EBPbeta (3 times), c-Ets-1 (2 times), c-Fos (3 times), c-Jun (3 times), c-Myb (2 times), E2F-1 (2 times), Elf-1 (2 times), HNF-3beta (2 times), IPF1 (2 times), MyoD (3 times), NF-1 (2 times), NF-AT1 (2 times), Pax-5 (2 times), Sp1 (6 times), Sp3 (2 times), and USF2b (2 times) are repeated. These repeats depict predicted binding of transcription factors of different species to the promoter, for example, c-Fos is repeated three times, suggesting c-Fos of mouse, human, and rat origin can recognize and interact with the given sequence.               89  Table 3.3 Putative transcription factors of the mouse parkin promoter identified by PROMO. Transcription factors along with their lowest dissimilarity score are shown.   Transcription Factor Lowest Dissimilarity Score Transcription Factor Lowest Dissimilarity Score ABI4 3.5 IA-1 14.8 Adf-1 2.7 IPF1 3.0 AhR 0.8 IRF-1 7.9 AhR:Arnt 9.3 IRF-3 1.2 AIRE 11.9 Isl-1 7.0 ALF1B 5.4 JunB 2.9 Alfin1 9.1 JunD 8.6 ANT 14.1 Kr 13.7 Antp 2.2 LF-A1 9.7 AP-2 1.2 LVc 0.0 AP-2alphaA 0.0 LyF-1 5.6 AP-4 3.7 MATalpha2 2.0 Arnt 7.2 Max-1 0.0 ATF3 9.9 MAZ 14.3 BR-C Z2 9.2 MCM1 12.7 BR-C Z3 14.6 MF3 8.0 BTEB3 1.2 MNB1a 1.5 BTEB4 1.0 MYB2 0.0 C/EBP 0.0 MYBAS1 1.2 C/EBPalpha 0.0 Myf-3 0.0 C/EBPbeta 0.6 Myf-5 9.5 C/EBPbeta(p20) 14.0 MyoD 0.0 C/EBPdelta 0.0 myogenin 0.0 CAC-BP 4.6 MZF-1 0.1 Cdx-1 0.9 Ncx 0.0 c-Ets-1 5.7 NF-1 0.7 c-Ets-1 54 0.0 NF-1/L 14.5 c-Fos 5.7 NF-AT1 1.7 c-Jun 2.6 NF-AT2 4.9 c-Myb 3.5 NF-AT3 6.5 COE1 5.6 NF-ATI 1.1 COE2 4.7 NF-E4 9.2 CP2 5.5 Nkx2-1 1.4 c-Rel 3.0 NRF-1 0.0     90  Table 3.3 Cont'd.  Transcription Factor Lowest Dissimilarity Score Transcription Factor Lowest Dissimilarity Score CREMtau 0.0 Nrf2:MafK 9.3 CREMtau1 0.0 p300 0.0 CREMtau2 0.0 p53 3.9 Crx 1.9 Pax-2 0.3 Cutl1 9.5 Pax-2a 0.0 D1 11.3 Pax-5 2.7 DBP 7.1 Pax-6 0.0 DEF:GLO 9.4 Pax-9a 10.2 DEF:GLO:SQUA 0.0 Pax-9b 10.2 DP-1 11.6 PBF 0.0 DRF1.1 14.3 POU1F1a 6.7 DRF1.3 14.3 PR A 4.3 DSXF 14.5 PR B 4.3 DSXM 14.5 Prd 9.5 E12 9.4 PUR alpha 12.1 E2F 14.2 PUR beta 12.1 E2F-1 0.0 R2 4.8 E2F-1:DP-1 7.2 RC2 0.0 E2F-5 13.7 RelA 12.3 E47 4.3 RFX1 8.9 E4F1 1.9 Sp1 0.0 E74A 8.3 Sp3 1.0 Egr-1 8.0 Spz1 0.1 EIIaE-A 8.7 STAT1beta 12.7 Elf-1 1.9 STAT3 13.1 Elk-1 0.0 STAT4 1.5 ENKTF-1 12.6 STAT5A 0.0 ETF 0.6 StuAp 1.8 Eve 8.6 SUT1 11.6 f()-f() 0.0 T11 5.9 FACB 0.0 TAF 8.0 FOXN2 13.9 T-Ag 3.1 FOXO3a 13.9 TCF-2 10.4 GAL4 6.0 TRMI 7.1 GCF 12.6 Ubx 1.2 GKLF 9.3 unc-86 5.8     91  Table 3.3 Cont'd.  Transcription Factor Lowest Dissimilarity Score Transcription Factor Lowest Dissimilarity Score GR-alpha 13.5 USF 8.2 GT-1 6.2 USF-1 5.6 HELIOS 1.4 USF2b 1.7 HES-1 0.0 VDR 0.0 HMG 1(Y) 2.7 Vpr 12.6 HNF-1A 4.5 VSF-1 12.7 HNF-1B 9.8 WTI I 10.5 HNF-3 11.3 YY1 0.0 HNF-3 beta 5.3 ZF5 0.0 HOXA3 2.5 Zic1 0.0 HSF1 (long) 14.3 Zic2 0.0 HSF1 (short) 14.3 Zic3 0.0                  92  Discussion In addition to the possible effects epigenetics may have, transcription factors could have a crucial role in the differential regulation of parkin. A myriad of potential transcription factors of parkin identified by PATCH 1.0, TFSearch, and PROMO could act as regulators of the parkin promoter. Comparing the predicted transcription factors from each site, PROMO had 128, PATCH 1.0 had 60, and TFSearch had 12 transcription factors unique to only that program (Figure 3.19). PROMO and PATCH 1.0 both had 24 transcription factors in common (NRF-1, E47, ETF, AP-2, AP-2alphaA, PR B, c-Myb, C/EBP, C/EBPbeta, Sp3, E12, Arnt, AhR, Pax-5, Isl-1, STAT3, NF-1, AP-4, MAZ, STAT5A, IPF1, Myogenin, Pax-2, and f(alpha)-f(epsilon)) (Figure 3.19). PROMO and TFsearch had 2 transcription factors in common (Nkx-2 and p300) (Figure 3.19). PATCH 1.0 and TFSearch had 2 transcription factors in common (GATA-1 and SRY) (Figure 3.19). Transcription factors were narrowed down to the 6 transcription factors are predicted by all 3 sites (AP-1, USF, Sp1, MyoD, E2F, and LyF-1) (Figure 3.20). Additional transcription factors of interest were selected from the three predictive binding programs based on the literature. Additional transcription factors of interest are SRY, NRF-1 and NRF-2 (Figure 3.20).  E2F is a family of transcription factors found in eukaryotes. The E2F family is composed of both transcriptional activators (E2F1, E2F2, E2F3a) and repressors (E2F3b, E2F4, E2F5, E2F6, E2F7, E2F8) (Muller and Helin,   93  2000). The role of E2F transcription factors is in cell cycle control, DNA repair and synthesis, and apoptosis. This suggests E2F would be most active during cell division or death (Liu and Greene, 2001; Muller and Helin, 2000). Since neurons do not divide once terminally differentiated from neural progenitor cells, E2F expression in neurons would be expected to be tied to apoptosis. E2F in this case would be promoting apoptosis, which is not observed with the TIDA neurons after MPTP, suggesting E2F is probably not increasing parkin expression (Liu and Greene, 2001). In addition, E2F1 expression via gene chip data has been previously shown to increase in the SN after MPTP, since E2F1 is traditionally a transcriptional activator and parkin does not increase in the SN, E2F1 is probably not acting on the Park2 promoter (Behrouz dissertation).        LyF-1 (Lymphoid transcription Factor 1), also known as Ikaros, is a tumor suppressor protein commonly found in immune cells, such as B cells, T cells, NK cells, and granulocytes (Merkenschlager 2010, Kastner et al., 2013). Being as such, it is unlikely it is localized to neurons in the ARC or SN. In addition, there are no results for in situ hybridization of LyF-1 in the brain under normal conditions (Allen Brain Atlas). MyoD (Myogeneic Differentiation) is a transcription factor that regulates muscle differentiation (Buckingham and Rigby, 2014). Since it is involved in muscle development, it is unlikely to be expressed in the brain. In situ hybridization results from the Allen Brain Atlas show no of MyoD in the   94  midbrain under normal conditions, and low levels present in the olfactory bulb and cerebellum. Though there is evidence that suggests MyoD has a role in motor and retinal neuron development, there is no evidence to suggest MyoD is present in nigrostriatal or tuberoinfundibular DA neurons of adult mice (Baguma-Nibasheka et al., 2016; Kablar 2004; Wang et al., 2003). The NRF-1 indicated in the searches could refer to Nuclear Respiratory Factor 1 or Nuclear Factor Erythroid 2-Like 1. For purposes of clarification, Nuclear Respiratory Factor 1  will be abbreviated NRF1 and Nuclear Factor Erythroid 2-Like 1 abbreviated Nrf1. Nuclear Respiratory Factor 1 (NRF1) is a transcriptional activator of cytochrome C. Expression of NRF1 is induced by PGC-1, which is also a coactivator that works with NRF1 and regulates mitochondrial biogenesis (Wu et al., 1999). Parkin has also been shown to regulated PGC-PARIS (Shin et al., 2011). NRF1 is also known to interact with the parkin promoter, as well as Pink1, DJ-1 and PAELR via ChIP-Seq experiments (Satoh et al., 2013). Nuclear Factor Erythroid 2-Like 1 (Nrf1) a known transcription factor of proteasome subunits. Inhibition or decreased activity of the proteasome allows the 120 kDa form of Nrf1 located in the endoplasmic reticulum, to be cleaved into a 65 kDa active form that localizes to the nucleus, and increases expression of proteasome subunits (Radhakrishnan et al., 2010, 2014). Nrf1 is also a basic leucine zipper   95  transcription factor, and has been shown to interact with members of the AP-1 family of transcription factors (Novotny et al., 1998). Both appear relevant to neurons and roles parkin is known to play in mitochondrial maintenance and the unfolded protein response (UPS).   Nuclear Factor Erythroid 2-Like 2 (Nrf2) is involved in the response of cells to oxidative stress, and has a role in the regulation of glutathione S-transferase and NAD(P)H:quinone oxidoreductase, as well as other antioxidant response proteins (Venugopal and Jaiswal, 1996; Hayes et al., 2000). As with Nrf1, Nrf2 can dimerize with activator protein-1 (AP-1) transcription factors (Novotny et al., 1998). MPP+ is a mitochondrial Complex I inhibitor which can lead to the production of reactive oxygen species and can also displace DA from vesicles and produce toxic DA metabolites, including DA quinones (Hayes et al., 2000). Nrf2 in relation to antioxidant response proteins alone would be expected to be beneficial in response to MPP+, although Nrf2 could have the additional benefits that parkin provides if it regulates parkin expression.      Specificity protein 1 (Sp1) is constitutively expressed in neurons, and post-translational modification and interactions between other transcription factors regulate the activity rather than the expression of Sp1 (Hung et al., 2006; Tan and Khachigian 2009; Miras-Portugal et al., 2015). MPTP and its metabolite MPP+ however, have been shown to increase Sp1 expression (Chen et al., 2015). Caveats to this apparent discrepancy are that the mice   96  were treated for seven days via intraperitoneal injection and tissue was collected from the axon terminal region, where the presence of transcription factors would be in medium spiny neurons, rather than the DA neurons (Chen et al., 2015). In addition, Chen et al (2015) used primary human MPP+. These cells would not be expected to possess the dopamine transporter (DAT) to uptake MPP+ and would most likely require a higher concentration of MPP+ to illicit an effect (Chen et al., 2015). In addition, Sp1 has been predicted to act as a scaffold for other transcription factors, such as ATF3, STAT3, and c-Jun, linking Sp1 potentially to the AP-1 transcription factors (Kiryu-Seo et al., 2008).  Upstream stimulatory factor (USF) is a ubiquitously expressed transcription factor with a basic helix-loop-helix motif. The USF family is composed of USF1 and USF2, which can homo- or hetero-dimerize. The USF family plays a role in ultraviolet stress response, immune response, cell cycle, lipid metabolism, and has been shown to regulate some calcium dependant transcription factors in neurons (Corre and Galibert, 2005). USFs are ubiquitously expressed throughout the brain and other tissue making these potential regulators of parkin (Gregor et al., 1990; Sirito et al., 1992, 1994; Allen Brain Atlas).   Sex determining Region on chromosome Y (SRY) or Testis Determining Factor (TDF) is a transcription factor encoded by the SRY gene on the Y   97  chromosome. SRY is expressed for a short time in progenitor cells (pre-Sertoli cells), SRY directs these cells to become Sertoli cells, leading to the formation of the testis (Bradford et al., 2007). In addition to the expression of SRY during development of the testis, SRY is also present in the substantia nigra (SN) of adult male rodents (Dewing et al., 2006).  Specifically, SRY is found in NSDA neurons expressing TH in the SN (Dewing et al., 2006). It has also been shown that SRY acts on the TH promoter (at a AP-1 site), and leads to the up-regulation of TH (Milsted et al., 2004). When SRY is knocked-down, there is no loss in neurons, but there are fewer neurons that express TH in the SN (Dewing et al., 2006). SRY has been shown to act as a repressor in some situations and shown to be present in NSDA neurons, as such, it could act as a repressor of parkin (Dewing et al., 2006; Rath et al., 2008). SRY mRNA has also been shown to be present in the ventral tegmental area, cortex, locus coeruleus and hypothalamus, and could act on both TH and parkin (Lee and Harley, 2012). Activator Protein 1 (AP-1) is composed of homo or heterodimeric complexes that contains a combination of proteins from either the Jun, Fos, activating transcription factor (ATF), or Jun dimerization protein families (Hai and Currathe ST following MPTP exposure, but remain unchanged in the SN (Pérez-Otaño et al., 1998; Potashkin et al., 2007). Expression of c-Fos and c-Jun in the SN are reported to increase in response to MPTP, however, these   98  changes require multiple doses or high doses of MPTP (Duchemin et al., 1992; Nishi, 1997; Chen et al., 2001;). In addition, ATF has been shown in cell culture (SH-SY5Y, HEK293T, PC12, and primary mouse cortical neurons) to be linked to parkin expression and endoplasmic reticulum (ER) stress, though there were no studies in animals to support the cell culture experiments (Bouman et al., 2011; Sun et al., 2013).  Taken together, the majority of the predicted transcription factors are related to the putative AP-1 binding site. This site is located approximately -160 bp upstream of the transcriptional start site of Park2 and is conserved in Homo sapiens, Mus musculus, Bos taurus, and Equus caballus (Bouman et al., 2011).  In addition to the AP-1 family of proteins that can bind to the site, SRY has been shown to bind AP-1 sites, as well as Nrf1 and Nrf2, which can dimerize with AP-1 proteins (Milsted et al., 2004; Novotny et al., 1998). For these reasons, the transcription factors that will be initially studied will be those that can interact with AP-1 sites, as well as NRF1, which has been shown via ChIP-Seq to interact with the Park2 gene.   99    Figure 3.19 Putative transcription factors of parkin identified by one or more program. Venn diagram shows overlap within transcription factors identified from PROMO, Patch 1.0, and TFSearch.     100   Figure 3.20 Predicted transcription binding sites on the mouse and human Park2 promoters. Coding strand sequences of the human Park2 (green) and   101  Figure 3.20 cont'd mouse Park2 (black) promoters. Sequences were aligned, bases identical in each are indicated by lines, gaps in the sequence when aligned are indicated by dashes. Putative binding sites are color-coded by species and shown overlaying their respective sequences. Rows are 50 bases long.                       102  Chapter 4: Assessment of Putative Transcription Factors of Parkin  Introduction The differential expression of parkin between brain regions is in response to the metabolite of MPTP, MPP+. This being the case, the effects of MPTP provides insight into what pathways may be activated following acute oxidative stress. MPP+ is known to damage mitochondria, elicit oxidative stress via the production of reactive oxygen species (ROS), destabilize microtubules, directly inhibit the proteasome in vitro, and cause endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) (Lansdell unpublished, Cartelli et al., 2013; Sun et al., 2013; Chan et al., 1991; Reinhart et al., 1987; Liu et al., 1992; Hung and Lee, 1997). Putative parkin transcription factors identified by both the literature and predictive software based on the sequence of Park2 promoter, were selected to be examined based on if they were known to respond to a known effect of MPP+.    ATF4 and Nrf1 both are involved in pathways responding to ER stress and the UPR, with Nrf1 also related to proteasome dysfunction. ATF4, also known as CREB2, is a transcription factor that falls loosely into the CREB (cAMP Response Element Binding protein) family. As well as being in the CREB family, ATF4 also can dimerize with proteins in the AP-1 complex (Hai and   103  Curran, 1991). ATF4 has been shown to up-regulate parkin expression in cell culture including SH-SY5Y cells in response to the mitochondrial protein gradient uncoupler CCCP or ER stress inducer tunicamycin, and PC12 cells in response to ER stress inducer thapsigargin or MPP+ (Sun et al., 2013; Bouman et al., 2011).  ER stress activates PERK (PKR-leads to the phosphorylation of eImethionine, this complex then interacts with other initiation factors to bind to the 40S (small) ribosomal subunit, this active complex then scans the mbind the tRNA-methionine, which means that it will not complex with the 40S -regulation of ATF4 via translational control involving a pair of uORFs (Upstream Open Reading Frames) (Vattem and Wek, 2004).  complex scans the mRNA for the start codon in the first uORF, once found, the 60S (large) subunit binds and translation of uORF1 begins and ends. The -tRNA-methionine interacts with the 40S subunit. This re-initiates translation at the second pho-  104  methionine. After translation of the first uORF, the 40S subunit continues to scan the mRNA, but re--tRNA-methionine. This delay leads to the 40S subunit missing the start codon in the second uORF, and instead finding the start codon in the ATF4 ORF (Vattem and Wek, 2004).  After it is translated, ATF4 leads to increased transcription of multiple genes, one of which is GADD34 (Growth Arrest and DNA Damage inducible protein 34) (Pons et al., 2007). GADD34 complexes with PP1 (Protein resulting in a feedback loop that shuts down the production of ATF4. After it has served its purpose, ATF4 can then be degraded (30-60 min half-life) (Pons et al., 2007).  Nrf1 and ER Stress Like ATF4, Nrf1 is a basic leucine zipper transcription factor that must dimerize with another basic leucine zipper protein to function. Nrf1 is required for the production of proteasome subunits after proteasome inhibition (Radhakrishnan et al., 2010). Nrf1 starts as a 120 kDa protein localized to the ER lumen and under conditions of ER stress is retrotranlocated to the cytosol and bound by its N-terminus to the ER membrane. Under normal conditions, retrotranslocated Nrf1 is rapidly degraded by the proteasome. When the proteasome is inhibited, Nrf1 is   105  stabilized and can be cleaved by proteases (producing a 95 kDa protein) and translocated into the nucleus to up-regulate proteasome subunit genes (Radhakrishnan et al., 2014).   Nrf2 and Oxidative Stress  Nrf2 is related to Nrf1 and is involved in the response to oxidative stress. Nrf2 is involved in the regulation of glutathione S-transferase and NAD(P)H:quinone oxidoreductase, along with other antioxidant response proteins (Venugopal and Jaiswal, 1996; Hayes et al., 2000). Mice deficient in Nrf2 are more sensitive to MPTP (Chen et al,. 2009) and activation of Nrf2 in cell culture can protect against MPP+ induced damage (Wruck et al., 2007). In the absence of oxidative stress, Nrf2 is bound to Keap1, which is an adapter for Cul3 E3 ligase complexes (Itoh et al., 1999; Kobayashi et al., 2004). When bound to Keap1, Nrf2 is rapidly ubiquitinated and subsequently degraded by the proteasome. In the presence of oxidative stress, key cysteines are oxidized, changing the conformation of Keap1 and thereby decreasing its affinity for Nrf2 (Taguchi et al., 2011). Loss of Keap1 binding allows Nrf2 to accumulate (Taguchi et al., 2011). Nrf2 is then phosphorylated by casein kinase 2 and this phosphorylated form can enter the nucleus and act as a transcription factor (Apopa et al., 2008). Both Nrf1 and Nrf2 recognize the Antioxidant Response Element (ARE) found in DNA sequences.    106   AP-1 Transcription Factors Another set of transcription factors with basic leucine zipper motifs are the AP-1 family of transcription factor. AP-1 transcription factors recognize 12-O-tetradecanoylphorbol-13-acetate response elements (TRE) and TRE-like sites, which in some cases are contained within an antioxidant response element (ARE) (Xie et al., 1995). AP-1 transcription factors can form homo or heterodimeric complexes that contain a combination of proteins from either the Jun, Fos, ATF, or Jun dimerization protein families (Hai and Curran, 1991). Nrf1 and Nrf2 have also been show to be able to dimerize with AP-1 transcription factors (Novotny et al., 1998).  AP-1 transcription factors have previously been shown to respond to MPTP. Expression of c-Fos and c-Jun were reported to increase in the SN, multiple or high doses of MPTP (Perez-Otano et al., 1998; Duchemin et al., 1992; Nishi, 1997; Chen et al., 2001). JunD is the binding partner that predominantly dimerizes with FosB. The dominant negative mutation of JunD, DNA (Been et al., 2013; Berton et al., 2009; Struhl, 1988).      107  NRF1, Parkin and Mitochondrial Biogenesis Not directly related to Nrf1 or Nrf2, but abbreviated the same is NRF1. NRF1 is a factor in mitochondrial biogenesis, more specifically is a transcriptional activator of cytochrome C, also known as mitochondrial Complex IV. Peroxisome proliferator-activated receptor-(PGC-NRF1 protein to stimulate mitochondrial biogenesis (Russell et al., 2004; Wu et al., 1999). NRF1 also has direct links to parkin, where NRF1 has been shown via ChIP-Seq results to bind to the Park2 promoter (Satoh et al., 2013). Parkin also regulates the expression of NRF1 through the degradation of the transcriptional repressor of PGC--interacting substrate (PARIS) (Shin et al., 2011).  SRY and the TH Promoter Sex determining Region on chromosome Y (SRY) is a transcription factor encoded by the SRY gene on the Y chromosome. SRY is the gene responsible for testis development. SRY interestingly has been found in the NSDA neurons of adult male rodents and to interact with an AP-1 site on the TH promoter (Dewing et al., 2006; Milsted et al., 2004). The loss of SRY also results in fewer TH expressing neurons in the SN of male rodents (Dewing et al., 2006). In addition to the SN, SRY has been found in the VTA, cortex, locus coeruleus and hypothalamus (Lee and Harley, 2012). This unusual   108  expression pattern and link to DA neurons makes SRY an interesting transcription factor to study.   Transcription Factor Regulation of the Parkin Promoter   All of these transcription factors have the potential to respond to the effects of MPP+. In the examination of these transcription factors, certain criteria are expected of a transcription factor of parkin. A transcription factor of parkin should: 1) be up-regulated after MPTP in the ARC and not the SN (if a transcriptional activator), or be down-regulated in the ARC and not the SN (if a transcriptional repressor); 2) expression should change prior to an increase in parkin; 3) highly expressed for approximately as long as parkin is highly expressed (though this depends on if it is a early, intermediate or late responder); 4) present (and increase) in the nuclei of DA neurons and interact with the parkin promoter; 5) when expression is experimentally manipulated, there should be a corresponding effect on parkin expression.   Acute MPTP-induced Parkin Expression in the ARC To initially examine these transcription factors, a 6 h time-point after MPTP administration was selected. Initial time-courses of parkin after MPTP shows parkin protein in the ARC increases by 12 h post-MPTP and is maintained at elevated levels at least until the 24 h time-point, and parkin mRNA is elevated at 8 h post-MPTP (Benskey et al., 2012). RNA polymerase   109  II elongation rates have been estimated to average between 1.3 and 4.3 kb per min., though have been shown to be as high as 50 kb per min. (Maiuri et al., 2011; Darzacq et al., 2007; Femino et al., 1998). Translation rates have been estimated at approximately 6 amino acids per sec (Ingolia et al., 2011; Bostrom et al., 1986). Since Park2 is approximately 1.2 Mb and 464 amino acids, transcription is expected to range from 4.6 h (rate of 4.3 kb per min.) to 0.4 h (rate of 50 kb per min.), and about 1.3 min. for translation. This suggests that increased transcription of parkin probably starts somewhere between 3 and 7 h post-MPTP, this places 6 h post-MPTP in this window. The 6 h time-point also splits two previously examined time-points; 4 h were MPP+ levels are high and 8 h where mRNA is elevated (Benskey et al., 2012). For all of the transcription factors examined, an increase in the ARC exclusively (or decrease in the SN exclusively) are sufficient for the transcription factor to be further studied.       ATF4 Results Expression of ATF4 protein was unchanged at 6 h post-MPTP in both the ARC and SN. In the ARC, fold change for ATF4 protein in the saline control mice was 1.00 ± 0.09, and 0.85 ± 0.07 for 6 h post-MPTP treated mice (Figure 4.1). In the SN, fold change for ATF4 protein in the saline control mice was 1.00 ± 0.08, and 0.96 ± 0.10 for 6 h post-MPTP treated mice   110  (Figure 4.2in both the ARC and SN. In the ARC, fold change foprotein in the saline control mice was 1.00 ± 0.13, and 0.71 ± 0.23 for 6 h post-MPTP treated mice (Figure 4.3). In the SN, fold change for and 1.27 ± 0.21 for 6 h post-MPTP treated mice (Figure 4.4).  Due to the lack of effect observed in mice, the dopaminergic neuronal MN9D cell line was used to further examine the effects of MPP+ and ER stress. To induce ER stress tunicamycin was used to inhibit N-linked glycosylation and cause a UPR. In the vehicle control cells fold change for ATF4 was 1.00 ± 0.37 and 3.50 ± 0.33 at 12 h after the addition of MPP+ (Figure 4.5). Parkin fold change in these cells was 1.00 ± 0.22 in the vehicle control cells and 1.14 ± 0.26 at 12 h after the addition of MPP+ (Figure 4.6). The same vehicle controls were used for the tunicamycin treated cells. ATF4 in the tunicamycin treated cells was 9.73 ± 0.48 relative to the control, and parkin in the tunicamycin treated cells was 0.68 ± 0.15 (Figures 4.7 and 4.8).    111   Figure 4.1 Comparison of the effects of MPTP on ATF4 in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify ATF4 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.   112   Figure 4.2 Comparison of the effects of MPTP on ATF4 in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify ATF4 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    113   Figure 4.3 ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.   114   Figure 4.SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    115   Figure 4.5 Comparison of the effects of MPP+ on ATF4 in MN9D dopaminergic neuronal cell culture. MPP+ was added to DMEM media to a concentration of (n=3, with 3 technical repliates). Western blots were used to quantify ATF4 normalized to GAPDH. Columns indicate the group means, error bars -test) is indicated with an asterisk.    116   Figure 4.6 Comparison of the effects of MPP+ on parkin in MN9D dopaminergic neuronal cell culture. MPP+ was added to DMEM media to a concefor Western blots (n=3, with 3 technical repliates). Western blots were used to quantify parkin normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    117   Figure 4.7 Comparison of the effects of tunicamycin on ATF4 in MN9D dopaminergic neuronal cell culture. tunicamycin was added to DMEM media processed for Western blots (n=3, with 3 technical repliates). Western blots were used to quantify ATF4 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean ,and -test) is indicated with an asterisk.    118   Figure 4.8 Comparison of the effects of tunicamycin on parkin in MN9D dopaminergic neuronal cell culture. tunicamycin was added to DMEM media processed for Western blots (n=3, with 3 technical repliates). Western blots were used to quantify parkin normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.        119  Discussion It was expected that if ATF4 acts as a transcriptional activator of parkin following acute neurotoxicant exposure, then it would increase in the ARC and not the SN in response to MPTP administration. The lack of expression change of ATF4 is unexpected and contradictory to what was previously reported in the literature. There was also no change in a key regulator of -5Y and PC12 neuronal cell lines to act as transcriptional activators of parkin (Sun et al., 2013; Bouman et al., 2012). In addition, MPP+ was shown in PC12 cells to increase ATF4 expression, as well as induce ER stress (Sun et al., 2013). The lack of ATF4 expression could be explained by differences in the model used in these studies. PC12 cells are derived from a pheochromocytoma from a rat adrenal medulla and used in a pure neuronal culture, whereas the brain regions examined in the present study are a mixed population of cells that contain DA neurons, more complex than the cell culture model.  Another caveat is the amount of neurotoxicant exposure. In the ME and ST, MPP+ at its highest reported levels (4 h post-MPTP) are around 400 comparison, Sun et al. used 1 mM of MPP+. The formula weight of MPP+ is 170.23 g/mol, which equates to cellular exposure of 170.23 ng MPP+ per ml of solution. In comparison, the MN9D dopaminergic neuronal cell line shows   120  circulation in the cell culture model, where over time MPP+ would be flushed from the tissue in vivo. To further explore the use of a cell culture model, the MN9D cell line was used. MN9D cells when differentiated synthesize, release and reuptake DA (Balasooriya and Wimalasena, 2007; Choi et al., 1992). Unlike the ARC and the SN, ATF4 expression in MN9D cells did change in response to MPP+, an approximately 3 fold increase, however there was no change in parkin. To focus directly on ER stress and ATF4, tunicamycin was used to cause an UPR. Tunicamycin caused an almost 9 fold increase in ATF4, a more drastic response than MPP+ caused. Even with high levels of ATF4, parkin expression remains unchanged. Taken together this suggests that MPTP is not causing sufficient ER stress in the brain regions of interest, and that at least in MN9D cells, ER stress and ATF4 are not driving parkin expression.    Nrf1 and Nrf2 Results Expression of Nrf1 protein was unchanged after MPTP in both the ARC and SN. In the ARC, fold change of the ER bound 120 kDa Nrf1 protein in the saline control mice was 1.00 ± 0.09, at 6 h post-MPTP it was 0.77 ± 0.12 and 0.85 ± 0.13 in 24 h post-MPTP (Figure 4.9). Fold change in the SN of the active 95 kDa Nrf1 protein in the saline control mice was 1.00 ± 0.13, at 6 h post-MPTP it was 0.87 ± 0.12 and 0.77 ± 0.14 in 24 h post-MPTP   121  (Figure 4.10) In the SN, fold change of the ER bound 120 kDa Nrf1 protein in the saline control mice was 1.00 ± 0.19, at 6 h post-MPTP it was 0.82 ± 0.23 and 0.68 ± 0.26 in 24 h post-MPTP (Figure 4.9). Fold change in the SN of the active 95 kDa Nrf1 protein in the saline control mice was 1.00 ± 0.17, at 6 h post-MPTP it was 1.08 ± 0.13 and 0.70 ± 0.22 in 24 h post-MPTP (Figure 4.10). Expression of Nrf2 protein was unchanged at 6 h post-MPTP in both the ARC and SN. In the ARC, fold change Nrf2 protein in the saline control mice was 1.00 ± 0.06, and 1.16 ± 0.13 in 6 h post-MPTP treated mice (Figure 4.11). In the SN, fold change Nrf2 protein in the saline control mice was 1.00 ± 0.22, and 1.35 ± 0.24 in 6 h post-MPTP treated mice (Figure 4.12). Due to questions regarding the reliability of the Nrf2 antibody, Nrf2 knock-out (KO) mice were explored as a control and parkin protein was measured in the ARC and SN. Fold difference for parkin protein in the ARC of wild-type (WT) mice was 1.00 ± 0.45, and parkin in the Nrf2 KO mice was 8.98 ± 0.08 (Figure 4.13). Fold difference parkin protein in the SN of wild-type (WT) mice was 1.00 ± 0.16, and parkin in the Nrf2 KO mice was 3.13 ± 0.02 (Figure 4.14).       122   Figure 4.9 Comparison of the effects of MPTP on Nrf1 (120 and 95 kDa) in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify Nrf1 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.   123    Figure 4.10 Comparison of the effects of MPTP on Nrf1 (120 and 95 kDa) in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify Nrf1 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.   124    Figure 4.11 Comparison of the effects of MPTP on Nrf2 in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify Nrf2 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    125    Figure 4.12 Comparison of the effects of MPTP on Nrf2 in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify Nrf2 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    126    Figure 4.13 Parkin expression in the ARC of WT and Nrf2KO mice. Male C57Bl/6J and Nrf2KO mice were injected with saline (10 ml/kg; s.c.) and killed by decapitation 24 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify parkin normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.    127   Figure 4.14 Parkin expression in the SN of WT and Nrf2KO mice. Male C57Bl/6J and Nrf2KO mice were injected with saline (10 ml/kg; s.c.) and killed by decapitation 24 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify parkin normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.       128  Discussion Expression of Nrf1 was measured at 6 h and 24 h post-MPTP with no observable change in the ARC or SN. These results suggest that Nrf1 is not a transcription factor mediating neurotoxicant-induced activation of parkin promoter activity in the ARC. The role of Nrf1 in response to ER stress and proteasome dysfunction, and the lack of change in Nrf1 at either time-point suggest the extent of these stressors is not sufficient to increase Nrf1 or cause a shift to the 95 kDa active form (Digaleh et al., 2013; Steffin et al., 2010; Radhakrishnan et al., 2014). Based on the lack of change in ATF4,  appear to cause significant ER stress. MPTP does cause differential effects on proteasome activity, with decreased activity in the ST and not in the ME (Lansdell, et al., in preparation). Similarly, this loss of proteasome function in the ST following acute neurotoxicant exposure is most likely not severe enough to elicit a Nrf1 response, nor does it alter ST parkin expression (Benskey et al., 2013). The focus of Nrf2 as a transcription factor driving neurotoxicant-induced parkin expression is based on the assumption of increased oxidative stress caused by MPTP. At 6 h post-MPTP the only region where ROS accumulates is the ME, though there is no change in Nrf2 in the ARC. Since Nrf2 reacts to oxidative stress, the lack of change in Nrf2 is unexpected. Similarly there was no change in the SN, but this is congruent with a lack of change in ROS   129  in the ST and SN 6 h post-MPTP. The interpretation of these results comes with a few caveats. Changes in Nrf2 expression could be short-lived, as Nrf2 normally has a half-life of less than 20 min (Itoh et al., 2004). In order to measure an accumulation of Nrf2 a proteasome inhibitor would be needed, however this causes its own problems, as a proteasome inhibitor would alter other transcription factors, such as Nrf1. There is also the issue that available Nrf2 antibodies are non-specific, with additional bands present on Western blots. In the case of the antibody used in the present studies, there was one clear band present that was slightly smaller than the 75 kDa weight ladder marker. The predicted size of Nrf2 being 68 kDa, this appeared to be the correct band.  Additional follow-up experiments using available Nrf2KO mice compared to WT C57Blj6 mice was performed to further examine if there is a link between Nrf2 and parkin. In these animals parkin expression was determined to be significantly higher in the Nrf2KO mice in both the ARC and SN. This contradicts earlier predictions that Nrf2 is a transcriptional activator of parkin, but would support the role of Nrf2 as a transcriptional repressor. This is not unprecedented since Nrf2 has been observed to act as a transcriptional repressor of the RON tyrosine kinase receptor in MDA MB 231 cells, derived from mammary tissue (Thangasamy et al., 2011). Though this could be the case, alternatively higher parkin expression in Nrf2KO mice   130  could be a compensatory mechanism in animals with a life-long deficit of functional Nrf2.    NRF1 Results Expression of NRF1 protein was unchanged at 6 h post-MPTP in both the ARC and SN. In the ARC, fold change for NRF1 protein in the saline control mice was 1.00 ± 0.24, and 0.84 ± 0.22 for 6 h post-MPTP treated mice (Figure 4.15). In the SN, fold change for NRF1 protein in the saline control mice was 1.00 ± 0.19, and 1.06 ± 0.17 for 6 h post-MPTP treated mice (Figure 4.16).  Since NRF1 is related to mitochondrial biogenesis and MPP+ inhibits mitochondrial Complex I activity and produces oxidative stress, ROS was measured as an index of oxidative stress after neurotoxicant exposure using a 2', 7'-dichlorofluorescin diacetate (DCF-DA) assay. In measuring the production of ROS, the only change at 6 h post-MPTP occurred in the ME. At At 6 h post-MPTP, the ME had 6700.40 ± 1249.46 aFigure 4.171114.14 ± 127.69 at 6 h post-MPTP (Figure 4.18). In the ST, basal levels -MPTP   131  (Figure 4.19). In tand 572.65 ± 33.84 at 6 h post-MPTP (Figure 4.20).  In addition to comparisons made between vehicle and MPTP treated mice, comparisons were also made between brain regions at basal levels in control mice. Comparing the axon terminal region (ME) to the cell body region (ARC) of the TIDA neurons, the ARC have lower levels of ROS (Figure 4.21). Comparing the axon terminal region (ST) to the cell body region (SN) of the NSDA neurons, there was no difference in ROS levels (Figure 4.22). In comparing the two sets of axon terminal regions, the ME had higher levels of ROS than the ST (Figure 4.23). Comparing the two sets of cell body regions, the ARC had higher levels of ROS than the SN (Figure 4.24).                                                          132    Figure 4.15 Comparison of the effects of MPTP on NRF1 in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify NRF1 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.      133    Figure 4.16 Comparison of the effects of MPTP on NRF1 in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify NRF1 normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.   134    Figure 4.17 Comparison of the effects of MPTP on ROS formation and accumulation in the ME of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ME were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 standard error -test) is indicated with an asterisk.    135    Figure 4.18 Comparison of the effects of MPTP on ROS formation and accumulation in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ARC were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 standard error of the mean.     136    Figure 4.19 Comparison of the effects of MPTP on ROS formation and accumulation in the ST of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ST were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 standard error of the mean.    137    Figure 4.20 Comparison of the effects of MPTP on ROS formation and accumulation in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). SN were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 standard error of the mean.    138    Figure 4.21 Comparison of ROS presence in brain regions containing TIDA neurons. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ME and ARC were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 standard error of the mean, and -test) is indicated with an asterisk.    139    Figure 4.22 Comparison of ROS presence in brain regions containing NSDA neurons. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ST and SN were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 standard error of the mean.    140    Figure 4.23 Comparison of ROS presence in brain regions containing axon terminals of the TIDA or NSDA neurons. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ME and ST were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error bars represent +1 t-test) is indicated with an asterisk.    141   Figure 4.24 Comparison of ROS presence in brain regions containing cell bodies of the TIDA or NSDA neurons. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). ARC and SN were microdissected, protein isolated and a DCF-DA assay was used to quantify fluorescence generated by ROS. Fluorescence was measured in arbitrary units and normalized to protein. Columns indicate the group means, error paired t-test) is indicated with an asterisk.          142  Discussion The lack of any observed change of NRF1 in either the ARC or SN does not rule out the possibility of NRF1 being a transcription factor of parkin. Rather, it suggests that NRF1 is probably not involved in initiating the increased expression of parkin in the ARC in response to acute neurotoxicant exposure. NRF1 was measured at 6 h post-MPTP, though this is enough time for MPP+ to accumulate in the axon terminals and for DA stores to be depleted (Behrouz et al., 2007; Benskey et al., 2012), it may not be enough time for mitochondrial damage and induction of mitochondrial biogenesis to occur. Since mitochondria damaged by MPP+ should lead to increased ROS accumulation and oxidative stress, ROS levels were examined in the present study. The only region where ROS levels increase at 6 h post-MPTP is the ME. This is possibly because the axon terminals of the TIDA neurons are located outside the blood-brain barrier and as such   were exposed to higher levels of MPP+ than the NSDA neurons (Benskey et al., 2012). Interestingly, levels of ROS are higher in both the cell body and axon terminal regions of the TIDA neurons as compared to the NSDA neurons. This suggest that the TIDA neurons are normally predisposed to compensate for higher levels of oxidative stress and expression of parkin may play a role in this process. It is possible that NRF1 expression changes at later time-points after MPTP, possibly due to mitochondrial biogenesis or as a downstream effect of parkin. Rather than NRF1 initiating parkin expression, it is possible that the   143  changes in parkin expression could alter NRF1 via PARIS-PGC-2011). In the ARC increased parkin could lead to decreased PARIS, increased PGC-expected to occur in the SN, where decreased parkin could lead to increased PARIS, which would negatively regulate PGC-maintain NRF1 expression. If this is the case, it is possible that parkin and NRF1 are acting in a feed-forward loop, where parkin is initiating NRF1 expression, and NRF1 acts to increase parkin, until either parkin or NRF1 expression returns to basal levels.   SRY Results Expression of SRY protein was unchanged at 6 h post-MPTP in both the ARC and SN. In the ARC, fold change for SRY protein in the saline control mice was 1.00 ± 0.21, and 3.18 ± 0.30 for 6 h post-MPTP treated mice (Figure 4.25). In the SN, fold change for SRY protein in the saline control mice was 1.00 ± 0.07, and 0.72 ± 0.21 for 6 h post-MPTP treated mice (Figure 4.26).         144   Figure 4.25 Comparison of the effects of MPTP on SRY in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and -actin. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.    145    Figure 4.26 Comparison of the effects of MPTP on SRY in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and -actin. Columns indicate the group means, error bars represent +1 standard error of the mean.         146  Discussion SRY had potential to be a transcriptional regulator of parkin, as it has been shown to be in DA neurons and interact with the same DNA sites as AP-1 transcription factors (Dewing et al., 2006; Milsted et al., 2004). Expression of SRY appears to mirror parkin expression, i.e., SRY increases in the ARC and not the SN 6 h post-MPTP exposure. The specificity of the antibody however does pose an issue. Initial trials of the antibody for Western blots used tissue from both male and female specific mouse tissue (testis, ovaries and brains from each examined), with a faint band at the predicted size of 24 kDa only present in the tissue from male mice. The issue is the antibodies commercially available were raised in mice and the secondary antibody recognizes mouse antibodies. The band at 24 kDa assumed to initially be SRY also was present in controls where the primary antibodies were not added to the blots. This also is the approximate weight of light chain IgG in mice, which is most likely what was measured in these experiments, instead of SRY.  Although this suggests that SRY appears to be absent or not at a measureable level in the brain regions examined, it does suggest that there is an immune response that occurs in the ARC at 6 h post-MPTP that does not occur in the SN. If the band in light chain IgG, the increase in the ARC may be due to its position outside of the blood-brain barrier, where the TIDA axon terminals are exposed to circulating blood in the hypophyseal portal   147  system. Regardless, SRY does not appear to be a strongly expressed protein and there are better candidates available, additionally significantly more optimization of SRY would be required for further studies involving this protein.   AP-1 Results The AP-1 transcription factors examined were c-Fos, c-JunD, -Fos protein in the saline control mice was 1.00 ± 0.06, and 1.14 ± 0.12 for 6 h post-MPTP treated mice (Figure 4.27). In the SN, fold change for c-Fos protein in the saline control mice was 1.00 ± 0.05, and 0.95 ± 0.18 for 6 h post-MPTP treated mice (Figure 4.28). Fold change for c-Jun protein in the the ARC of saline control mice was 1.00 ± 0.16, and 0.96 ± 0.20 for 6 h post-MPTP treated mice (Figure 4.29). In the SN, fold change c-Jun protein in the saline control mice was 1.00 ± 0.18, and 1.07 ± 0.22 in 6 h post-MPTP treated mice (Figure 4.30).  Fold change for FosB protein in the saline control mice was 1.00 ± 0.11, and 4.67 ± 0.14 for 6 h post-MPTP treated mice (Figure 4.31). In the SN, fold change for FosB protein in the saline control mice was 1.00 ± 0.06, and 1.13 ± 0.07 for 6 h post-MPTP treated mice (Figure 4.32  148  protein in the ARC of saline control mice was 1.00 ± 0.07, and 2.60 ± 0.12 for 6 h post-MPTP treated mice (Figure 4.33). In the SN, fold change for for 6 h post-MPTP treated mice (Figure 4.34).  In the ARC, fold change for JunD protein in the saline control mice was 1.00 ± 0.15, and 0.88 ± 0.12 for 6 h post-MPTP treated mice (Figure 4.35). In the SN, fold change for JunD protein in the saline control mice was 1.00 ± 0.22, and 0.86 ± 0.21 for 6 h post-MPTP treated mice (Figure 4.36). Fold and 0.63 ± 0.11 for 6 h post-MPTP treated mice (Figure 4.37). In the SN, he saline control mice was 1.00 ± 0.22, and 0.77 ± 0.20 for 6 h post-MPTP treated mice (Figure 4.38).  dominant negative protein and will inhibit the function of any basic leucine zipin the ARC was 0.23 ± 0.04 in the saline control mice and 0.19 ± 0.02 at 6 h post-mice and 0.42 ± 0.02 at 6 h post-MPTP (Figure 4.39 and 4.40). Though there was no change in the ratio after MPTP administration within each ARC (Figure 4.39).    149    Figure 4.27 Comparison of the effects of MPTP on c-Fos in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify c-Fos normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    150    Figure 4.28 Comparison of the effects of MPTP on c-Fos in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify c-Fos normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    151    Figure 4.29 Comparison of the effects of MPTP on c-Jun in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify c-Jun normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    152    Figure 4.30 Comparison of the effects of MPTP on c-Jun in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify c-Jun normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    153    Figure 4.31 Comparison of the effects of MPTP on FosB in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify FosB normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.   154    Figure 4.32 Comparison of the effects of MPTP on FosB in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify FosB normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.   155     Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and  GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.   156    Figure 4.34 Comparison of the effects of MPTP o Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western indicate the group means, error bars represent +1 standard error of the mean.   157    Figure 4.35 Comparison of the effects of MPTP on JunD in the ARC of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and Western blots used to quantify JunD normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    158    Figure 4.36 Comparison of the effects of MPTP on JunD in the SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantify JunD normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean.    159    Figure 4.37  Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, ARC were microdissected, protein isolated and indicate the group means, error bars represent +1 standard error of the mean.     160    Figure 4.38  mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h after injection (n=8/group). Brains were sectioned, SN were microdissected, protein isolated and Western blots used to quantiindicate the group means, error bars represent +1 standard error of the mean.       161     Figure 4.39 the ARC and SN of mice. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and sacrificed 6 h after injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify JunD anColumns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.      162  Discussion Within the group of AP-1 proteins examined in the present study, only -activation of parkin expression and sparing of TIDA neurons located in this brain region (Behrouz et al, 2007; Benskey et al., 2012; 2013; 2015). following MPTP, which corresponds with a lack of effect of this neurotoxicant on parkin expression and the higher susceptibility of NSDA neurons located in this region to degeneration following neurotoxicant exposure and degeneration in PD (Behrouz et al, 2007; Matzuk et al., 1985; Heikkila et al., 1984; Benskey et al., 2012, 2013, 2015)      Previous studies reported thaare increased in the ST following MPTP exposure (Perez-Otano et al., 1998). MPTP exposure paradigms used in these studies required more aggressive toxicant exposure (e.g., multiple repeated doses of MPTP) to observe any -Otano et al., 1998; Potashkin et al., 2007). Most likely the changes in FosB occur in medium spiny neurons of the striatum, perhaps in response to axonopathy of NSDA neurons and loss of DA receptor-mediated regulation of these neurons (Gerfen et al., 2011; Albin et al., 1989).   163  The expression of c-Fos and c-Jun were previously reported to increase the SN following multiple or high doses of MPTP (Duchemin et al., 1992; Nishi, 1997; Chen et al., 2001). These high dose MPTP paradigms are more severe than the acute single injection paradigm used in these experiments, which could explain why no increases in c-Fos or c-Jun were observed in the -Fos or c-Jun does not change in the ARC at any of the times examined in response to MPTP. It is possible however that expression of these immediate early transcription factors increases prior to the 6 h time-point. Additionally, phosphorylation of c-Fos, c-Jun, or both may occur to modulate activity independent of steady state levels of these transcription factors to promote parkin transcription. -MPTP in the ARC or the SN, they are still relevant proteins that are potential produce a functional transcription factor. Lack of change in JunD suggests bind to the FosBs, however this blocks the ability of the dimer to function as a transcription factor (Berton et al., 2009; Struhl, 1988). Because of this the holds true for in vehicle and MPTP (6h post-injection) samples. These results suggest that the higher   164  FosBs have in cells in the SN.   Overall Conclusions From the eleven transcription factors examined at 6 h-post MPTP, FosB neurotoxicant-differentially expressed between the ARC and SN, mirroring parkin expression (only increasing in the ARC and not the SN). Expression of both increase prior to parkin, as would be expected of a transcription factor that the potential to last as long as parkin expression. The half-in vitro is greater than eight days and MPTP-elevated for at least seven days (Chen et al., 1997; Perez-Otano et al., 1998). The long-s well as its nature to accumulate could potentially explain long-term parkin expression in the ARC after chronic MPTP exposure (Nestler, 2001; Kelz et al., 1999; Benskey et al., activators of parkin, further analysis of their temporal expression patterns and cellular localization is required.          165  ARC and SN   Introduction In the analyses of putative parkin promoter transcription factors 6 h post-with parkin expression, i.e., increasing in the ARC but not in the SN. FosB ultiple doses of MPTP, but not in the SN (Pérez-Otaño et al., 1998; Potashkin et al., 2007). The caveat to this is that these changes are most likely not occurring in the DA neurons, since the cell bodies of these neurons are located in the SN. More likely the changes occur in the post-synaptic medium spiny neurons in response to the disruptive effects of MPTP on the axon terminals of the NSDA neurons in the ST.  FosB gene. While a full-length transcript produces FosB, alternative splicing lacking 101 amino acids on the C-terminal end results in increased stability and accumulation following multiple stimuli can also be increased through phosphorylation by casein kinase 2 (Ulery et al., 2006; Ulery and Nestler, 2007). The stability and known target genes of   166  ve a role in drug addiction and desensitization (Nestler, 2008).  -1 transcription factors at 6 h post MPTP, a more complete temporal profile of these transcription factors is required. Additionally, as potential transcription factors of parkin, an increase in the nuclei of TIDA neurons in the ARC is predicted, as a transcription factor must be present in the nucleus to interact with DNA.            Temporal Expression of AP-1 Transcription Factors Results As with the 6 h post-differentially expressed between brain regions. In the ARC, FosB expression increased approximately 3-fold by 4 h post-MPTP and declined to two-fold by 12 and 24 h post-MPTP (Figures 5.1 and 5.3increased by approximately 3-fold by 4 h post-MPTP and this increase was maintained up to 24 h post-MPTP (Figures 5.2 and 5.3). Although both FosB  in expression of either protein at any time following MPTP treatment (Figures 5.1, 5.2 and 5.4). As for the other potential components of the AP-1 transcription factors that were not observed to change at 6 h post-MPTP, there was also no   167  change at any of the times examined. There was no change in c-Fos measured in the ARC or in the SN at 4, 6, 8, 12, or 24 h post-MPTP (Figures 5.5-5.7). There was no change in c-Jun measured in the ARC or in the SN at 4, 6, 8, 12, or 24 h post-MPTP (Figures 5.8-5.10). JunD -1 displayed no change in protein at any of the times measured (Figures 5.11-5.14though as with the previous results at 6 h post-higher in the SN (Figure 5.15). These results demonstrate that FosB and -1 transcription factors that show differential response to neurotoxicant exposure between brain regions.              168    Figure 5.1 Time course effects of MPTP on FosB expression in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify FosB normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.    169    Figure 5.2 SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.          170    Figure 5.3 6, 8, 12, or 24 h post-MPTP.                     171    Figure 5.4 6, 8, 12, or 24 h post-MPTP.                  172    Figure 5.5 Time course effects of MPTP on c-Fos expression in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify c-Fos normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.           173    Figure 5.6 Representative Western blots for c-Fos in the ARC at 4, 6, 8, 12, or 24 h post-MPTP. c-Fos was normalized to GAPDH.                               174    Figure 5.7 Representative Western blots for c-Fos in the SN at 4, 6, 8, 12, or 24 h post-MPTP. c-Fos was normalized to GAPDH.                  175    Figure 5.8 Time course effects of MPTP on c-Jun expression in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify c-Jun normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.             176    Figure 5.9 Representative Western blots for c-Jun in the ARC at 4, 6, 8, 12, or 24 h post-MPTP. c-Jun was normalized to GAPDH.                                   177    Figure 5.10 Representative Western blots for c-Jun in the SN at 4, 6, 8, 12, or 24 h post-MPTP. c-Jun was normalized to GAPDH.            178   Figure 5.11 Time course effects of MPTP on JunD expression in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify JunD normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.   179    Figure 5.12 and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.          180    Figure 5.13 4, 6, 8, 12, or 24 h post-MPTP.                  181   Figure 5.14 6, 8, 12, or 24 h post-MPTP.                182    Figure 5.15 the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 4, 6, 8, 12, or 24 h post-injection (n=8/group). Brains were sectioned, ARC and SN were standard error of the mean.           183  Time-courses shown in Figures 5.1 and 5.2 expression both peak as early as 4 h post-MPTP. An early time-course expression increase after MPTP administration. In the ARC, FosB expression increased approximately 3-fold by 1 h post-MPTP and this increase was maintained at 2 and 4 h post MPTP (Figures 5.16 and 5.18). An increase in -MPTP, where expression levels were approximately 2-fold and 4-fold higher than control at 2 and 4 h post-MPTP treatment (Figures 5.17 and 5.18). In the SN, there was no change in Figures 5.16, 5.17 and 5.19).     184   Figure 5.16 Early time course effects of MPTP on FosB expression in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 2, or 4 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify FosB normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.     185    Figure 5.17 ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 2, or 4 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.       186    Figure 5.18 Repr1, 2, or 4 h post-MPTP.               187   Figure 5.19 2, or 4 h post-MPTP.                   188  Discussion Similar to parkin and consistent with the 6 h post-MPTP results, FosB and induces FosB expression as early as 1 h post-h post-precedes increases in parkin mRNA at 8 h and protein at 12 h post-MPTP (Benskey et al., 2012). Activation of differential expression of FosB and tween brain regions is consistent with the expectations of transcription factors mediating neurotoxicant-induced differential parkin expression.  -1 proteins does not change at 4, 6, 8, 12, or 24 h post-MPTP. This does not rule out the possibility that expression changes occur earlier than 4 h, or onset and return to basal levels was too rapid and occurred between time-points measured. As activity of the proteins can be altered independent of expression via post-translational modifications, such as phosphorylation, it is possible that phosphorylated c-Fos or c-Jun, could be possible transcription factors of parkin. Though not initially measured, this offers a future direction to explore in addition to FosB and    189  transcriptional activators of parkin. However, an increase in JunD would be cells, expression of JunD may be sufficient to pair with the FosBs. Comparably, in Sprague-Dawley rats there has been shown to be moderate expression of JunD at basal levels in the ARC and even lower levels in the SN JunD (Jorissen et al., 2007). Regardless, the expression patterns of FosB warrants further examination of the FosB gene.          Sub- Results Transcription factors need to localize to the nucleus in order to interact fractions were examined in the ARC and SN. In the ARC, both FosB and - fold 6 h at 6 h post-MPTP (Figures 5.20 and 5.22). Expression of FosB in the nuclear 3-fold in the nuclear fraction 6 h following treatment (Figures 5.21 and 5.22). ytoplasmic or nuclear fractions of the SN following MPTP did not change (Figures 5.23-5.25).   190    Figure 5.20 derived from the ARC. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h post-injection (n=8/group). The ARC was microdissected from fresh/unfrozen tissue, cytoplasmic and nuclear fractions separated, and Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.   191    Figure 5.21 from the ARC. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h post-injection (n=8/group). The ARC was microdissected from fresh/unfrozen tissue, cytoplasmic and nuclear fractions separated, and Western blots used to the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.   192    Figure 5.22 and nuclear fractions derived from the ARC at 6 h post-MPTP. FosB and protein H3 in the nuclear fraction.    193    Figure 5.23 derived from the SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h post-injection (n=8/group). The SN was microdissected from fresh/unfrozen tissue, cytoplasmic and nuclear fractions separated, and Western blots used group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.   194    Figure 5.24 from the SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 6 h post-injection (n=8/group). The SN was microdissected from fresh/unfrozen tissue, cytoplasmic and nuclear fractions separated, and Western blots used to the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from vehicle-treated controls.   195    Figure 5.25 and nuclear fractions derived from the SN at 6 h post-MPTP. were normalized to GAPDH in the cytoplasmic fraction and histone protein H3 in the nuclear fraction.            196  Discussion  neurotoxicant exposure they must localize to the nucleus. Based on Western be present in both the cytoplasm and nuclei in the ARC and SN. As observed changes in the ARC, with no change observed quantitative change in the amount of these transcription factors in whole SN tissue. There was also no change in localization in the SN, i.e., no increases or decreases in either both the cytoplasm and nuclei, with FosB only increasing in the cytoplasm by 6 h post-MPTP.  Themay act as a transcription factor of parkin mediating neurotoxicant-induced activation of the parkin promoter.  Elevated levels in cytoplasmic FosB (in the absence of nuclear localization) suggests that this transcription factor does not regulate parkin expression for prolonged periods (i.e. 6 h) after neurotoxicant exposure.  It is likely that FosB is an early initiator of the prolonged regulator of the parkin promoter. This hypothesis is consistent with the known function of the FosB family of transcription factors in addiction (Nestler 2008).    197  at serine 27 by cNestler et al., 2007). When casein kinase 2 is inhibited, translocation of t al., 2007). By nuclear fraction at 6 h. Additionally, high concentrations of dimers containing ding in the nuclei may prevent additional translocation of FosB into the nucleus.    Localization of Total FosB in DA Neurons, Total Neurons, and Glial Cells  Results cell bodies of TIDA located in nuclei of TIDA and NSDA neurons immunofluorescent staining was  an antibody against TH, the rate limiting enzyme in DA synthesis and a marker for DA neurons in the ARC and SN. Sections through the ARC and SN of vehicle and 6 h post-MPTP mice were stained and imaged (Figure 5.26). Total numbers of TH-immunoreactive neurons with and without FosB in the nuclei were counted.   198  The percentage of TH-immunoreactive neurons with FosB stained nuclei were calculated for each brain region of individual mice and averaged across treatment groups. In the ARC of vehicle-treated mice approximately 90% of TH neurons had total FosBs localized to nuclei and this did not change 6 h post-MPTP treatment (Table 5.1). In the SN of vehicle-treated mice approximately 90% of TH neurons had total FosBs localized to the nuclei and this did not change at 6 h post-MPTP (Table 5.1).      199   Figure 5.26 -immunoreactive neurons in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and perfused 6 h immunofluoresence used to co-localize total FosB in TH positive neurons. -immunoreactive cells with and without total FosB in the nuclei were quantified. Scale ba   200  potentially be occurring in non-DA neurons. To examine this possibility, an antibody against NeuN was used. NeuN is a neuronal splicing factor and a phenotype independent neuronal marker. A majority (>95%) of all ARC and SN neurons in vehicle and 6 h post-MPTP mice exhibited nuclear staining for FosB, with no change after MPTP (Figure 5.27 and Table 5.1). -neuronal cells, such as glial cells. The glial marker, GFAP (glial fibrillary acidic protein), was used with the antibody for the FosBs to stain sections from the ARC and SN of vehicle and 6 h post-MPTP treated mice. Only a small percentage (~10%) of cells stained with GFAP co-localized with total FosB (~10%) in the ARC and SN of both vehicle and MPTP treated animals (Figure 5.28 and Table 5.1).      201   Figure 5.27 neurons in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and perfused 6 h later co-part were imaged and NeuN-immunoreactive cells with and without total FosB in the     202    Figure 5.28 and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and perfused 6 h later (n=5/group). Sections 20 -localize total FosB in -immunoreactive cells with and without total FosB in the nuclei were     203  Table 5.1 Mean percentage of TH-immunoreactive neurons, total neurons, and glial cells containing total FosB.  Cells Treatment ARC SN DA Neurons Vehicle 90.6% ± 0.4% 92.5% ± 0.4% MPTP 88.8% ± 1.3% 90.5% ± 0.8% Total Neurons Vehicle 98.7% ± 0.9% 97.7% ± 0.9% MPTP 98.5% ± 0.6% 97.9% ± 0.6% Glial Cells Vehicle 8.0% ± 2.7% 11.7% ± 4.6% MPTP 7.6% ± 1.4% 11.1% ± 3.4%                    204  Discussion   measurements in cytoplasmic and nuclear fractions in isolated brain regions only partially addresses what is happening in DA neurons. To further resolve whether these changes occur in DA neurons dual immunofluorescent staining was used to locate total FosB in TH immunoreactive neurons at 6 h post-MPTP.  An overwhelming majority (i.e. 90%) of TH immunoreactive neurons contained FosB in there nuclei. There was no change in the percentage of TH neurons with total FosB in their nuclei after MPTP in either NSDA neurons in the SN or TIDA neurons in the ARC. The lack of increase in the numbers of DA neurons with total FosB in protein occurs within individual TIDA neurons, rather than a recruitment in  Alternatively, the number of non-TH neurons or glia expressing total FosB could be increasing. Both NeuN and GFAP staining for total neurons and glia show no change in the number of cells expressing total FosB 6 h post-MPTP. In the ARC and SN, almost all of the neurons observed express FosB to some degree. Though other neurons express total FosB in the ARC, the selectivity of MPP+ for the DA transporter (Bezard et al., 1998; Javitch et al., 1985). As with neurons, both astrocytes and microglia have been shown to   205  However, in the ARC and SN, there is a low percentage of glia co-localizing with FosB. Considering the low number of total FosB expressing glia, the to changes in glial cells.   Long-Expression Results -life and is constant with the expectations of a transcription factor of parkin, and extended time-course was performed. Effects of MPTP on DA vesicular stores, DA synthesis, release, and re-uptake pathwat 1, 3, and 7 d post-MPTP were determined. DA concentrations (an index of vesicular neurotransmitter storage) in the ST were decreased to about 25% of same day vehicle controls at 1, 3, and 7 d after a single injection of MPTP (Figure 5.29). Comparably, DA concentrations in the ME after MPTP were not significantly different from the respective same day vehicle controls, showing full recovery of neurotransmitter stores in the ME (Figure 5.30). In addition to the depleting effect of MPTP on DA ,TH in the ST was decreased to approximately 50% of controls at 1, 3, and 7 d post-MPTP with no recovery (Figures 5.31 and 5.32). Concentrations of the DA uptake transporter DAT in the ST was decreased 50% of the control (or lower) at 1, 3, and 7 d post-MPTP (Figures 5.33 and 5.35). In contrast, levels of the DA vesicular   206  transporter VMAT2 in the ST did not change at 1, 3, and 7 d post-MPTP (Figures 5.34 and 5.35).    207    Figure 5.29 Extended time course effects of MPTP on DA concentration in the ST. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ST was microdissected, and samples were prepared for neurochemistry. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.   208    Figure 5.30 Extended time course effects of MPTP on DA concentration in the ME. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ME was microdissected, and samples were prepared for neurochemistry. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.      209    Figure 5.31 Extended time course effects of MPTP on TH expression in the ST. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ST was microdissected, protein isolated, and Western blots used to quantify TH normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.            210    Figure 5.32 Representative Western blots for TH in the ST at 1, 3, or 7 d post-MPTP. TH was normalized to GAPDH.                  211    Figure 5.33 Extended time course effects of MPTP on DAT expression in the ST. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ST was microdissected, protein isolated, and Western blots used to quantify DAT normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.    212    Figure 5.34 Extended time course effects of MPTP on VMAT expression in the ST. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ST was microdissected, protein isolated, and Western blots used to quantify VMAT normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.         213    Figure 5.35 Representative Western blots for DAT and VMAT in the ST at 1, 3, or 7 d post-MPTP. DAT and VMAT were normalized to GAPDH.                    214  In addition to the DA pathway, there were also changes in body weight. Animals were weighed just prior to neurotoxicant administration and then just prior to sacrifice either 1, 3 or 3 days later. MPTP treated animals at 1 and 3 d post-MPTP lost weight compared to same day vehicle controls.  By 7 d post-MPTP, MPTP and vehicle animals had gained weight, with no difference in weight gain between groups (Figure 5.36).     Consistent with Figure 5.1, FosB at 1 d post-MPTP was approximately 2-fold higher in the ARC than in same day vehicle mice (Figure 5.37 and 5.39). FosB levels at 3 and 7 d post-MPTP were not different than same day vehicle controls (Figure 5.37 and 5.39-fold higher in the ARC than the same day vehicle control at 1 d post-MPTP, but drops to about 1.5 fold higher than same day vehicle control at 7 d post-MPTP (Figure 5.38 and 5.39). Parkin, a potential downstream target of the FosBs, was found to be approximately 1.5 fold higher in the ARC than same day vehicle controls at 1, 3, and 7 d post-MPTP (Figure 5.40 and 5.41). In the SN there was no significant change in parkin protein at 1, 3, or 7 d post-MPTP (Figure 5.42 and 5.43).     215    Figure 5.36 Extended time course effects of MPTP on weight change. Male C57Bl/6J mice were weighed prior to being treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and reweighed prior to being killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Change from the starting weight was calculated for each animal. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.    216   Figure 5.37 Extended time course effects of MPTP on FosB expression in the ARC. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ARC was microdissected, protein isolated, and Western blots used to quantify FosB normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.     217    Figure 5.38 the ARC. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ARC was microdissected, GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.         218    Figure 5.39 1, 3, or 7 d post-MPTP.                 219    Figure 5.40 Extended time course effects of MPTP on parkin expression in the ARC. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, ARC was microdissected, protein isolated, and Western blots used to quantify parkin normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.       220   Figure 5.41 Representative Western blots for parkin in the ARC at 1, 3, or 7 d post-MPTP. Parkin was normalized to GAPDH.            221    Figure 5.42 Extended time course effects of MPTP on parkin expression in the SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 3, or 7 d post-injection (n=8/group). Brains were sectioned, SN was microdissected, protein isolated, and Western blots used to quantify parkin normalized to GAPDH. Columns indicate the group means, error bars represent +1 standard error of the mean. An asterisk indicates values that are significantly different (p < 0.05; t-test) from the same day vehicle-treated controls.           222    Figure 5.43 Representative Western blots for parkin in the SN at 1, 3, or 7 d post-MPTP. Parkin was normalized to GAPDH.                     223  Correlation tests were performed comparing neurotoxicant-induced calculated for FosB and parkin were 0.463 for the vehicle control, 0.517 at 1 d post-MPTP, -0.912 at 3 d post-MPTP, and 0.396 at 7 d post-MPTP (Table 5.2). Spearman correlation coefficients calculated for FosB and parkin were 0.381 for the vehicle control, 0.786 at 1 d post-MPTP, -0.874 at 3 d post-MPTP, and 0.667 at 7 d post-MPTP (Table 5.2). Pearson correlation co-0.349 for the vehicle control, 0.885 at 1 d post-MPTP, 0.899 at 3 d post-MPTP, and 0.894 at 7 d post-MPTP (Table 5.2). Spearman correlation coefficients calculated for -0.0476 for the vehicle control, 0.929 at 1 d post-MPTP, 0.922 at 3 d post-MPTP, and 0.738 at 7 d post-MPTP (Table 5.2). In addition to Pearson and Spearman correlation coefficients, linear regressions -MPTP (Figures 5.44 and 5.45). The r2 values for FosB and parkin were 0.214 for the vehicle control, 0.267 at 1 d post-MPTP, 0.832 at 3 d post-MPTP, and 0.157 at 7 d post-MPTP. The r2 control, 0.782 at 1 d post-MPTP, 0.807 at 3 d post-MPTP, and 0.799 at 7 d post-MPTP.      224  Table 5.2 Pearson and Spearman correlation statistics in the comparison of   Treatment Protein Compared  Pearson Coefficient Pearson P-value Spearman Coefficient Spearman P-value Vehicle FosB 0.463 0.248 0.381 0.321 deltaFosB -0.349 0.397 -0.0476 0.885 1 day post-MPTP FosB 0.517 0.19 0.786 0.015 deltaFosB 0.885 0.0035 0.929 0.001 3 days post-MPTP FosB -0.912 0.0016 -0.874 0.001 deltaFosB 0.899 0.0024 0.922 0.001 7 days post-MPTP FosB 0.396 0.332 0.667 0.059 deltaFosB 0.894 0.0028 0.738 0.029         225    Figure 5.44 Linear regressions plotted for FosB as compared to parkin at 1, 3, and 7 days post-MPTP. FosB and parkin were normalized to GAPDH, fold change calculated based on same day vehicle control. Vehicle control shown was from 1 day post-injection, which does not statistically differ from 3 or 7 day controls.    226    Figure 5.45 Linear regressions 3, and 7 days post-MPTP. change calculated based on same day vehicle control. Vehicle control shown was from 1 day post-injection, which does not statistically differ from 3 or 7 day controls.            227  Discussion  Acute MPTP has long-lasting effects in brain regions containing the NSDA and TIDA neurons. Similar to the differential recovery in the ST and ME at 1 d post-MPTP, there was no recovery in the ST of stored DA and complete recovery in the ME. These effects on DA stores in the ST are long lasting, with no sign of recovery up to 7 d post-MPTP. In addition to DA stores affected in the ST, deficiencies in proteins involved in DA synthesis and re-uptake persist. Both TH and DAT in the ST decrease with no signs of recovery up to 7 d post-MPTP. The losses observed in both of these proteins that are normally located in axon terminals suggest that MPTP administration may have resulted in axonopathy. A caveat to this is the steady-state expression of VMAT2 protein. As VMAT2 is found on synaptic vesicle membranes, no change in this protein suggests that the number of synaptic vesicles in the terminals does not change. Therefore either an axonopathy occurs and VMAT2 is up-regulated to compensate for axon loss in unlesioned neurons, or axonopathy does not occur and MPTP-induced decreases in TH and DAT are due to long-term down-regulation.  Decreases in DAT have been shown to precede cell death and loss of axon terminals of the NSDA neurons in response to MPTP (Muroyama et al., 2011; Kurosaki et al., 2003). Long-term decreases in DAT and TH support the hypothesis that axonopathy to some degree occurs after acute MPTP exposure. VMAT2 levels have been shown to correspond to neuron survival   228  in response to MPTP, where higher levels of VMAT2 results in resilience to neurotoxicant exposure (Lohr et al., 2016). Taken together, the results suggest axonopathy occurs and VMAT2 is a compensator mechanism that aids the remaining neurons in survival. To further examine axonopathy, a silver stain could be used to show axon density, or an additional axon marker could be used, such as synaptobrevin, synapsin, or synaptosomal associated protein of 25 kDa (SNAP25). The finding that increased parkin in the ARC is maintained up to 7 d post-transcription factors of parkin. The normal half-life of parkin is about 8 h, so transcription factors of parkin would need to be constantly present over all 7 7 d post-expectations of a transcription factor of parkin (Nestler, 2001). These findings also may explain the observation that parkin protein is elevated following chronic MPTP treatment. In one chronic MPTP experiment mice treated every 3.5 d for 35 d were allowed to recover 21 d, and even after the recovery period, parkin remained elevated in the ARC (Benskey et al., why parkin expression is maintained over 21 d. Within individual mice there is a strong correlation between FosB and parkin expression only at 1 d post-MPTP, consistent with a role for early regulation of the parkin promoter by   229  is highly correlated with elevated parkin protein at 1, 3, and 7 d post-MPTP. These results further strengthen the support for Folong-term expression of parkin.    Overall Conclusions Consistent with the results presented in the previous chapter for 6 h post-MPTP exposure, data presented in this chapter reveal that the temporal In the ARC, expression of both FosBs increases by 2 h post-MPTP (well st 7 d post-MPTP, similar to the long-term parkin expression caused by MPTP.  and a correlation between parkin.        230  Expression in Cell Culture and in the ARC and SN  Introduction The data presented in the previous chapter is consistent with the stimulatory effects of MPTP on parkin expression. Between both proteins, osB is not only located in TIDA neurons in extracts after MPTP. Long-expression of parkin and could account for long-term elevated parkin levels in the ARC in both acute and chronic models of MPTP (Benskey et al., 2013). MPTP has many potential deleterious effects in central DA neurons including generation of ROS and ER stress, but it is not known what causes the differential expression of the FosBs that results in the corresponding regional differences in the pattern of parkin expression after acute neurotoxicant exposure.  The FosB gene is regulated by both cAMP response element-binding protein (CREB) and serum response factor (SRF) (Morgan and Curran, 1995; Vialou et al., 2012). Multiple stimuli, such as growth factors, serum, neurotransmitters, and calcium, can lead to phosphorylation and therefore increased activity of CREB and SRF. CREB can be phosphorylated by protein   231  kinase A (PKA), calmodulin-dependent protein kinase II (CaMKII), calmodulin-dependent protein kinase IV (CaMKIV), protein kinase B, and p90 ribosomal S6 kinase (Sun et al., 1994; Delghandi et al., 2005; Du et al., 1998; Bohm et al., 1995). SRF can be phosphorylated by Casein kinase 2, calmodulin-dependent protein kinase IV (CaMKII), protein kinase A, and mitogen-activated protein kinase-activated protein kinase 2 (Chang et al., 2013; Miranti et al., 1995; Heidenreich et al., 1999; Blaker et al., 2009). Common pathways between phosphorylation of CREB and SRF are PKA and the CaMKs. PKA is activated by cAMP, which can accumulate due to adenylate cyclase and the G-protein coupled receptors. The CaMKs are regulated by the presence of Ca2+. Changes in Ca2+ are common in neurons, as an influx of Ca2+ is required in neurons for neurotransmitter release into the synapse. In addition, ER stress and mitochondrial dysfunction can both cause Ca2+ accumulation in cells and subsequent CaMK activation (Deniaud et al., 2008; Arnould et al., 2002). In total, many of the stresses that MPTP is known to cause are potential induces of CREB and/or SRF phosphorylation. Through studying the pathways that may be involved in the differential induction of transcription of the FosB gene in different brain regions, further upstream information on parkin regulation may be revealed. In addition to possible mechanisms that may induce expression of the FosBs and parkin in response to neurotoxicant induced stress, expression of the FosBs can be   232  manipulated to determexpression.   Expression of the FosBs can be induced through known means, such as PMA (phorbol 12-myristate 13-acetate, also called 12-O-tertradecanoylphorbol-12-acetate or TPA). PMA exposure of cells in culture induces expression of AP-1 proteins, including FosB (Limb et al., 2009; protein can also be expressed in vivo via the use of adeno-associated viruses (AAV), recapitulating the downstream effects underlying addiction, hippocampal-dependent learning and memory, and levadopa induced dyskinesia (Hedges et al., 2009; Cao et al., 2010; Zhang et al., 2014; Eagle et al., 2015).  JunD that lacks 48 amino acids on the N-terminal end (Yazgan and Pfarr, 2002; Struhl, 1988). As the N-terminal end contains the DNA binding can form, but these are not functional as transcription factors. The experiments described in this chapter utilize PMA in vitro, and AAV-induced overin vivo methods to determine if there is a causal link between FosBs and parkin expression under basal conditions and following neurotoxicant exposure.      233  Transcriptional Activators of the FosB Gene Results CREB and SRF, known transcriptional activators of FosB were measured at 1, 2 and 4 h after MPTP as a strategy to identify mechanisms underlying the MPTP-CREB (Ser133), increased over 2-fold by 1 h post-MPTP in the ARC and remained elevated at 4 h post-MPTP (Figures 6.1 and 6.5). In the SN, phosphorylated CREB decreased by 1 and 2 h post-MPTP, and returned to basal levels by 4 h post-MPTP (Figures 6.1 and 6.5). The expression of total CREB protein in the ARC or SN did not change at 1, 2, or 4 h post MPTP (Figures 6.2 and 6.6). Phosphorylated SRF (Ser103) increased to approximately 2-fold at 1 and 2 h post-MPTP in the ARC and returned to basal levels by 4 h post-MPTP (Figures 6.3 and 6.5). Phosphorylated SRF did not change in the SN at any of the time points examined (Figures 6.3 and 6.5), There was no change in total SRF in the ARC or SN (Figures 6.4 and 6.6).   234    Figure 6.1 Early time course effects of MPTP on phosphorylated CREB (Ser133) in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 2, or 4 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify phosphorylated CREB normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.    235    Figure 6.2 Early time course effects of MPTP on total CREB in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 2, or 4 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify CREB normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean.     236    Figure 6.3 Early time course effects of MPTP on phosphorylated SRF (Ser103) in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 2, or 4 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify phosphorylated SRF normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled shapes.    237    Figure 6.4 Early time course effects of MPTP on total SRF in the ARC and SN. Male C57Bl/6J mice were treated with either saline (10 ml/kg; s.c.) or MPTP (20 mg/kg; s.c.) and killed by decapitation 1, 2, or 4 h post-injection (n=8/group). Brains were sectioned, ARC and SN were microdissected, protein isolated and Western blots used to quantify SRF normalized to GAPDH. Mean fold change are represented by shapes with ± 1 standard error of the mean.        238    Figure 6.5 Representative Western blots for phosphorylated CREB (Ser133) and phosphorylated SRF (Ser103) in the ARC and SN at 1, 2, or 4 h post-MPTP. Phosphorylated CREB and SRF were normalized to GAPDH.               239   Figure 6.6 Representative Western blots for total CREB and SRF in the ARC and SN at 1, 2, or 4 h post-MPTP. CREB and SRF were normalized to GAPDH.                  240  Discussion  In order to elucidate a potential mechanism for differential expression of parkin following acute MPTP exposure known transcriptional activators of the FosB gene, SRF and CREB, were examined (Vialou et al., 2012). Both CREB and SRF have been shown to have a potential role in the response to oxidative stress in neurons (Lee et al., 2009; Rieker et al., 2012). Phosphorylated SRF and CREB both increase early (by 1 h) after MPTP in the ARC but not the SN. Total SRF and CREB expression is not effected by MPTP, suggesting that increased kinase activity, rather than transcription leads to the increases the phosphorylated forms. CREB expression are consistent with these transcription factors acting on the FosB gene. Though FosB is already elevated by 1 h post-are not elevated until 2 h and do not peak until 4 h post-is still in the process of increasing, this suggests that transcription factors are acting at the FosB promoter during this timeframe. At both 1 and 2 h post-MPTP phosphorylated SRF levels are elevated and at 1, 2 and 4 h post-MPTP phosphorylated CREB is elevated. Though none of the time points measured show the increase in phosphorylated SRF or CREB preceding FosB expression, the differential expression between the ARC and SN and the temporal expression of phosphorylated SRF and CREB are consistent with their known roles as transcriptional activators of FosB.   241  In addition to interactions with the FosB gene, phosphorylation of SRF and CREB are potentially activators of other genes that could aid in the recovery of the TIDA neurons. The loss of SRF for example, has been shown to increase the sensitivity of NSDA neurons to MPTP (Rieker er al., 2012). This suggests that the increase of phosphorylated SRF could be beneficial to    Results neuronal population, the neuroblastoma-derived MN9D cell line was used. As with NSDA neurons in the brain in response to MPTP administration in vivo, addition of its active metabolite MPP+ in vitro results in decreased stored DA in MN9D cells (Figure 6.7). MPP+ however did not decrease MN9D cell viability at least out to 24 h post-MPP+ (Figure 6.8). As anticipated, MPP+ did not cause a change -MPP+ (Figure 6.9-6.11). Parkin expression in MN9D cells at 6 and 12 h post-MPP+ also did not change (Figure 6.12 and 6.13). PMA was used to induce expression of AP-1 transcription factors FosB and at 6 and 12 h post-PMA. FosB had a fold change of 2.61 ± 0.29 at 6 h and 2.48 ± 0.05 at 12 h post-PMA (Figure 5.14 and 5.16  242  change of 3.68 ± 0.16 at 6 h and 3.11 ± 0.32 at 12 h post-PMA (Figure 5.15 and 5.16was no change in parkin expression (Figure 5.17 and 5.18).                              243   Figure 6.7 Effects of MPP+ on stored DA in MN9D cells. MN9D cells were + in DMEM media. Cells were collected at 0.5, 1, 2, 4, 8, 16, and 24 h post-MPP+ and prepared for neurochemical analysis. Time-points are plotted as percent of control and are represented by circles with ± 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with filled circles.      244    Figure 6.8 Cell viability of MN9D cells at 24 h post MPP+. MN9D cells were + in DMEM media. Cells were collected at 24 h post-MPP+ and a trypan blue assay used to assess cell viability. Mean percent of viable cells are plotted as columns with error bars representing + 1 standard error of the mean.           245    Figure 6.9 FosB expression in MN9D cells at 6 and 12 h post MPP+. MN9D + in DMEM media. Cells were collected at 6 and 12 h post-MPP+, protein isolated, and Western blots used to quantify FosB normalized to GAPDH. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean.    246    Figure 6.10 cells at 6 and 12 h post MPP+. MN9D + in DMEM media. Cells were collected at 6 and 12 h post-MPP+, protein isolated, and Western blots used to quantify represented by columns with error bars representing + 1 standard error of the mean.    247    Figure 6.11 at 6 and 12 h post-MPP+.      248    Figure 6.12 Parkin expression in MN9D cells at 6 and 12 h post MPP+. MN9D + in DMEM media. Cells were collected at 6 and 12 h post-MPP+, protein isolated, and Western blots used to quantify parkin normalized to GAPDH. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean.          249    Figure 6.13 Representative Western blot for parkin in MN9D cells at 6 and 12 h post-MPP+. Parkin was normalized to GAPDH.              250    Figure 6.14 FosB expression in MN9D cells at 6 and 12 h post PMA. MN9D and 12 h post-PMA, protein isolated, and Western blots used to quantify FosB normalized to GAPDH. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with asterisks.    251    Figure 6.15  at 6 and 12 h post PMA. MN9D and 12 h post-PMA, protein isolated, and Western blots used to quantify represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; one-way ANOVA) from the vehicle controls are denoted with asterisks.    252    Figure 6.16 cells at 6 and 12 h post-PMA.      253    Figure 6.17 Parkin expression in MN9D cells at 6 and 12 h post PMA. MN9D and 12 h post-PMA, protein isolated, and Western blots used to quantify parkin normalized to GAPDH. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean.         254     Figure 6.18 Representative Western blot for parkin in MN9D cells at 6 and 12 h post-PMA. Parkin was normalized to GAPDH.                  255  Discussion Results from the in vitro experiments reveal that MN9D cells closely resemble what has been previously found for NSDA neurons in the SN. Indeed, MPP+ (the active metabolite of MPTP) leads to a rapid, time-dependent decrease in cellular stored DA that does not recover by 24 h after treatment. With no change observed in cell viability, this loss in DA is not the result of cell loss. It is also important to note that there is no increase in ROS in MN9D cells at the concentration of MPP+ used (Choi et al., 1999). in these transcription factors in the SN. Moreover, then was no there was no change in parkin in these cells. This is slightly different than the SN, where parkin in the SN decreases by 24 h after MPTP (Benskey et al., 2012). Comparing the MN9D cells to the ARC, the two are completely different in regards to DA stores, and expression of FosBs and parkin. MN9D cells do not recover DA stores by 24 h after neurotoxicant exposure nor do these cells have increased expression of FosBs or parkin as is seen in the ARC (Benskey et al., 2012). Accordingly, MN9D cells responses are more like those of NSDA neurons than the TIDA neurons, as such, MN9D cells are not a good model to study transcription factor mediated parkin expression following neurotoxicant exposure.     PMA is a phorbol ester commonly used in vitro to stimulate expression of AP-1 transcription factors including FosBs (Lee et al., 1997).  To determine if   256  induction of AP-1 transcription factor expression is linked to activation of parkin expression MN9D cells were treated with PMA. With PMA, FosB and ere is no increase in parkin, parkin in this neuroblastoma cell line. There are a few caveats to this interpretation.  While PMA was used to induce AP-1 transcription factors in these to global up-regulation of AP-1 transcription factors, and in other neuronal cell lines such as SHSY-5Y cells, is used to terminally differentiate cells (Balasooriya and Wimalasena, 2007). MN9D cells are a fusion of mouse embryonic ventral mesencephalic neurons with neuroblastoma cells (Choi et al., 1992). As such the MN9D cells are an immortalized cell line that are continually dividing. MN9D cells will continue to divide until terminally differentiated, at which time the cells  express DAT, become able to uptake DA, and take on the appearance of a typical neuron (cell body with projections) (Balasooriya and Wimalasena, 2007; Choi et al., 1992).  Differentiation of MN9D cells can be achieved through exposure to sodium butyrate over a period of days. Sodium butyrate is a histone deacetylase inhibitor, and should cause increased expression of genes not usually expressed as highly. The differentiation processes itself has the potential to alter both parkin and AP-1 expression. In some cell lines sodium butyrate   257  increases the activation of AP-1 transcription factors, while in other cells lines it decreases activity (Alva-Murillo et al., 2015; Andoh et al., 1999; He et al., 2016). If AP-1 misregulation caused by sodium butyrate in the differentiation process increases parkin, then parkin expression could be high enough that a ceiling effect is reached. As parkin is both an E3 ligase and a substrate of itself, parkin levels should only be able to increase to a certain extent before expression plateaus (Zhang et al., 2000; Imai et al., 2001). Overall, the MN9D cells do not appear to be the best model in which to study the relationship between parkin, the FosBs, and differential susceptibility of neurons.   AAV- Results parkin protein in the ARC an SN, an AAV-bilaterally injected into the ARC or unilaterally injected in the SN. In the ARC of mice receiving AAV-AAV-GFP control at 1.00 ± 0.12 (Figures 6.19 and 6.20). In the ipsilateral SN that received AAV-the AAV-GFP control contralateral SN at 1.00 ± 0.08 (Figures 6.21 and 6.22). Unilateral injection of the AAV-neurochemical changes in NSDA neurons terminating in the ST. DA in the ST   258  was 271.19 ± 15.95 ng/mg of protein in the AAV-GFP control side and 245.56 ± 22.36 ng/mg of protein in the AAV-Figure 6.23). DOPAC in the ST was 24.85 ± 1.29 ng/mg of protein in the AAV-GFP control side and 23.52 ± 1.69 ng/mg of protein in the AAV-Figure 6.24). DOPAC/DA ratio in the ST was 0.089 ± 0.0035 in the AAV-GFP control side and 0.091 ± 0.0036 ng/mg in the AAV-Figure 6.25). On contrast, bilateral injection into the ARC altered DA stores axon terminals of TIDA neurons in the ME. DA in the ME was 56.08 ± 8.60 ng/mg of protein in the AAV-GFP control mice and 188.02 ± 21.73 ng/mg of protein in mice that received AAV-Figure 6.26).       259    Figure 6.19 Parkin in the ARC after AAV-- mediated overexpression. An AAV-AV-GFP control vector was stereotaxically injected into the ARC of male C57Bl/6J mice. Injections were bilateral, with mice receiving either AAV-GFP or AAV-four weeks and killed by decapitation (n=8/group). Brains were sectioned, ARC was microdissected, protein isolated and Western blots used to quantify parkin normalized to GAPDH. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; t-test) from the vehicle controls are denoted with asterisks.    260    Figure 6.20 ARC of mice injected with either AAV-GFP or AAV- Parkin, FosB, and alized to GAPDH.    261    Figure 6.21 Parkin in the SN after AAV--mediated overexpression. An AAV--GFP control vector was stereotaxically injected into the SN of male C57Bl/6J mice. Injections were unilateral, with one hemisphere receiving AAV-GFP and the other AAV-to recover for four weeks and killed by decapitation (n=8/group). Brains were sectioned, SN was microdissected, protein isolated and Western blots used to quantify parkin normalized to GAPDH. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; paired t-test) from the vehicle controls are denoted with asterisks.    262    Figure 6.22 Representative WSN of mice injected with either AAV-GFP or AAV-Parkin, FosB, and      263    Figure 6.23 DA concentration in the ST after AAV- An AAV-vector or AAV-GFP control vector was stereotaxically injected into the SN of male C57Bl/6J mice. Injections were unilateral, with one hemisphere receiving AAV-GFP and the other AAV-for four weeks and killed by decapitation (n=8/group). Brains were sectioned, ST was microdissected and processed for neurochemical analysis. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; paired t-test) from the vehicle controls are denoted with asterisks.     264    Figure 6.24 DOPAC concentration in the ST after AAV- An AAV-vector or AAV-GFP control vector was stereotaxically injected into the SN of male C57Bl/6J mice. Injections were unilateral, with one hemisphere receiving AAV-GFP and the other AAV-for four weeks and killed by decapitation (n=8/group). Brains were sectioned, ST was microdissected and processed for neurochemical analysis. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean.      265    Figure 6.25 DOPAC/DA ratio in the ST after AAV- An AAV-vector or AAV-GFP control vector was stereotaxically injected into the SN of male C57Bl/6J mice. Injections were unilateral, with one hemisphere receiving AAV-GFP and the other AAV- allowed to recover for four weeks and killed by decapitation (n=8/group). Brains were sectioned, ST was microdissected and processed for neurochemical analysis. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean.     266    Figure 6.26 DA concentrations in the ME after AAV- An AAV-vector or AAV-GFP control vector was stereotaxically injected into the ARC of male C57Bl/6J mice. Injections were made bilaterally with mice receiving either AAV-GFP or AAV-and killed by decapitation (n=8/group). ME were microdissected and processed for neurochemical analysis. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; paired t-test) from the vehicle controls are denoted with asterisks.     267  Discussion  The neurochemical analysis after AAV-provides another difference between neurons. In the ST, AAV-injection in the SN does not alter DA stores, nor does it effect DA metabolism, as shown by the lack of change in the DOPAC/DA ratio. In the ME, AAV-TIDA axon terminals, possibly through increased DA synthesis. TH is the rate-limiting enzyme in DA synthesis in TIDA neurons (Lookingland and Moore, 2005). FosB as well as other AP-1 transcription factors are known to bind to TPA-response elements within the TH promoter and regulate the expression of TH (Gizang-Ginsberg and Ziff, 1994; Yukimasa et al., 1999;). The way AP-1 transcription factors effect TH expression varies in different tissues, for example in PC12 cells, AP-1 represses TH expression, where in F9, NIH3T3 cells and a rodent model it can increase TH expression (Ghee et al., 1998; Gizang-Ginsberg and Ziff, 1994; Chae et al., 1996). Though same binding partners. It is reasonable to assume that the increase in DA in  TH promoter, though future studies should be performed to examine TH expression and activity in these neurons.  As for the lack of increase in DA or DA metabolism in the ST, this may be explained by tissue specific differences (Ghee et al., 1998). The NSDA neurons have D2 presynaptic autoreceptors located on their axon terminals   268  in the ST that can directly regulate and inhibit TH and DA synthesis. If over-autoreceptors may repress DA synthesis back to basal levels. Reports of D2 autoreceptor regulation of the TIDA neurons varies (Timmerman et al., 1995; Berry and Gudelsky, 1991). The lack of D2 autoreceptor inhibition of DA synthesis in axon terminals of TIDA neurons in the ME could explain how DA stores could increase in the ME.  and SN. The AAV-effect neurons, and has been shown to not infect glial cells (Zachariou et al., 2006). This suggests that changes caused by AAV-occur in neurons in these regions, of which TIDA and NSDA neurons comprise a large portion. The increase in parkin in both regions is consistent  a transcription factor of parkin, and that increased expression of this transcription factor is sufficient to increase in regulating parkin in response to neurotoxicant exposure       269  AAV- Results An AAV-as a method to block parkin expression in the ARC and DA recovery in the ME after acute MPTP exposure. In the ARC of mice that received AAV-GFP and saline, mean fold change FosB was 1.00 ± 0.17, in mice that received AAV-GFP and MPTP, mean fold change FosB was 3.75 ± 0.19, in mice that received AAV- saline, mean fold change FosB was 1.10 ± 0.16, and in mice that received AAV-2.46 ± 0.80  (Figures 6.27 and 6.29received AAV-0.18, in mice that received AAV-0.10, in mice that received AAV-was 0.99 ± 0.28, and in mice that received AAV-Figures 6.28 and 6.29).  Parkin in the ARC increased with MPTP in AAV-GFP control mice, but did not change in the mice that received AAV-present. In the ARC, mice that received AAV-GFP and saline, mean fold change parkin was 1.00 ± 0.08, in mice that received AAV-GFP and MPTP, mean fold change parkin was 2.21 ± 0.11, in mice that received AAV-and saline, mean fold change parkin was 1.10 ± 0.07, and in mice that   270  received AAV-0.14 (Figures 6.30 and 6.31). Injection of the AAV-axon terminals of TIDA neurons in the ME. DA in the ME was 56.08 ± 8.60 ng/mg of protein in the AAV-GFP control mice that received saline, and 42.19 ± 4.60 ng/mg of protein those that received MPTP. In mice that AAV-7.41 ng/mg of protein those that received MPTP (Figure 6.32).           271    Figure 6.27 FosB in the ARC after AAV-sion and 24 h post-MPTP. An AAV--GFP control vector was stereotaxically injected into the ARC of male C57Bl/6J mice. Injections were bilateral, with mice receiving either AAV-GFP or AAV-for four weeks and killed by decapitation (n=8/group). Brains were sectioned, ARC was microdissected, protein isolated and Western blots used to quantify FosB normalized to GAPDH. Mean fold change from AAV-GFP vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; two-way ANOVA) from the vehicle controls are denoted with asterisks.     272    Figure 6.28 --MPTP. An AAV-AAV-GFP control vector was stereotaxically injected into the ARC of male C57Bl/6J mice. Injections were bilateral, with mice receiving either AAV-GFP or AAV-for four weeks and killed by decapitation (n=8/group). Brains were sectioned, ARC was microdissected, protein isolated and Western blots used to quantify -GFP vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; two-way ANOVA) from the vehicle controls are denoted with asterisks.     273    Figure 6.29 mice injected with either AAV-GFP or AAV- were normalized to GAPDH.     274    Figure 6.30 Parkin in the ARC after AAV--MPTP. An AAV--GFP control vector was stereotaxically injected into the ARC of male C57Bl/6J mice. Injections were bilateral, with mice receiving either AAV-GFP or AAV-for four weeks and killed by decapitation (n=8/group). Brains were sectioned, ARC was microdissected, protein isolated and Western blots used to quantify parkin normalized to GAPDH. Mean fold change from AAV-GFP vehicle control is represented by columns with error bars representing + 1 standard error of the mean. Values significantly different (p<0.05; two-way ANOVA) from the vehicle controls are denoted with asterisks.    275    Figure 6.31 ReprARC of mice injected with either AAV-GFP or AAV- Parkin,      276    Figure 6.32 DA concentrations in the ME after AAV--MPTP. An AAV--GFP control vector was stereotaxically injected into the ARC of male C57Bl/6J mice. Injections were bilateral, with mice receiving either AAV-GFP or AAV-for four weeks and killed by decapitation (n=8/group; two-way ANOVA). ME were microdissected and processed for neurochemical analysis. Mean fold change from vehicle control is represented by columns with error bars representing + 1 standard error of the mean.       277  Discussion  nD in the ARC does not affect DA in the ME. DA levels in the ME of mice that were injected with AAV-GFP or AAV-not different from each other. DA was not different in the ME 24 h after MPTP consistent with previous published reports from our laboratory (Behrouz, et al., 2007; Benskey, et al., 2012), even in the presence of -induced increase in parkin is not required for DA recovery in the ME after MPTP. The caveat to these results is that the data appears to be trending towards there not being a recovery of DA in MPTP-treated mice that received AAV-with the requirement of parkin for recovery of the TIDA neurons (Benskey et al., 2015). expression of parkin, however it does not effect MPTP-expression. -regulation of parkin following acute neurotoxicant exposure, and the preferential dimerization of pathway that directly regulates parkin.      278  Overall Conclusions Phosphorylation of CREB and SRF upstream transcriptional activators of FosB is consistent with the regional differences in expression patterns of and parkin, phosphorylated CREB and SRF increase early in the ARC and not the SN. CREB and SRF add an additional level to the potential regulation of parkin, allowing for some speculation on a parkin activation pathway in response to acute neurotoxicant exposure. Where one or more of the stresses caused by MPTP leads to the phosphorylation of CREB and SRF, which of these causes increased transcription of the FosB proteins and parkin is not known. Identification of these pathways can be used as the basis of future experiments designed to discover which kinase or kinases are involved in MPTP-induced CREB and SRF phosphorylation, and whether both CREB and SRF are required for activation of FosB and parkin.  Identification of the specific stress or combination of stresses caused by MPTP that are crucial to activate the FosB/parkin pathway in TIDA neurons could help explain why this pathway does not appear to function in the same way in NSDA neurons.   Whether the FosBs directly interact with the parkin promoter is not known. Chromatin immunoprecipitation (ChIP) followed by RTPCR would be the best approach to answer this question, but the in vitro model used in these experiments did not show a relationship between expression of the FosBs   279  and parkin. The MN9D cell model does not show MPP+ induction of the FosBs or parkin, nor does PMA induced expression of the FosBs induce parkin expression. This is completely different than what is observed in vivo, so the best alternative option would be to use ChIP paired with RTPCR in the ARC. One issue, however, is that the ARC is a small brain region in mice that does not yield much protein. For an average Western blot, protein extracted from that at least 1 mg of protein be used, which is not a problem in cell culture, but would require pooled ARC tissue from up to 40 mice. The use of such a large sample size of one is not financially practical. An alternative would be to find an in vitro cellular system that better models neurotoxicant induced activation of FosB and parkin expression in the ARC, possibly through one created by the fusion of TIDA neurons with a neuroblastoma cell line.             280  Chapter 7: General Discussion   Differences Between TIDA and NSDA Neurons The differential expression of parkin is associated with the susceptibility of the NSDA neurons and the resilience of the TIDA neurons to neurotoxic insult (Benskey et al., 2012, 2015). Additional to how parkin regulation varies in these neurons, there are other inherent differences. The TIDA neurons are part of a circumventricular system within the brain that is not protected by the blood-brain barrier. The axons of the TIDA neurons terminate in the hypophyseal portal system and cell bodies are located along the third ventricle. Through their location, the TIDA neurons are exposed to blood in the ME and cerebrospinal fluid from the ventricular system of the brain. This compares to the NSDA neurons that are protected by the blood-brain barrier and are not directly exposed to substances carried in the blood. The difference in location of these neurons suggests that the TIDA neurons are more prone to exposure to stressors than the NSDA neurons. This is supported by the nearly 4 fold higher concentration of MPP+ found in the ME as compared to the ST after MPTP as well as the higher basal levels of ROS via a DCF-DA assay that exist in the ME and ARC, as compared to the ST and SN (Benskey et al., 2012). It is possible that the TIDA neurons induce the increase in parkin as an adaptive mechanism acquired due to constant exposure to stress.     281  Additionally, the TIDA neurons have substantially shorter axons than the NSDA neurons. This could play a role in the signaling pathways that regulate parkin expression in response to acute MPTP exposure. Since MPP+ is primarily thought to be taken into DA neurons via DAT located on axon terminals, the shorter axons in the TIDA neurons could facilitate a more rapid response, both in modifying gene expression in the cell bodies and delivery of cytoprotective proteins to the site of neurotoxicant insult in the axon terminals. As the axons of the NSDA neurons are significantly longer than the axons of the TIDA neurons, there may be an problem of stress responses present in the terminals signaling back to the cell bodies in the NSDA neurons. In addition, MPP+ is known to destabilize microtubules, therefore vesicles and proteins transported via the microtubules would be expected to be hindered more in longer axons in the NSDA neurons as compared to the TIDA neurons (Cartelli et al., 2013; Cappelletti et al., 2005).  Another possible difference between the TIDA and NSDA neurons that could be a contributing factor is the presence of D2 autoreceptors at the axon terminals. The NSDA neurons have D2 autoreceptors that respond to synaptic DA and lead to a decrease in cAMP in presynaptic DA axon terminals (Kebabian and Greengard, 1971). Unlike the NSDA neurons, the presence of D2 autoreceptors on axon terminals of TIDA is not quite clear, with evidence both for and against (Timmerman et al., 1995; Berry and Gudelsky, 1991). Since DA from the TIDA neurons is released into the portal   282  blood, rather than a synapse, and is transported away from the presynaptic axon terminals it is likely that D2 autoreceptors (if present) do not have as robust a function as in the NSDA neurons.  D2 autoreceptors may factor into the differential response between these neurons by affecting the CREB pathway. MPTP has been shown to cause not only the depletion in DA stores, but an increase in DA release from NSDA neuron axon terminals in the ST (Ozaki et al., 1987; Serra et al., 2008). An increase in released DA in the ST would interact with the D2 autoreceptor, leading to the inhibition of adenylate cyclase and a subsequent decrease in cAMP and PKA activity. As a consequence, phosphorylation of CREB would be expected to decrease. This is consistent with the observed decrease in phosphorylated CREB (Ser133) in the SN after MPTP in the present studies. Assuming D2 autoreceptors do not have a strong control over the TIDA neurons, increased DA release would not be expected to effect cAMP or phosphorylation of CREB. Indeed, the present study shows that MPTP increases (rather than decreases) phosphorylated CREB in the ARC. This potential inherent difference between TIDA and NSDA neurons adds context to a potential activation pathway that results in increased parkin expression in response to acute neurotoxicant exposure.           283  Potential Pathway Involved in Parkin Regulation Transcription factors and indices of oxidative and ER stress, mitochondrial damage, and proteasome impairment examined in the present series of experiments yield some insight into the stress response pathways following acute MPTP exposure. MPTP does not elicit effects on ATF4 or Nrf1, transcription factors known to respond to ER stress and proteasome impairment. Likewise, any damage caused to mitochondria at the times measured in these experiments, was not sufficiently robust to induce NRF1 in the mitochondrial biogenesis pathway. This is not to say these pathways are not involved in regulating parkin expression, but the results do not support the conclusion that the single MPTP exposure paradigm used in these studies engages these pathways. The only pathway that appears to be affected is the oxidative stress pathway, where ROS levels increase only in the ME at 6 h post-MPTP.  Oxidative stress can act through multiple pathways, two of which involve CREB and SRF, transcription factors of the FosB gene (Vialou et al., 2012). ROS can lead to an increase in intracellular Ca2+ as well as cAMP (Ermak and Davies 2002; Li et al., 2011). Ca2+ is a cofactor of calmodulin kinases and cAMP is a cofactor of PKA, both of which can phosphorylate CREB and SRF. Pexpression and nuclear localization are consistent with the expectations of a parkin transcription factor (Figure 7.1).    284   in both expression after MPTP, it is likely that dimers containing a FosB protein directly interacts with the Park2 promoter. If the FosBs do not directly interact with the protein, they are at least at a point where the CREB and SRF pathways converge. Additionally, CREB has been linked to neuron survival in response to ROS and loss of SRF increases the susceptibility of the NSDA neurons to MPTP (Lee et al., 2009; Rieker et al., 2012). This suggests that CREB and SRF are probably acting in other neuroprotective pathways, in addition to activating parkin through the FosBs. Another possibility is that CREB acts directly to regulate parkin, as it could interact with the known AP-1 site on the Park2 promoter (Bouman et al., 2011).     285   Figure 7.1 Predicted parkin regulatory pathway involved in the response to acute MPTP exposure. Oxidative stress caused by the effects of the metabolite of MPTP, MPP+, causes increases in cAMP and Ca2+. The intracellular Ca2+ acts as a cofactor for both CaMKII and CaMKIV, which can phosphorylate CREB and SRF, respectively. Likewise, cAMP would act as a cofactor of PKA and phosphorylate both CREB and SRF. The phosphorylated CREB and SRF act at the promoter of the FosB gene and increase expression expression.    286  Another potential transcription factor of parkin that was not examined or identified by predictive binding software is parkin. Parkin is already known to autoubiquitinate, but it may also regulate its own expression at the transcriptional level (Shimura et al., 2000; Zhang et al., 2000; Chaugule et al., 2011). The promoter region of Park2 contains a perfect consensus binding sequence (GCCGGAG) of parkin -116 to -123 upstream of the transcriptional start site of parkin (Dupaln et al., 2013). Parkin has been shown to act as both a transcriptional activator of presenilin-1 and a repressor of presenilin-2 (Duplan et al., 2013). As these are the only known cases of parkin acting as a transcription factor, it is unclear whether parkin would act as an activator or repressor of itself. However, parkin acting as its own repressor would make the most sense as a mechanism to maintain parkin levels in the cells.   FosBs Beyond Parkin AP-1 transcription factors, especially those with JunD containing dimers are present in neurons that survive brain injury (Pennypacker 1997, 1998, 2000). Increased expression of parkin in response to the FosBs is just one of in addiction and memory through altering synaptic plasticity. Indeed, known transmission (synaptotagmin 6, synaptogyrin 1, glutamate receptor 2, glutamate receptor   287  NMDA zeta 1, glutamate receptor AMPA 2 alpha 2, glutamic acid decarboxylase 2), cell signaling (adenylate cyclase activating peptide receptor, G-protein alpha o, protein kinase C beta, protein kinase II alpha, calmodulin 3), cell adhesion and motility (cadherin 2, kinesin family 1B and 5C, microtubule associated protein 2) and cellular stress responses (heat shock protein 40 and nuclear factor kappa-light chain-enhancer of activated B cells) (McClung and Nestler, 2003; Pitchers et al., 2013; Nestler et al., 2001; Nestler 2012, 2015).  neurotrophic factor (BDNF) expression, induces the formation of dendritic spines, and alters the composition of neurotransmitter receptors (Robison et al., 2013; McClung and Nestler, 2003; Nestler et al., 2001; Nestler 2012, 2015; Nikulina et al., 2012; Krasnova et al., 2013; Wang et al., 2013). Changes in dendritic spines and receptors can alter how the neurons function, altering the sensitivity and reactivity of the neurons.  in L-DOPA-ST of post-L-DOPA-induced dyskinesia in primates (Lindgren et al., 2011; Cao et al., 2010; Berton et al., 2009). This could cause increased activity of the MSN, resulting in the dyskinesia. In comparison to the apparent negative effects of   288  to 6-hydroxydopamine and MPTP induced axonal damage (Singh et al., as a potential therapeutic target, the multiple downstream targets and effects make them unsuitable candidates for gene therapy.     289  Potential Future Directions The link between the FosBs and regulation of parkin expression after MPTP is only part of a larger picture. MPTP can only be used to recapitulate some of the damage to DA neurons in PD. Other models examining the FosBs and parkin should be used to determine if the relationship between the two still holds true in other contexts. Two other procedures used to -synuclein and the use of pre--synuclein fibrils. Both methods lead to the formation of Lewy bodies, a key pathology associated with PD that is lacking with the use of MPTP. In regards to parkin expression, whether there is the same differential -synuclein based models is not known, let alone other proteins that may be neuroprotective. Additional to other models to induce effects similar to that observed in PD, potential regulators of parkin other than transcription factors should be examined.  One potential difference in responses between susceptible and resistant DA neurons could be the levels of microRNAs (miRNAs) present that can bind and suppress the expression of parkin mRNA. Algorithms from microrna.org and mirbase.org have shown 36 potential parkin mRNA binding miRNA candidates in mice and 49 in humans. In addition to examining miRNAs that are predicted to directly interact with parkin mRNA, some miRNAs, such as mir-34b and mir-34c have also been shown to regulate parkin expression (Minones-Moyano et al., 2011).  Interestingly, PD brain   290  samples show concomitant decreases in mir-34b, mir-34c and parkin expression (Minones-Moyano et al., 2011).  Since miRNAs only down-regulate expression, the decrease in both parkin and mir-34b and mir-34c; along with mir-34b and mir-34c not predicted to bind parkin mRNA, indicate that these miRNAs are most likely not functioning in the repression of parkin or binding parkin mRNA directly.  Instead, these miRNAs may function in parkin activation by repressing an upstream transcription factor that is inhibitory to parkin expression.  Similar to the regulation of parkin via transcription factors, there could transcriptional coactivators involved. The histone acetyl transferase (HAT) p300 is one such transcriptional coactivator that has been shown to interact with members of the CREB family, the AP-1 family, HIF1, E2F, p53, MyoD, basal transcriptional machinery and others (Chan and La Thangue, 2001). There are five protein-binding domains on p300, as well as a bromodomain, which allows p300 to function as a HAT (Chan and La Thangue, 2001). Through acetylation of histone tails, the DNA becomes less tightly bound to the histones and the chromatin relaxes. This relaxation of the chromatin could be responsible for an increase in transcription and result in long-term changes in transcription.  In addition to acting as a HAT, p300 is also predicted to act as a bridge between transcription factors and basal transcriptional machinery, or as a scaffold recruiting other proteins that could modify transcription (Chan and   291  La Thangue, 2001). One of the ways that p300 is regulated is based on stability. Under normal conditions, p300 autoacetylates itself and can be destabilized by deacetylated via sirtuin 2 (dependent on the presence of NAD+ as a cofactor) (Black et al., 2008). In the presence of oxidative stress and mitochondrial damage, p300 is not deacetylated and is a more stable protein and accumulates (Jain et al., 2012). As MPTP inhibits mitochondrial Complex I (which normally converts NADH to NAD+), MPTP should decrease the amount of free NAD+ present, decreasing SIRT2 activity and causing p300 accumulation. Since p300 has been shown via reported ChIP-Seq results on ENCODE to bind the Park2 promoter and can be regulated by oxidative stress, it is an interesting potential avenue to explore.    Concluding Remarks The differential susceptibility of the TIDA and NSDA neurons offers a unique tool to study how different populations of DA neurons react to stress in response to neurotoxic insult. The experiments in this dissertation have lead to the identification of potential transcriptional regulators involved in the differential regulation of parkin after acute MPTP exposure. Of these, of parkin, i.e. expression precedes parkin and is maintained as long as parkin, it is localized to the nuclei of DA neurons, and manipulations of the transcription factor or its actions produces corresponding alterations in   292  a differentially expressed transcriptional activator of parkin. Furthermore, phosphorylated CREB and SRF, activators of the FosB gene, are consistent following acute MPTP exposure. Though the small amount of protein each the PARK2 promoter via ChIP-RTPCR, results from AAV-mediated therapeutic solution to make the NSDA neurons more like the TIDA neurons, it is a first step to at least examining how parkin may be regulated.                 293             REFERENCES               294  REFERENCES   Agirre X, Roman-Gomez J, Vazquez I, Jimenez-Velasco A, Garate L, Montiel-Duarte C, Artieda P, Cordeu L, Lahortiga I, Calasanz MJ, Heiniger A, Torres A, Minna JD, Prosper F (2006) Abnormal methylation of the common PARK2 and PACRG promoter is associated with downregulation of gene expression in acute lymphoblastic leukemia and chronic myeloid leukemia. International journal of cancer 118:1945-1953.  Albin RL, Young AB, Penney JB (1989) The functional anatomy of basal ganglia disorders. Trends in neurosciences 12:366-375.  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