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REGIONAL DISPARITIES IN EXPRESSING ALZHEIMER’S
DISEASE NEUROPATHOLOGY - AN INFERENTIAL STUDY

presented by

DEEBIKA BALU

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______ . ——_—

REGIONAL DISPARITIES IN EXPRESSING ALZHEIMER’S
DISEASE NEUROPATHOLOGY — AN INFERENTIAL STUDY

By

DEEBIKA BALU

A THESIS
Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Chemical Engineering and Materials Science

2007

 

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ABSTRACT

REGIONAL DISPARITIES IN EXPRESSING ALZHEIMER’S DISEASE
NEUROPATHOLOGY — AN INFERENTIAL STUDY

By
DEEBIKA BALU

Alzheimer’s disease (AD) is a progressive, memo-degenerative disease triggered by
multiple factors. Modification of two major neuronal proteins, Amyloid Beta and Tau,
contributes to formation of senile plaques and neuro-fibrillary tangles in the brain. Such
pathological changes lead to neuronal dysfunction. It is interesting to note that the part of
the brain involved in cognition and memory i.e. the cortex and the hippocampus are the
ones that are severely affected compared to the cerebellum. The cerebellum (another part
of the brain) is mostly involved in motor neuronal activities such as muscle movements,
posture and equilibrium.

Saturated free fatty acids such as palmitic acid and stearic acid have been shown to
induce the pathological changes observed in AD through astroglia mediated oxidative
stress in cortical neurons. It is known that the de novo synthesized ceramides signals that
cascade of events that eventually lead to oxidative stress in cortical astrocytes. Post-
mortem brain analysis studies in AD patients suggest higher levels of ceramides in cortex
and hippocampus compared to cerebellum. The results of the current study suggest that
the cerebellar astrocytes proliferate in the presence of free fatty acids by production of
lower levels of ceramides. The study was undertaken to study the variation in the
behavior of the neurons and astrocytes from two different brain regions and there is
indeed a marked difference in the astrocytic cell fate (cell death or proliferation) in the

presence of free fatty acids.

COPYRIGHT by
DEEBIKA BALU
2007

ACKNOWLEDGEMENTS

Mom and Dad, I could not have accomplished this without you; thank you for the
support and encouragement throughout the years. I thank my family and my friends for
supporting me through tough times.

I sincerely thank my advisor, Dr. Christina Chan for her help and guidance. I
cannot convey how much I appreciate your dedication, constructive criticism and
motivation for my scientific training over the past 2 years. I would also like to thank the
members of my guidance committee, Drs. Patrick Walton and Mark Worden for their
thoughtful advice and critique in the preparation of this work.

I wish to thank the faculty and students of the Department of Chemical
Engineering for contributing to my scientific development by providing an excellent
training atmosphere here at MSU. Last, but certainly not the least, to the members of the
lab, whose friendship and scientific feedback has been very supporting over the past few
years. All of you have made this an amazing graduate school experience.

This research was made possible by the help and support I received from many
people. It might be possible that I may not have explicitly named all the people without
whose help I would not have been able to present my research as a thesis, yet I do

remember their contributions to my research and my life.

iv

TABLE OF CONTENTS

LIST OF FIGURES ......................................................................................................... vii

LIST OF ABBREVIATIONS .......................................................................................... ix

1. INTRODUCTION
1. 1 Alzheimer’s disease and its impact ......................................................................... 2
1.2 Pathophysiological and metabolic changes in Alzheimer’s disease ........................ 2
1.3 AD and free fatty acids ............................................................................................ 4
1.4 AD and its effect on the different brain regions ...................................................... 5
1.5 Aims of the study ..................................................................................................... 6
1.6 Organization of the thesis ........................................................................................ 7

2. EFFECT OF FREE FATTY ACID (PALMTIC ACID) ON CEREBELLAR AND
CORTICAL: NEURONS AND ASTROCYTES

2.1 INTRODUCTION ................................................................................................... 9
2.1.1 Free fatty acid metabolism: role of acyl CoA synthetases ........................... 10
2.1.2 Fatty Acyl CoA Isomers .............................................................................. 11

2.2 MATERIALS AND METHODS ........................................................................... 13

2.2.1 Cell Culture ................................................................................................... 13
2.2.2 Free fatty acid treatment ............................................................................... 15
2.2.3. Western Blotting .......................................................................................... 15
2.2.4 Measurement of Reactive Oxygen Species ................................................... 16
2.2.5 Cell Proliferation Assay ................................................................................ 17

2.3 RESULTS AND DISCUSSION ............................................................................ 18

2.3.1 Effect of palmitic acid on cerebellar neurons ............................................... 18
2.3.2 Regional disparities in expressing AD-pathological changes ....................... 23

3. EFFECT OF PALMITIC ACID IN GLUCOSE METABOLISM IN CORTICAL
AND CEREBELLAR ASTROCYTES

3.1 INTRODUCTION ................................................................................................. 31
3.1.1 The concept of energy metabolism in brain .................................................. 31
3.1.2 Alzheimer’s Disease and Type-2 Diabetes ................................................... 33
3.1.3 Abnormalities in brain glucose metabolism and its impact on cellular
and molecular mechanisms in sporadic dementia of Alzheimer type .................... 34

3.2 MATERIALS AND METHODS ........................................................................... 35
3.2.1 Cell Culture and free fatty acid treatment ..................................................... 35

3.2.2 Measurement of glucose uptake by the astrocytes ........................................ 35
3.2.3 Western Blotting ........................................................................................... 35

3.3 RESULTS AND DISCUSSION ............................................................................ 36

4. CONCLUSIONS AND FUTURE DIRECTIONS

4.1 CONCLUSIONS .................................................................................................. 41
4.2 FUTURE DIRECTIONS TO THE STUDY .......................................................... 43
4.2.1 Invivo and metabolic studies ........................................................................ 43

4.2.2 Insulin signaling in response to FFAs in different regions ........................... 43
4.2.3 Ceramide synthesis as regulatory machinery ................................................ 44

4.3 SHORTCOMINGS IDENTIFIED IN THE STUDY ............................................ 45
APPENDIX A: QUANTIFYING THE PROTEIN LEVELS IN WESTERN BLOT ...... 47

APPENDIX B: PHASE CONTRAST IMAGES OF NEURONS AND
ASTROCYTES FROM THE CEREBELLUM AND CORTEX OF THE

RAT BRAIN .......................................................................................... 49
APPENDIX C: SUPPLEMENTARY IMAGES ................................................. 50
APPENDIX D: CAVEATS IN MEASUREMENT OF REACTIVE OXYGEN

SPECIES ............................................................................................. 56
BIBLIOGRAPHY ........................................................................................................... 58

vi

LIST OF FIGURES

Fig 1.1 Protein abnormalities in AD ............................................................... 3
Fig 2.1 Free fatty acid metabolism ............................................................... 10
Fig 2.2 Direct treatment of cerebellar neurons with increasing concentration

of palmitic acid ...................................................................................... 18
Fig 2.3 Treatment of cerebellar neurons with conditioned media from

cerebellar astrocytes ................................................................................. 19
Fig 2.4 F FA induced tau hyperphosphorylation in cerebellar neurons ....................... 20
Fig 2.5 FFA induced tau hyperphosphorylation in cortical neurons ......................... 21
Fig 2.6 FFA induced BACEI upregulation in cortical neurons ............................... 22

Fig 2.7 Relative expression levels of ACS3 in cortical, cerebellar astrocytes
and neurons .......................................................................................... 24

Fig 2.8.A Fluorescence microscopy images of intracellular accumulation of ROS

in neurons ............................................................................................. 25
Fig 28.3 Quantification of ROS produced in cortical and cerebellar neurons ............. 26
Fig 2.9 Proliferation of cerebellar astrocytes in the presence of palmitic acid .............. 29

Fig. 3.1 Positron Emission Tomography images of brain glucose utilization
in a normal and an AD patient .................................................................... 32

Fig 3.2 PA downregulates glucose uptake by cortical and not cerebellar astrocytes.. . ....37

Fig 3.3 GLUT-l proteins in cellular membranes ................................................ 38
Fig 3.4 FF A induced GLUT-l downregulation in cortical but not in cerebellar

astrocytes .............................................................................................. 39
Fig a Direct treatment of cerebellar neurons with increasing concentration of palmitic
acid ..................................................................................................................................... 47
Fig b Phase contrast images .............................................................................................. 49
Fig c.1 Direct treatment of cerebellar neurons with palmitic acid ................................... 50
Fig c.2. FFA induced tau hyperphosphorylation in cerebellar neurons ............................. 51

vii

Fig c.3 FFA induced tau hyperphosphorylation in cortical neurons .................................. 52
Fig c.4 F F A induced BACEl upregulation in cerebellar neurons ..................................... 53

Fig c.5 Relative expression levels of ACS3 in cortical, cerebellar astrocytes and
neurons ............................................................................................................................... 54

Fig c.6 F F A induced GLUT-l downregulation in cortical but not in cerebellar
astrocytes ............................................................................................................................ 55

viii

LIST OF ABBREVIATIONS
ACh : Acetyl Choline
ACS : Acetyl Co-enzymeA Synthetase
AD : Alzheimer’s disease
APP : Amyloid Precursor Protein
ATP : Adenosine Triphosphate
BACE : Beta Amyloid Cleaving Enzyme
Co-A : Co-enzyme A
FACL : Fatty Acyl CoA Ligase
FFA : Free fatty acid
NFT : Neuro-fibrillary Tangles
PA : Palmitic Acid
PET : Positron Emission Tomography
ROS : reactive oxygen species

PBS : phosphate buffered saline

ix

CHAPTER I

1. INTRODUCTION

1. 1 Alzheimer’s disease and its impact

In the recent report released by the Alzheimer’s disease Association, it has been
estimated that there are about 5.1 million Americans suffering from Alzheimer’s disease
and the number is expected to increase to 11 million by 2050 if no cure would be found
by then (Alzheimer’s Association Report 2007). The report reveals the financial, social
and emotional impact that the disease has on the patients, their families, the society and
the nation. The United States Federal Government spends about $148 million annually on
the direct and indirect costs associated with Alzheimer’s disease (AD) and other related
dementia. There is no cure or vaccine therapy available for the disease that devastates the
life not only of the patient but also changes the life of their immediate family. Hence, it is
necessary to find a cure in the near future or at least outline the high risk factors for this

disease.

1.2 Pathophysiological and metabolic changes in Alzheimer’s disease

Alzheimer’s disease is a pathological condition marked by two hallmark protein
abnormalities the Amyloid Plaques commonly known as the Senile Plaques and Neuro

Fibrillary Tangles (Mattson M.P., 2004). The arnyloid plaques are deposits of the
Amyloid Beta (AB) Protein formed outside the neurons in the brain. This protein is
derived from a long, transmembrane protein, arnyloid precursor protein (APP). APP is
first cleaved by B secretase (BACEl) forming the smaller, membrane bound C-terminal
fragments of APP (CTFs), which are further cleaved by y-secretase leading to the

formation of AB protein (Vassar et a1 1999).

The neurofibrillary tangles (NFT) are paired helical filaments that are composed of the
tau protein. The Tau proteins are the microtubule-associated proteins expressed in
neuronal cells. They play an essential role in the assembling the microtubules and in
regulating axonal transport. Aggregation of specific sets of these Tau proteins is the
pathological symptom of many neuro-degenerative diseases such as Alzheimer’s disease.
When hyperphosphorylated, the Tau proteins form the tangles that disrupt basic cell
fimctions and ultimately cell death. Furthermore, the density of the NFT’s has been

shown to correlate well with the severity of dementia in AD (Bierer et al 1995).

 

Hyper-Phosphorylated Tau Disintegrating microtubule

FIglJ Protein abnornmllties in AD (Beta Amyloid formation image Courtesy of Alzheimer’s Disease
Education and Referral center). Images in thesis are presented in color.

In addition to the above mentioned pathological changes, AD is also characterized by
certain metabolic changes such as decreased cerebral glucose metabolism (46% decrease)
and reduced Adenosine Triphosphate (ATP) formation (about 19% decrease) compared
to the age matched healthy controls (Hoyer et a1 1991). Positron Emission Tomography
(PET) scans that allow in vivo imaging of the brain metabolism show decreased cerebral
glucose metabolism in patients suffering from AD. Further, this decrease correlates with
the regions that are severely affected in AD (Munch et al 1998). Few studies have
suggested that glucose hypometabolism could be an important marker for early diagnosis
of AD (Mosconi 2005). The activities of various metabolic enzymes have been shown to
be decreased in AD, mainly of pyruvate dehydrogenase (Sorbi et a1 1983) and
cytochrome oxidase (Parker et a1 1994). Furthermore, autopsy studies in AD brain shows
decreased cytochrome oxidase activity specifically in NFT—bearing neuron (Mutisya et al
1994). Finally, metabolic changes in the cerebral cortex have been suggested as one of

the early markers of AD (Peter et a1 2004).

1.3 AD and free fatty acids

Most of the research that has been conducted so far has determined certain risk factors for
AD. The prime risk factors that have been identified for AD include high fat diet, brain
trauma, Type 2 Diabetes, and ageing. High fat diet is a significant risk factor for the
development of AD and the degree of saturation of fatty acids is an important criterion in
determining the risk (Grant 1999). Further, few in vivo studies where the rats fed with
high fat diet developed AD like changes in their brain (Levin et a1 2002). Also, diabetes

mellitus is a significant risk factor for AD (Arvanitakis et a1 2002), and is characterized

by elevated plasma levels of saturated free fatty acids (F FAs). Similarly, brain trauma has

been established as an independent risk factor for AD (Guo et a1 2000).

All of these risk factors except ageing (which is a natural process accompanied by
increased oxidative stress levels) is well marked by the elevated levels of free fatty acids
in the plasma. Hence free fatty acids have a direct or indirect effect in causing AD. It is
interesting to note that in AD brain, the NFTs are enriched in fatty acids such as Palmitic
and Stearic acids. Recent finding suggests that saturated free fatty acids such as Palmitic
Acid and Stearic Acid cause pathological changes such as tau hyperphosphorylation and

BACE-l upregulation in cortical neurons (Patil et a1 2005, 2006).

1.4 AD and its effect on the different brain regions

The pathophysiological changes such as presence of Amyloid plaques and neurofibrillary
tangles are mainly confined to those regions of the brain involved in cognition, thinking
and memory i.e. the cortical and the hippocampal regions. The cerebellum is the part of
the brain involved in involuntary muscle co-ordination, equilibrium, posture and motor
learning. It is relatively spared from the changes that occur during the pathogenesis of
AD. Although there are few reports that suggest that cerebellum does get affected in
severe form of AD and there are Amyloid plaques scattered in the cerebellum in the late

stages of the disease (Y amaguchi et a1 1989).

In addition, glucose metabolism in the cerebellar region also has been shown to be

reduced depending on severity of the disease (lshii et a1 1998). Free fatty acids, before

they enter the cellular membranes are activated to their respective Co-Enzyme A (Co-A)
derivative by a group of membrane associated enzymes called acyl CoA synthetases
(Lehninger Principles of Biochemistry, Fourth Edition). The activity of the acyl CoA
synthetases has been shown to ten times higher in the cortex compared to the cerebellum
(Szutowicz et a1 1983). Interestingly, this might explain the relatively lesser effect of flee

fatty acids on cerebellar neurons in causing AD-like changes.

1.5 Aims of the study

The present study is an effort to understand the effect of saturated flee fatty acids
specifically flee fatty acids in causing pathological and metabolic changes occurring AD
(if any) in the cerebellum. It is a comprehensive study explaining the regional disparities
in expressing AD-like symptoms in the brain. It is shown in this study that the cerebellar
astrocytes are metabolically less active than the cortical astrocytes and the cerebellar
neurons also show changes similar to cortical neurons on exposure to Palmitic acid at a
higher concentration. We have investigated the mechanism through which the cerebellar
astrocytes remain less susceptible to the effect of flee fatty acids. This has led us to
determine the differences in flee fatty acid metabolism in the cortex and the cerebellum.
This could be used to determine the exact point at which the flee fatty acid metabolism

differs and hence could be used to determine right therapeutic drug targets for AD.

1.6 Organization of the thesis

In the current study, we were interested in focusing on two different aspects of cerebellar
metabolism. In chapter 2, the results of treating the neurons and astrocytes (both flom the
cortex and flom the cerebellum) are presented. Additionally, a hypothetical experiment to
demonstrate the higher activity of cortical astrocytes compared to the cerebellar
astrocytes has been described. The dose response of the cerebellar neurons to palmitic
acid conditioned astrocytes media was also studied. There exists a difference in flee fatty
acid metabolism between the astrocytes and the neurons flom the cortical and cerebellar
regions. It was identified the difference arises due to differential expression of Acyl CoA
Synthetase 3 (ACS3) — an isomer group enzymes called Acyl CoA Synthetases that

activate the flee fatty acids to enter the metabolic cycle inside the neurons and astrocytes.

Chapter 3 described the experiments that were performed to elucidate the metabolic
differences between the cortical and cerebellar astrocytes. Interestingly, since glucose
metabolism is being suggested as an early marker for AD, we studied the glucose
metabolism in cortical and cerebellar astrocytes treated with varying concentration of
Palmitic acid. It was also identified that the difference in the glucose metabolism is due to
the differential expression of GLUT 1 receptor protein — a set of glucose transporter
proteins that regulate the entry of glucose molecules through the cell walls of the

astrocytes.

CHAPTER 2

2. EFFECT OF SATURATED FREE FATTY ACIDS ON CORTICAL
AND CEREBELLAR NEURON S AND ASTROCYTES

2.1 Introduction

Elevated levels of free fatty acids is a common feature in most of the prime risk factors of
AD that include Type 2 Diabetes, hyperlipidemia and brain trauma. Few studies have
suggested a role for astroglia in mediating the oxidative stress pathway in causing AD
like pathophysiological changes upon exposure to palmitic and stearic acids (Patil et a1
2005, 2006). These studies mostly focus on the cortex or the hippocampus : the regions
of the brain that are severely affected in AD. The cerebellum : the part of the brain
involved in motor learning, equilibrium and body posture is the least affected, and this
might explain as to why the AD patients can maintain their posture and equilibrium

although the have severe problems in cognition and memory.

Hence we decided to elucidate the reason behind this regional disparity and the
mechanism by which the cerebellar neurons do not undergo such changes upon exposure
to saturated flee fatty acids. It was determined that this is due to the difference in
expression of Acyl CoA Synthetase 3 (ACS3): an isomer of acyl CoA synthetase (an
enzyme that activates flee fatty acids before its catabolism / anabolism inside a cellular

membrane).

2.1.1 Free fatty acid metabolism: role of acyl CoA synthetases

Cells cannot metabolize flee fatty acids directly. Free fatty acids are first activated to
their respective CoAcyl derivatives by acyl CoA synthetase. This step is usually a branch
point after which the FFAs could be metabolized ether anabolically or catabolically. The
anabolic pathway leads to production of Triglycerides, ceramides, phospholipids and
cholesterol synthesis, and the catabolically pathway leads to beta-oxidation in
mitochondria or the peroxisomes. Formation of a thiol ester bond between the Fatty acid
and the (thiol group of) Coenzyme A forms acyl CoA, and this reaction usually takes
place in the outer membrane of the mitochondria. Various isoforms of this enzyme has
been shown to be expressed in various levels in different parts of the body e.g liver,
kidney, brain, and muscle cells. The regulation of flee fatty acid uptake by the Fatty
Acid Transporter protein (FATP) and the long chain acyl CoA Synthetase ligase (ACSL)
has been well studied in adipocytes, and liver cells.

Cytoplasm

Fatty Acid + CoA Acyl- CoA

Outer
mitochondrial
membrane

 

Acyl- CoA

Inter mitochondrial space

Inner
mitochondrial

Fig 2.1 Free fatty acid metabolism. Images in this thesis are presented in color.

2.1.2 Fatty Acyl CoA Isomers:

Long-chain acyl-coenzyme A l synthetases (ACSs) catalyze the formation of acyl-CoA
thioesters from fatty acids, ATP and CoA (Tanaka et al., 1979). Various groups have
cloned and characterized the different isoforms in different species including humans
(Ghosh et al., 1995; Minekura et al., 1997). Interestingly, five different enzymes with a
common structure have been characterized in the rat, and each ACS appears to have a

marked tissue distribution and a completely different regulation flom those of the others

(Suzuki et al., 1995).

For example, the rat ACSZ and ACS3 are predominantly expressed in brain (Fujino et al.,
1996) whereas the Rat ACSl is cormnonly found in liver, heart, and adipose tissue
(Suzuki et al., 1990). Rat ACS4 and ACSS are highly expressed in steroidogenic tissues
and in small intestine, respectively (Kang et al., 1997). Irrespective of the organs or
tissues in which they are highly expressed, the biological significance of all the five
isoforms cannot be overlooked; their importance lies in the fact that all of them are
essential in lipid metabolism in the each of the tissues listed above. The isoforms that
have different substrate preferences are expressed in different tissues and are present in
different subcellular locations. The expression of each of the rat isoforms is likely to be
regulated independently. For example, ACSl mRNA in different tissues is variously
upregulated by PPARa and PPARy (Suzuki et A]. 1995, Martin et al 1997) and by
insulin, ACSZ in brain is upregulated by PPARB (Kansara, et al 1996). These findings
suggest that each of these isomers have distinct functions, possibly directing fatty acids to

different metabolic pathways.

11

In human skin fibroblasts, triacsin C, which inhibits ACSl and 4 but not ACS5, blocks de
novo glycerolipid synthesis but does not affect lysophospholipid reacylation (Igal et a1,
1997), suggesting that different ACS isoforms generate independent acyl-CoA pools
which have specific fates. In humans, mutations in the FACL4 gene inactivate the
enzyme are linked to mental retardation (Meloni et al, 2002), reasserting the unique

function of this isoforrn in brain.

In our study we first determined if there is indeed any variation in the expression of tau
and BACE-1 between the two regions of the brain namely the cortex and the cerebellum.
If there does exist a difference in the expression, we could conclude that this could be
only due to the differential regulation of flee fatty acid metabolism (specifically saturated
flee fatty acids) in the two brain regions. Since, it has been shown that astrocytes and not
neurons regulate the flee fatty acid metabolism in the brain we determined the effect of
saturated flee fatty acids (Pahnitic Acid) on the neurons and the astrocytes of both the

brain regions.

12

2.2 Materials and Methods

2.2.1 Cell Culture

2. 2.1.1 Cerebellar Neurons and Astrocytes Culture

Cultures of dissociated cerebellar granule cells were isolated flom seven-day-old Sprague
Dawley rat pups and cultured according to the methods established by Trenkner with
some modifications (Trekner 1991). Cerebella were dissected out, fleed of meninges, and
incubated 15 minutes at room temperature in Ca-and Mg—flee Hanks' balanced salt
solution (CMF-HBSS) containing 1% trypsin. The cell solution was washed with 0.016%
Deoxyribose Nuclease (Dnase) solution. The tissues were centrifiiged for at 3000 rpm
and the supernatant was discarded. The cerebella pieces were resuspended in the DNase
solution and triturated using a glass Pasteur pipette. The cell solution was centrifuged
through a gradient buffer containing 4% bovine serum albumin (BSA) in CMF-HBSS for
5 min at 3000 rpm. The supernatant was discarded and the cells were resuspended in
DMEM (Invitrogen) supplemented with 10% Fetal Bovine Serum, 25 mM KCl and
0.001mM Insulin flom Bovine Pancreas (Sigma, MO). The cells were plated at a density
of 1 x 106 cells per well in poly —l-Lysine (Trevigen Inc, MD) coated 6-well plates.
Cultures were kept in a humidified atmosphere of 5% C02, 95% air at 37°C in a granule
cell medium. Three days after incubation (37°C, 5% C02), the medium was subsequently
replaced with 2ml of granule cell medium supplemented with 5 uM cytosine-B-
arabinofuranoside (Arac flom Calbiochem, CA, USA). After 2 days, the neuronal culture
was switched back to granule cell medium without Arac. The experiments were
performed on 6- to 7-day-old culture. One-half of the culture medium was exchanged

with flesh medium twice a week.

13

To obtain astroglial cultures the cells were replaced with the medium without addition of

Arac on the second day from the day of isolation.

2.2.1.2 Cortical Neurons and Astrocytes Culture

Cultures of primary cortical neurons and astrocytes were obtained flom one-day-old
Sprague—Dawley rat pups according to the published methods as described by Chandler
et a1 with some modifications (Chandler et al 1993). The cells were plated on poly-l-
lysine-coated, six-well plates at the concentration of 2 X 106 cells per well in flesh
cortical medium DMEM (Invitrogen, CA) supplemented with 10% horse serum (Sigma,
M0), 25 mM glucose, IOmM HEPES (Sigma), 2mM glutamine (Sigma), 100 IU/ml
penicillin, and 0.1 mg/ml streptomycine. For neuronal cultures two days after incubation
(37 °C, 5% C02), the medium was replaced with 2 ml of cortical medium supplemented
with 5 uM cytosine-B-arabinofirranoside (Arac flom Calbiochem, CA, USA) to prevent
the proliferation of astrocytes and glial cells. After 2 days, the neuronal culture was

switched back to cortical medium without Arac.

The experiments were performed on 6- to 7-day-old culture. To obtain primary cultures
of astroglial cells, the cortical cells flom one-day-old Sprague—Dawley rat pups were
cultured in DMEM/Ham's F12 medium (1:1), 10% fetal bovine serum (Invitrogen),
100 IU/ml penicillin, and 0.1 mg/ml streptomycine. The cells were plated on poly-l-
lysine coated, 6-well plates at the concentration of 2 X 106 cells per well. Cells were

grown for 8—10 days (37 °C, 5% CO2) and culture medium was changed every 2 days.

14

2.2.2 Free fatty acid treatment

Palmitic acid was used for the experiments as the representative fatty acids for the
saturated fatty acids since it is the most abundant fatty acids of their class in the plasma.
In case of severe brain injury, the levels of palmitic acid and Stearic acid in the brain
increase to approximately 180 M and 350 M respectively (Lipton 1999). Traumatic
brain injury has been established as a risk factor for AD and hence a concentration of 0.2-
0.25 mM has been used in the present study and a control of 4% BSA in the cortical

neuronal medium was used. The media in the seven to ten day old cultures

2.2.3. Western Blotting

To examine the effects of flee fatty acids induced changes in BACEl expression on the
cerebellar neurons, the cells were treated either with 0.2mM and 0.5mM palmitic acid, or
4% BSA (control) for 24 hours. For western blotting, the cells were washed three times
with ice-cold TBS (25mM Tris, pH 8.0, 140mM NaCl, and 5mM KCl) and lysed for 20
min by scraping into ice-cold Radioimmunoprecipitation assay (RIPA) buffer [1% (v/v)
Nonidet P40, 0.1% (w/v) SDS, 0.5% (w/v) Sodium deoxycholate, 20mM Tris, pH 7.4,
lSOmM NaCl, IOOmM NaF, lmM Na3V04, lmM EDTA, lmM EGTA, and lmM PMSF.
The cell lysate was obtained by centrifugation at 13,000 rpm for 20 min at 4°C. The total
protein concentration was measured by bichinconic acid assay (BCA). Equal amounts of
total protein flom each condition were run at 200V on 10% Tris-HCl gels for
phosphorylated tau and actin. The separated proteins were transferred to nitrocellulose
membranes for l h at 100V and incubated at 4°C overnight with the appropriate primary

antibodies [1:1000 BACE l (Chemicon, USA) 1:200 Actin]. Blots were washed three

15

times in PBS—Tween (PBS-T) and incubated with appropriate HRP-linked secondary
antibodies diluted in PBS-T for l h. After three washes in PBS-T, blots were developed
with the Pierce SuperSignal West Femto Maximum Sensitivity Substrate and imaged

with the Bio- Rad ChemiDoc.

To examine the F FA-induced hyperphosphorylation of tau, primary rat cerebellar neurons
were left untreated or treated with 0.25 mM of either palmitic or 4% BSA (control) for
24 h. After 24 h, the cells were lysed and Western blot analysis was performed to
determine the cellular levels of hyperphosphorylated tau using a specific primary
antibody AT8 (Pierce Biotechnology, IL). To determine the relative expression levels of
the ACS3, cerebellar astrocytes and neurons; cortical astrocytes were cultured, lysed and
Western blot analysis was done using primary polyclonal antibody for ACS3 (1:200

ACS3, Affinity Bioreagents Inc).

2.2.4 Measurement of Reactive Oxygen Species

Reactive oxygen species (ROS) were detected by staining with the dye 5-(6)-
chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCF A, Molecular Probes,
0R). Confluent cerebellar and cortical astrocytes were initially treated with 0.25uM
palmitic acid or 4% BSA and the conditioned media flom the respective astrocytes were
transferred to the neurons. In other words, the conditioned media flom the cortical
astrocytes were transferred to the cortical astrocytes and the media flom the cerebellar
astrocytes were transferred to the cerebellar neurons. The treated neurons were incubated

for 30 min at 37 °C with 2 uM H2DCFA in DMEM (Catalog number 10313, Invitrogen)

l6

without serum. The cells were then washed three times with PBS and the fluorescence
was read flom a fluorescence microscopy (Emission: 530 nm Excitation: 485 nm). To
minimize the background due to cell clustering, before the incubation with the dye, the
cells were kept in PBS and the fluorescence was noted (blank values). The absolute
values for the fluorescence due to ROS formation was obtained after subtracting the

blank values flom the ones obtained after dye incubation.

2.2.5 Cell Proliferation Assay

The cerebellar astrocytes cultured in a 6-well plate were trypsinized and the cells were
counted using a hemacytometer. The final density of the cell solution was made to be
equal to approximately 105 cells per ml by diluting the cell solution with the astrocytes
media. 100 [ll of this cell solution was plated on the 96 well plate and incubated for 6
hours so that the cells are completely attached to the surface. The media was carefully
removed and the cerebellar astrocytes were treated with 0.25, mM and 0.5 mM palmitic
acid (PA) or with 4% BSA (control). The cell numbers were quantified with a
Proliferation Assay (CyQuant NF Proliferation Assay, Invitrogen) using a fluorescence
microplate reader. The CyQUANT NF assay is based on measurement of cellular DNA
content via fluorescent dye binding. Because cellular DNA content is highly regulated, it
is closely proportional to cell number. The extent of proliferation is determined by
comparing the Absorbance values of the samples treated with Pahnitic acid with

untreated controls using a fluorescence spectrophotometer.

17

2.3 RESULTS AND DISCUSSION

2.3.1 Effect of palmitic acid on cerebellar neurons

There was no change in the levels of phosphorylated tan in rat cerebellar neurons treated
directly with pahnitic acid, as compared to controls (Fig 2.2). This could be because the
neurons do not actively take up and metabolize fatty acids compared to the astrocytes
(Blazquez et a1 2000). This shows that the physiological concentration of palmitic acid

during the progression of the disease i.e. at 0.25 mM do not have any effect on the

cerebellar neurons.
BSA 0.25mM
PA

‘u‘ AT8

____., “ Beta-Actin

Fig 2.2 Direct treatment of cerebellar neurons with increasing concentration of palmitic acid. Primary
rat cerebellar neurons were treated for 24 h with 0.25 mM of either palmitic acid (PA) or with 4% bovine
serum albumin (BSA), carrier for FFAs (control). Cell lysates flom fatty acid-treated and control cells were
imrnunoblotted with AT8 antibody, which recognize phosphorylated tau. B-actin is shown as a marker for
equal protein loading.

A number of studies suggests a central for astroglia mediated oxidative stress in causing
pathological changes observed in AD (Patil et al 2005). Therefore, in the present study,
we treated the rat cerebellar astrocytes with 0.25 mM palmitic or 0.5 mM pahnitic acid
for 24 hours and transferred the conditioned media to treat the cerebellar neurons for
24 hours. The conditioned media flom FF A-treated astrocytes did not cause
hyperphosphorylation of tau in the cortical neurons (Fig 2.3), as observed by Western

blotting with AT8 antibody.

18

AT8 recognizes tau phosphorylated at Ser202 and Thr205 (Goedert et al 1995). Ser202 is
one of the abnormal phosphorylation sites of the hyperphosphorylated tau associated with

NFTs in AD (Gong et a1 1994).

BSA 0.25mM 0.5mM
PA PA

.2 AT8

“ —- all. Beta-Actin

Fig 2.3 Treatment of cerebellar neurons with conditioned media from cerebellar astrocytes. Primary
rat cerebellar astrocytes were treated for 24 h with 0.25 mM , 0.5 mM palmitic acid (PA) or with 4%
bovine serum albumin (BSA), carrier for FFAs (control). The conditioned media flom the three conditions
were transferred to the neurons. Cell lysates flom fatty acid-treated and control cells were immunoblotted
with AT8 antibody, which recognize phosphorylated tau. B-actin is shown as a marker for equal protein
loading.

The conditioned media flom the cerebellar astrocytes did not cause any change in the tau
phosphorylation levels in cerebellar neurons. Previous studies show that conditioned
media flom pahnitic acid treated cortical astrocytes cause tau hyperphosphorylation and
BACE] upregulation in cortical neurons (Patil et a1 2005, 2006). Hence, we studied the
effect of conditioned media flom palmitic acid treated cortical astrocytes on cerebellar

neurons (Fig 2.4).

To further establish that the cortical astrocytes and not cerebellar astrocytes are involved
in causing AD-related pathological changes, we treated the cerebellar astrocytes with
0.25mM palmitic acid or with 4% BSA, and transferred the media to cortical neurons.
Tau phosphorylation levels (Fig 2.5) and BACEl levels (Fig 2.6) were determined by

Western blotting methods using specific primary antibodies.

19

CT-A

PA
CB-A
Control
CB-A
PA

Control

CT-A

AT-8

Beta-Actin

250

200~

1502

Phosphorylated Tau
(7. Control)
§

8

 

 

CT- 0 CT-Ffi CB- 0 CB- PA

Fig 2.4 FFA induced tau hyperphosphorylation in cerebellar neurons. Astrocytes from the cortical and
cerebellum were treated with 0.2 uM Palmitic Acid (PA) or 4% BSA (Control). The conditioned media
flom the astrocytes were transferred to the cerebellar neurons (24 hours treatment). A. Western Blot
Analysis of hyper-phosphorylated tau using AT-8 antibody in cerebellar neurons B. Quantification of
Western Blot Analysis of hyper-phosphorylated tau using phospho—specific AT-8 antibody in cerebellar
neurons. Histograms corresponding to AT8 blots represent quantification of the relevant bands' intensities.
All the histograms were nomialized based on the control data of each individual set of experiments. Data
represent meaniSD. of three independent experiments. Student’s t test was used to determine the
differences between treatment groups (* p < 0.01 compared to the control, # p < 0.05 compared within

treatment groups). Images in this thesis are presented in color.

20

Control
CT-A
PA

Control

C T-A
CB-A
PA

4
8
“an“. ~ ._ .2. p.‘

“--'¢'

AT-8

Beta-Actin

250
I ‘k

3

, ml

1-

'0:

§g150<

—=

be

°°1oo

fi.\’

my

2

a. so
or. .

 

 

Fig 2.5 FFA induced tau hyperphosphorylation in cortical neurons. Astrocytes from the cortical and
cerebellum were treated with 0.2 uM Palmitic Acid (PA) or 4% BSA (Control). The conditioned media
flom the astrocytes were transferred to the cortical neurons (24 hours treatment). A. Western Blot Analysis
of hyper-phosphorylated tau using AT-8 antibody in cortical neurons B. Quantification of Western Blot
Analysis of hyper-phosphorylated tau using phospho-specific AT-8 antibody in cerebellar neurons.
Histograms corresponding to AT8 blots represent quantification of the relevant bands’ intensities. All the
histograms were normalized based on the control data of each individual set of experiments. Data represent
meaniSD. of three independent experiments. Student’s t test was used to determine the differences
between treatment groups ("‘ p < 0.01 compared to the control, # p < 0.05 compared within treatment
groups). Images in this thesis are presented in color.

21

Control

4
a:

 

 

BACE-1 expression level (% Control)
9 a a a a S E; 2% §
[ A .

l
i

   

CT-PA

Fig 2.6 FFA induced BACE-1 upregulation In cortical neurons. Astrocytes from the cortical and
cerebellum were treated with 0.2 uM Palmitic Acid (PA) or 4% BSA (Control). The conditioned media
from the astrocytes were transferred to the cortical neurons (24 hours treatment). A. Western Blot Analysis
of BACE-1 using specific antibody in cortical neurons B. Quantification of Western Blot Analysis of
BACE-1 in cerebellar neurons. Histograms corresponding to BACE-l blots represent quantification of
the relevant bands’ intensities. All the histograms were normalized based on the control data of each
individual set of experiments. Data represent meaniSD. of three independent experiments. Student‘s t test
was used to determine the differences between treatment groups (* p < 0.05 compared to the control, # p <
0.05 compared within treatment groups). Images in this thesis are presented in color.

22

2.3.2 Regional disparities in expressing AD-pathological changes:

Few studies report the activity of the fatty acyl CoA Synthetase enzyme to be 10 times
higher in the cortex compared to that of the cerebellum (Szutowicz et a1, 1980). This
suggests that the cerebellum is distinct flom the cortex and the hippocampus in
expressing the neuropathological changes because of its distinct activity in metabolizing
Free Fatty Acids (FFA). Activity and expression levels are two different aspects in
governing the reaction catalyzed by an enzyme. Therefore, we investigated the relative
expression levels of the acyl CoA synthetase in the neurons and the astrocytes of both the
cortex and cerebellum. It was found that the astrocytes generally have higher expression
levels of ACS3 compared to the neurons. It is interesting to note that the cortical
astrocytes have the highest expression levels of ACS3 (Fig 2.7), which explains its higher
activity in metabolizing flee fatty acids and causing the pathological changes in AD.
Hence, this could be a reason as to why cerebellum is less susceptible to oxidative stress

because of FFA metabolism.

C B-N CT-N CB-A CT-A

.. .- err-Ii ........-. I—

AC S3

~ — “- ’
Beta-Actin

23

ACS3 protein levels
8

CT-N CB-A

 

     

Fig 2.7 Relative expression levels of ACS3 in cortical, cerebellar astrocytes and neurons. A. Western
Blot Analysis of ACS3 using ACS3 specific antibody B. Quantification of Western Blot Analysis of ACS3
protein levels in cerebellar and cortical neurons, cerebellar and cortical astrocytes. Histograms
corresponding to ACS3 blots represent quantification of the relevant bands’ intensities. Beta-Actin was
used as a marker for equal protein loading. Images in this thesis are presented in color.

2.3.3 Involvement of Reactive Oxygen Species (ROS)

Fatty Acids induces the gene for fatty CoA synthetase in adipocytes (Gargiulo et a1,
1999). This would lead to activation of Palmitate to Palmitoyl-CoA. If this occurs in
neurons then it would increase the utilization of flee fatty acids. In pancreatic beta cell
line INS-1, Palmitate induces activation of Camitine Palmitoyltransferase gene thereby
increasing beta-oxidation and reducing the glucose oxidation. We know that Protein
Kinase-C activation may lead to activation of NADPH oxidase and increase ROS
production in vascular cells. Fatty acids are shown to be involved in PKC activation,
leading to ROS production (Duval et a1, 2003). If this enzyme is not activated in

cerebellum, this might spare the cerebellum flom the effects of oxidative stress.

24

There are few cell systems in which Ceramide synthesis has been shown to induce
apoptotic pathways independent of ROS generation. In human granulose cells, palmitic
and stearic acid has been shown to cause apoptosis independent of ROS production or
Nitric Oxide (NO) production. In insulin secreting cells, palmitic acid causes ROS
generation and apoptosis independent of inducible Nitric Oxide Synthase (iNOS)
upregulation (Shimabukuro et al, 1998). These results indicate that saturated fatty acids
induce apoptosis due to metabolism of the respective acyl CoA derivative. This is
important in our study, as the acyl CoA synthetase is 10 times more active in cortex
compared to the cerebellum. We have confirmed that the ROS species produced in the
cortical neurons treated with conditioned media from palmitic acid exposed astrocytes to
be higher than the cerebellar neurons treated in the similar manner with conditioned

media from cerebellar astrocytes (Fig 2.8).

Control PA-(‘A

Control

  

25

14 «

12 _ ICB-C ICB-PA
DCT—C OCT-PA .

10 T ..1

Fluorescence units

 

 

 

 

Time (hours)

Fig 2. 8 Intracellular ROS accumulation in cerebellar and cortical neurons. A. Fluorescence
Microscopy images of intracellular ROS accumulation in neurons. a. Cerebellar neurons treated with
media from control cerebellar astrocytes b. Cerebellar neurons treated with the media from PA conditioned
cerebellar astrocytes c. Cortical neurons treated with media from control cerebellar astrocytes d. Cortical
neurons treated with the media from PA conditioned cortical astrocytes B. Quantification of ROS produced
in cortical and cerebellar neurons. Cortical and cerebellar astrocytes were treated with 0.25mM palmitic
acid or 4% BSA (control). The conditioned media from cortical astrocytes were transferred to the cortical
neurons and the ROS produced was measured in fluorescence units by using an oxidant-sensitive dye
(H2=DCFA, Molecular Probes) for 48 hours in 6 hours intervals. Similarly the ROS levels were quantified
using intensity of fluorescence produced on incubation with the dye. Thus a time dependent ROS
production was investigated in the cortical and cerebellar astrocytes. Results presented are expressed as
mean +/- SD of two independent time series experiments. Images in this thesis are presented in color.

We have shown that cortical astrocytes and not cerebellar astrocytes cause tau
hyperphosphorylation and BACEl upregulation in cortical and cerebellar neurons. This is
due to the difference in relative expression of the enzyme ACS3 a free fatty acid
activator. We have shown that it is highly expressed in cortical astrocytes compared to
the cerebellar neurons. It is interesting to note that most of the isomers of this enzyme are

highly expressed in proliferating cells such as astrocytes, leydig cells and in lymphocytes.

26

So what causes this marked difference in astrocytes metabolism upon treatment with free
fatty acid. It has been shown that de novo synthesized ceramides causes apoptosis in
astrocytes in a time dependent manner (Blazquez et a1 2000). Further there a number of
studies that suggests that ceramide plays an important role in the control of cell fate in the
central nervous system under different pathophysiological situations. Thus, elevations of
intracellular Ceramide levels, which may in turn be related to the induction of apoptotic

cell death, have been shown to occur in neurodegenerative disorders.

The cerebellar astrocytes must possess a different mechanism to control and regulate the
levels of intra-cellular ceramide. On the other hand, cortical astrocytes have been
suggested to produce ceramides on treatment with pahnitic acid and this causes oxidative
stress in the neighboring neurons eventually leading to AD-like pathology (Patil et al
2006). The major metabolic routes that contribute to the maintenance and variation of the
cellular ceramide include ceramide biosynthesis, by de novo pathway or sphingosine
recycling, ceramide formation fi'om complex sphingolipids degradation and ceramide
catabolism. It is worthwhile to know that any mitogenic stimulus is followed by changes
in ceramide levels. This is usually one of the key step in a series of reactions that are to
follow. Furthermore, proliferation of astrocytes requires low cellular levels of ceramides
(Riboni et a1 2002). In particular, mitogenic stimuli, such as basic fibroblast growth
factor (bFGF), rapidly down regulate the cellular levels of ceramide by stimulating
sphingomyelin synthase. Moreover, although ceramide may significantly contribute to
cell death in neurological disorders, the possible involvement of ceramide synthesis de

novo in neural cell death / differentiation is yet unknown.

27

Hence, this suggests that the ceramides secreted by the cerebellar astrocytes could be at
very low levels and hence this might cause the astrocytes to proliferate instead of
inducing oxidative stress. This could be the reason as to why the cerebellum do not show
pronounced neuro pathological changes as that of the cortex and hippocampus in
progression of Alzheimer’s disease. Interestingly, The central role of ceramides in AD is
supported by the elevated levels of ceramides found in AD brain as compared to healthy
controls; ceramide levels are higher in the affected regions i.e. cortex and hippocampus

as compared to the unaffected areas i.e.cerebellum (Cutler et a1 2004).

To determine if the treatment of the cerebellar astrocytes do indeed cause proliferation as
opposed to cell death we treated the cerebellar astrocytes with 0.25mM and 0.5 mM
palmitic acid (PA) or with 4% BSA( control) and quantified the number of cells with a
Proliferation Assay (CyQuant NF Proliferation Assay, Invitrogen) using a fluorescence

microplate reader.

28

6000 — a

5000 ~

4000 -

3000 -

2000 -

Fluorescence units

1000 e

 

 

 

CB-C CB-PA-0.25 CB-PAQS

Fig 2.9 Proliferation of cerebellar astrocytes in the presence of palmitic acid. Astrocytes from the
cerebellum were treated with 0.2 uM Palmitic Acid (PA) or 4% BSA and the proliferation was measured in
fluorescence intensity units using the CyQuant NF Proliferation Assay (’p < 0.05 compared to the control).
Images in this thesis are presented in color.

The cerebellar astrocytes indeed proliferate on treatment with palmitic acid for 24 hours
(Fig 2.9). This explains the unique machinery of the cerebellum in preventing the
neuronal and astrocytic cell death in AD or rather when the brain is under oxidative

stress.

29

CHAPTER 3

30

3. EFFECT OF PALMITIC ACID IN GLUCOSE METABOLISM IN
CEREBELLAR ASTROCYTES

3.1 INTRODUCTION

3.1.] The concept of energy metabolism in brain

The brain consumes about 20% of the total energy produced in the body, which is
relatively a large compared to its size. Most of the available energy is spent on pumping
ions such as Sodium and Potassium across cellular membranes. This process is critical for
the proper functioning of the central nervous system (CNS) because any abnormalities in
energy metabolism in the brain will potentially have a harmful effect on the proper

functioning of the tissues and the other physiological systems.

Most of the energy supplied to the brain comes through metabolism of glucose.
Therefore, metabolic regulation and control of glucose levels is of central significance.
Oxidation of the glycolytic product pyruvate yields the energy rich compound acetyl-
CoA, which is used for production of Adenosine Tri-Phosphate (ATP), Acetyl Choline
(ACh) and cholesterol. ATP is produced when glucose is oxidized in the Tri-Carboxylic
Acid (TCA) cycle, which is used to drive the active ion transport across the membranes.
ATP guarantees most cellular and molecular processes such as protein synthesis,
maintenance of ion homeostasis, folding sorting and degradation of proteins and even
maintenance of synaptic transmission. ATP is known to inhibit Insulin-degrading enzyme
(lDE) activity, which is shown to be over-expressed in AD brain in accordance with

decreased ATP levels (Camberos et a1 2001).

31

Glucose is critical for the production of acetyl-CoA, a precursor of acetylcholine
(Ragozzino et al, 1996), which is further confirmed by the fact that a decrease in glucose
concentrations result in a decrease in brain acetylcholine (Gibson et a1 1976). Hence, it is

interesting to study the effect of glucose in affecting the proper functioning of the CNS.

Alzheimer's brain

 

Normal brain Alzheimer's brain
Pot scam (glueooo utilization)

Fig. 3.1 Positron emission Tomography images of brain glucose utilization in a normal and an AD
patient (Mattson et al, 2004). These images are currently used to distinguish AD from other types of
dernentias. The metabolic degeneration occurring in the brain of an AD patient could be accurately
determined by PET images. Higher metabolic rate is indicated by the red color and the violet indicates
lower metabolism. As shown above. in AD the cortex shows relatively lower metabolism compared to the
age matched control as indicated by the blue spread over the entire brain section. In AD patient, the lower
area of the brain (cerebellum) is shown in red indicating that the cerebellum rermins relatively unaffected
compared to the normal brain. Images in this thesis are presented in color.

32

3.1.2 Alzheimer’s Disease and Type-2 Diabetes
Epidemiological evidence has shown that type 2 diabetes is a risk factor for AD (Ott et
al., 1999). Since diabetes is a disease of glucose metabolism, what is the functional

relationship between the two and how does type 2 diabetes increase the risk of AD?

Until recently, it was thought that insulin was not relevant to brain glucose metabolism.
However, it is now agreed that insulin can cross the blood brain barrier (BBB) and that a
small subset of CNS neurons in areas involved in cognitive function express insulin
sensitive GLUT 4 and 8 receptors. The brain can also synthesize a small amount of
insulin in situ (Watson and Crafi, 2003). In addition, there is a physiological coupling
between glucose and steroid levels via the hypothalarnic—pituitary—adrenal axis, and
abnormalities in peripheral glucose metabolism can indirectly elevate glucocorticoid
levels. Glucocorticoids do enter the brain where they can alter both glucose metabolism

and nerve cell viability (Sapolsky, 2000).

Although the molecular mechanisms are not yet understood, a number of studies suggest
that elevated peripheral glucose levels and insulin resistance have a negative effect on
memory and nerve cell viability. (1) Insulin resistance leads to a higher mortality rate
from ischemia (LeMay et al., 1988). (2) AD patients with lower insulin levels have lower
cognitive skills than those with normal levels (Craft et al., 1996). (3) The administration
of insulin and glucose together enhances the memory of AD patients to a greater extent
than glucose alone (Manning et al., 1993; Craft et al., 1996). (4) People with type 2

diabetes have impaired learning and more rapid memory decline with age than controls

33

(Ryan and Geckle, 2000). If one makes the assumption that there is a causal link between
insulin and glucose utilization in the brain, then it follows that glucose metabolism is

associated with cognitive skills.

3.1.3 Abnormalities in brain glucose metabolism and its impact on cellular and
molecular mechanisms in sporadic dementia of Alzheimer type

We know that the onset of AD is usually associated with Amyloid plaques and
neurofibrillary tangles but recently its has been shown that reduced cerebral glucose
utilization is the most consistent feature of an AD brain (Schubert, 2005). Type 2
Diabetes which is a risk factor for AD is also characterized by reduced glucose uptake
and hence increased blood glucose concentration (Bergman, 2000). Type 2 Diabetes is
well studied compared to AD and it has been shown that there is a direct effect of Free
Fatty Acids on muscle metabolism. For example, elevated FFAs are shown to suppress
glucose uptake (Bergman, 2000), and the fact that Blood Brain Barrier (BBB) is not a
barrier for FF As (Abel, Handbook of Neurochemistry,l982) suggests a direct / indirect
link between elevated levels of plasma F F As and decreased cerebral glucose metabolism.
Also, free fatty acids stored in the from of triacylglycerols in adipocytes, can also serve
as energy sources (Lehninger Principles of Biochemistry, Edition 2). Thus, this form of
free fatty acid can be utilized for supplementing the energy demand from peripheral
tissues. Taking all these ideas together it is worthwhile to study effect of elevated levels

of FF As on the neuronal/ astroglial glucose uptake.

34

3.2 Materials and Methods :

3.2.1 Cell Culture and free fatty acid treatment

The astrocytes from two different brain regions were cultured as described in the
previous chapter (Chapter 2). For the experiments, seven to ten day old cultures were
used. The cortical and cerebellar astrocytes were treated with 0.2 mM, 0.5 mM palmitic

acid or with 4% BSA (control) for 24 hours.

3.2.2 Measurement of glucose uptake by the astrocytes

To calculate the glucose uptake in particular, their concentrations in the media were
measured by using enzymatic glucose assay (Stanbio Laboratories, TX). The glucose
uptake was calculated by using the differences between the glucose concentrations in the
media before and after the treatment. The media was collected and centrifuged for 5
minutes at 1730 rpm to eliminate the presence of any cell debris which might interfere

with the assay.

3.2.3 Western Blotting

F or Western blot analysis, cell lysates were obtained as described in previous chapter
(Chapter 2). The total protein concentration was measured by BCA protein assay. Equal
amounts of total protein from each condition were run at 200V on 10% Tris-HCl gels for
1 hour. The separated proteins were transferred to nitrocellulose membranes for 1 h at
100V and incubated at 4°C overnight with the appropriate primary antibodies [1 :1000
GLUTl, 1:200 Actin]. Blots were washed three times in PBS—Tween (PBS-T) and

incubated with appropriate HRP-linked secondary antibodies diluted in PBS-T for l h.

35

After three washes in PBS-T, blots were developed with the Pierce SuperSignal West

Femto Maximum Sensitivity Substrate and imaged with the Bio- Rad ChemiDoc

3.3 Results and discussion

The complexity of nutrient utilization in the Central Nervous System has been a major
field of research since brain utilizes about 20% of the glucose as a energy source. It was
believed that glucose was the neuronal fuel under normal conditions, whereas ketones
could substitute for glucose under hypoglycemic conditions (Cryer, 1981). This view has
been challenged recently when it was shown that lactate could substitute as a fuel, during
energy times of energy deficit. It has been shown that lactate is an energy substrate for
neuronal functional recovery during hypoxia-ischemia (Schurr et a1 1997). Insulin-
induced hypoglycemia increases lactate utilization in the brain (Hellman et al 1992). In
fact, the astrocyte-neuron lactate shuttle hypothesis suggests that neurons may actually
prefer glial-derived lactate to glucose as a firel for neuronal activity (Magistretti et al
1999). According to the hypothesis, neuronal activity increases extracellular glutamate,
which stimulates glial anaerobic glycolysis and converts glucose to lactate. Lactate is
transported out of glia by monocarboxylate transporter 1 and into neurons by
monocarboxylate transporter 2, where it is metabolized to pyruvate via lactate
dehydrogenase-1. This pyruvate then enters the neuronal tn'carboxylic acid cycle to
generate ATP (Chih et a1 1991). In support of this hypothesis, a recent study showed that
the human brain preferentially uses circulating lactate to sustain metabolism even at a
normal glucose concentration (Smith et al, 2003). Fatty Acids can act as neuronal fuels

(Kim et al, 2003).

36

We have hypothesized a correlation between the glucose metabolism and the AD related
pathological changes. To test the hypothesis we treated the astrocytes cultured from the
cortex and the cerebellum with palmitic acid and determined their glucose uptake. The
experiment results suggest a significant decrease in glucose uptake upon Palmitic Acid
treatment in cortical and not cerebellar astrocytes compared to control. We treated the

primary cortical and cerebellar astrocytes with increasing concentrations of palmitic acid:

    

 

 

 

 

 

0.25 mM and 0.5 mM.
12
#
#
1o —
'i'l
it
a as '-’
a i
r 6*
L!“
E 4 l J. ,]
.3371.
2 x
o “H ~ ‘2 .. . . , 7 .
CT-A-C C T-A-PA- CT—A-PA-0.5 CB-A-C C B-A-PA- CB-A-PA-
0.25 0.25 0.5

Fig 3.2 PA downregulates glucose uptake by cortical and not cerebellar astrocytes. The cortical and
cerebellar astrocytes were treated for 24 hours with 0.25 mM of PA, or 0.5 mM PA or 4% BSA (control).
PA treatment significantly decreased glucose uptake by cortical astrocytes and .did not affect the cerebellar
astrocytes, in a concentration dependent rmnner. Data represent meaniSD. of four independent
experiments and the glucose uptake values are normalized based on the protein concentration determined
by BCA Assay. Data represent meaniSD. of six independent experiments. Student’s t test was used to
determine the differences between treatment groups (" p < 0.05 compared to the control, # p<0.07
compared within treatment groups.) Images in this thesis are presented in color.

37

Mechanism of Palmitate induced Glucose hypometabolism

AD brain is characterized by significant reductions in GLUT] levels, and also the disease
severity is associated with progressive decline in GLUTl gene expression (Simpson et a1
1994, Jacobs et al 1997). We hypothesized that the observed, palmitic acid (PA)-induced
abnormal glucose metabolism may be due to the potential effect of PA on the level of
astroglial glucose transporter (GLUTl). Consequently, the level of astroglial glucose
transporter (GLUTl) was significantly down-regulated in PA-treated astroglia as
compared to the untreated ones. Thus, the observed downregulation in glucose uptake by
astroglia in presence of palmitic acid may be attributed to the PA-induced

downregulation of GLUTl levels in astroglia.

Astrocyte

     
  

GLUT- 1

Cellular membrane

Fig 3.3 GLUT-l proteins in cellular membranes. GLUT-1 are a member of transmembrane proteins
specifically expressed in cortical astrocytes that facilitate the transport of glucose into the cell. Images in
this thesis are presented in color.

38

GLUT-1

~ — - - '1 _ Beta-Actin

 

 

 

B.
_ 160
O
‘E 140
5
g 120 4
2’ 100 «
.2
g 80 «
I-
E‘ 60
Lil
v-i 40 _
‘5
6: 20
0 ~ . a
CT-C CT-PA=0.25 CT-PA=0.5 CB-C CB- CB-PA=0.5
PA=0.25

Fig 3.4 FFA induced GLUT-l downregulation in cortical but not in cerebellar astrocytes. Astrocytes
from the cortical and cerebellum were treated with 0.25 mM Palmitic Acid (PA) or 0.5 mM PA or 5%
BSA (Control). A. Western Blot Analysis of GLUTl expression using GLUTl specific antibody in cortical
and cerebellar astrocytes B. Histograms corresponding to GLUT! blots represent quantification of the
relevant bands’ intensities. All the histograms were normalized based on the control data of each individual
set of experiments. Data represent meaniSD. of three independent experiments. Images in this thesis are
presented in color.

As hypothesized, the cortical astrocytes show a PA dependent GLUT-l downregulation
which explains as how the cerebral glucose metabolism is regulated. Further studies

could be undertaken to study the regulation of GLUT-l expression by PA.

39

CHAPTER 4

4O

4. Conclusions and future work

4.1 Conclusions

Saturated free fatty acids such as Stearic acid, and Palmitic acid has shown to induce tau
hyperphosphorylation and BACEl upregulation (two key events in progression of AD) in
primary cortical neurons treated with the conditioned media from free fatty acid exposed
primary cortical astrocytes. Primary astrocytes from cerebellum do not induce such
protein abnormalities, which might explain as to why the cerebellum is relatively spared
from neuronal degeneration in AD. Thus, the astrocytes have emerged as the cells that

play an important role in neuronal metabolism irrespective of the region of the brain.

One of the key metabolites in free fatty acid metabolism are the ceramides and they have
emerged as one of the intra-cellular molecules that could potentially act as a therapeutic
drug target for AD. Hence, we focused on the effect of free fatty acid on the part of the
brain that is least affected. It interesting to note that the levels of ceramide in cerebellum
is low compared to the cortex and the hippocampus in AD brain. Our experiments show
that the primary neurons irrespective of the region they originate from behave in a similar
manner. We showed this in a hypothetical experiment in which the cerebellar neurons
show tau hyperphosphorylation and BACEl upregulation when treated with the
conditioned media from free fatty acid treated cortical astrocytes. Thus, the basic
difference in expressing the AD-like neuropathology is due to the difference in the

astrocytic metabolism.

41

It was shown that glucose uptake by the cells is decreased in the cortical and not the
cerebellar astrocytes. In addition, it was determined that the expression of ACSB- a key
enzyme in free fatty acid metabolism was higher in the cortical astrocytes compared to
the cerebellar astrocytes. The result of this is that the cortical astrocytes are much more
metabolically active that the cerebellar astrocytes in metabolizing saturated free fatty
acids. Hence, we could justify this regional difference in the neuronal/astrocytic behavior

by the higher levels of the ACS3 in cortex.

We wanted to examine the effect of free fatty acids on the cerebellar astrocytes to
ascertain that the cerebellar astrocytes indeed possess a different machinery to metabolize
free fatty acid. It has been shown that the cortical astrocytes undergo apoptosis upon
treatment with free fatty acids due to de novo synthesized ceramides. It has been shown
for the first time that the cerebellar astrocytes do indeed proliferate upon treatment with
palmitic acid. Thus lower levels of ceramides produced due to lower metabolic activity of
the ACS3 causes the astrocytes to shift to a proliferative phase compared to the expected

apoptotic phase of the cell fate as observed in cortical astrocytes.

42

4.2 Future directions to the study

4.2.1 Invivo and metabolic studies

Invivo studies could be undertaken to study the potential of the identified target-
ceramides as a therapeutic agent. Since the cerebellar astrocytes are possessed with a
different machinery to metabolize fiee fatty acids a metabolic model may be constructed
on the neuron glial interaction in the cerebellum to determine the pathways that differ in
the tow brain regions namely the cortex and cerebellum. It would more easy to identify
drug targets if we consider the neuron-glial interaction from the two different regions and

compare them step by step rather than analyze region specific interaction pathway.

4.2.2 Insulin signaling in response to FFAs in different regions

It has been shown that the cerebellum and cortex differ in even metabolizing glucose,
which is the primary energy substrate. Insulin is a hormone that regulates blood glucose
levels. Apart from being the primary agent in maintaining carbohydrate homeostasis, it
has effects on fat metabolism and it changes the liver's activity in storing or releasing
glucose and in processing blood lipids, and in other tissues such as fat and muscle. While
some amount of literature is available on the effect of free fatty acids on insulin signaling
in beta cells and myocytes, similar studies in neural cells are lacking. It is interesting to
note that Type 2 Diabetes is a risk factor for AD. Taking all these ideas together, it would
be worthwhile to study the effects of the different types of FFAs on insulin signaling in
neural cells. These studies might determine the association between insulin signaling and

the progression of AD.

43

4.2.3 Ceramide synthesis as regulatory machinery

Ceramide, a lipid molecule that increases the cellular oxidative state, has been shown to
have be involved in several apoptotic patterns including trophic factor withdrawal and
inflammation (Coroneos et al., 1995; Kyriakis and Avruch, 1996). One pathway of
ceramide formation involves sphingomyelin hydrolysis and the other pathway (denovo
synthesis) involves formation of ceramide by ceramide synthase from less complex

molecules (Bose et al., 1995).

The implications for lower ceramide levels in cerebellum may actually be beneficial in
terms of metabolic activity. Higher levels of ceramide synthesis in the cortex along with
oxidative stress that further elevates the ceramide levels in AD may be responsible for the
severe neuronal degeneration in cortex and hippocampus. Pharmacological studies
involving inhibitors of ceramide synthesis could be undertaken to study the role of
ceramide in causing neuronal degeneration in AD and further elucidate the role of

ceramides in causing regional disparities in neuronal and astrocytic metabolism.

44

4.3 Shortcomings identified in the study

The study was mainly focused on assimilating the information that was obtained on a
basis of an underlying hypothesis that the cerebellar region shows relatively less neuronal
degeneration and lower levels of glucose metabolism compared to the cortex. Although,
very few post-mortem studies have identified Amyloid beta plaques and fibrillary tangles
in cerebellum and PET imaging studies have shown cerebellar glucose metabolism to be
comparable to that of the cortical glucose metabolism, we could at least ascertain that in
the in vitro primary culture studies cerebellar metabolism is different from the cortical
metabolism. The study was done primarily on pure astrocytic and neuronal cultures.
Usually a cell behaves completely differently when removed from its neighboring cells
(also called a niche). In our study, a co-culture study (of neurons and astrocytes) might be
more relevant in elucidating the involvement of astrocyte-neuron interaction in neural

cell fate determination.

45

APPENDICES

46

APPENDIX A
QUANTIFYING THE PROTEIN LEVELS IN WESTERN BLOT
The l-D electrophoresis gel is imaged and analyzed using the Quantity One sofiware®
(Bio-Rad Inc, USA). The analysis is done by determining the intensity of the blot
obtained on exposure to X-Ray, by using a chemiluminescent substrate that detects

Horseradish Peroxidase (HRP) on immunoblots.

Western Blotting with Actin antibody is used to confirm that equal proteins are run on
each of the wells during the loading of the gel. Equal areas of the blots are compared for
their relative intensities using the volume analysis tool in the software. Each of the blot
intensity is normalized with their respective actin blot intensity run as a separate gel.
Finally, all the blots are normalized with the control band intensity of that particular gel.

An example has been discussed, to explain the analysis done throughout the study.

BSA 0.25 mM
PA

eating is»... 1‘ AT8

Beta-Actin

Fig a Direct treatment of cerebellar neurons with increasing concentration of palmitic acid. Primary
rat cerebellar neurons were treated for 24 h with 0.25 M of either pahnitic acid (PA) or with 4% bovine
serum albumin (BSA), carrier for F FAs (control). Cell lysates from fatty acid-treated and control cells were
immunoblotted with AT8 antibody, which recognize phosphorylated tau. B-actin is shown as a marker for
equal protein loading.

47

Using the volume analysis tool the respective intensities for the AT8 and the actin blot
was obtained as shown in the tabulation. Each the condition, BSA (control) and PA

treatment was normalized with respective actin values

 

 

 

 

 

 

 

 

AT8 Actin Actin %
intensity intensity normal control

C 4594.37 7867.241 0.58398745 100
PA 4831.721 7486.841 0.64536178 110.5095

 

 

Since there was no change with respect to the control, we concluded that treatment of

cerebellar neurons directly with palmitic acid caused no changes in protein levels of tau

and BACEl.

48

APPENDIX B

PHASE CONTRAST IMAGES OF NEURONS AND ASTROCYT ES FROM THE
CEREBELLUM AND CORTEX OF THE RAT BRAIN

 

(3) (4)

Fig b Phase contrast images of (l) Cerebellar neurons, (2) Cerebellar astrocytes, (3) Cortical neurons
and (4) Cortical astrocytes

49

APPENDIX C

SUPPLEMENTARY IMAGES

Each of the experiments were done atleast thrice so that a good statistical analysis of the
data presented could be obtained. The main section of the thesis contains representative
blots for each of the protein level detection methods. This section puts together all the

blots obtained during the course of the study undertaken.

1. DIRECT TREATMENT OF CEREBELLAR NEURONS WITH PALMITIC
ACID

This experiment was done to confirm that cerebellar neurons like cortical neurons do not
take up free fatty acids such as palmitic acid. We observed no changes in the protein
levels of Tau and BACEl, which should be upregulated if the neurons actively

metabolize free fatty acids.

1. 2.
BSA 0.25mM BSA 0.25mM
PA PA
fi .1. BACE] .. .. .- ... AT8
- - Beta-Actin —~ Emma“

Fig c.1 Direct treatment of cerebellar neurons with palmitic acid. Primary rat cerebellar neurons were
treated for 24 h with 0.25 M of either palmitic acid (PA) or with 4% bovine serum albumin (BSA),
carrier for FFAs (control). Cell lysates from fatty acid-treated and control cells were irmnunoblotted with
(1)BACE1 and (2)AT8 antibody, which recognize phosphorylated tau. Beta-actin is shown as a marker for
equal protein loading.

50

2. CROSS CELL TYPE EXPERIMENTS

A. TAU PHOSPHORYLATION OF CEREBELLAR NEURONS

<3 4

83 a:

G— -I—- u..._ A"

CT-A
PA

Control

 

Beta-Actin
- i— vu- 1.
2 .
.3. we .3... ~ Q. .., '. a... AT8
’ Beta-Actin
94—- - i
3.
f f AT8
_ - _ 1-
Beta-Actin

u.“

Fig c.2. FFA induced tau hyperphosphorylation in cerebellar neurons. Astrocytes from the cortical and
cerebellum were treated with 0.2 uM Pahnitic Acid (PA) or 4% BSA (Control). The conditioned media
from the astrocytes were transferred to the cerebellar neurons (24 hours treatment). 1,2,3. Western Blot
Analysis of hyper-phosphorylated tau using AT-8 antibody in cerebellar neurons. Beta-Actin was used a
control for equal protein loading.

51

B. TAU PHOSPHORYLATION OF CEREBELLAR NEURONS

1. _.
a; I. 4% s
S o o 9* g (3 8 a
AT-8
“ —-—" ' Beta-Actin
2.
AT-8
7 Beta-Actin
- — — -
3.
“w... - ,_. _. ,, .. Ar.s
Beta-Actin

 

Fig c.3 FFA induced tau hyperphosphorylation in cortical neurons. Astrocytes from the cortical and
cerebellum were treated with 0.2 uM Palmitic Acid (PA) or 4% BSA (Control). The conditioned media
from the astrocytes were transferred to the cortical neurons (24 hours treatment). 1,2,3Western Blot
Analysis of hyper-phosphorylated tau using AT-8 antibody in cortical neurons. Beta-Actin was used a
control for equal protein loading.

52

C. BACEl UPREGULATION IN CEREBELLAR NEURONS

Control

Control

CT-A
CT-A
PA

CB—A

CB-A

PA

.1

u! ‘9: q " BACE]

Beta-Actin
_ - - .I—v

2.
—- - - — BACE]
Beta-Actin
3.
".m- ‘ w - 0"” BACE]

a—I- - ” Beta-Actin

Fig c.4 FFA induced BACE] upregulation in cerebellar neurons. Astrocytes from the cortical and
cerebellum were treated with 0.2 uM Palmitic Acid (PA) or 4% BSA (Control). The conditioned media
from the astrocytes were transferred to the cerebellar neurons (24 hours treatment). Western Blot Analysis
of BACEl using BACEl antibody in cerebellar neurons.

53

3. ACS3 EXPRESSSION IN NEURONS AND ASTROCYTES OF THE CORTEX
AND CEREBELLUM

1 . CB-N CT-N CB-A CT-A

ACS3
- ~ _
~_ 8 Beta Actin
2.
.. .. ru- .......... V... ACS3

Bt-Atl
“-_ °" M

Fig c.5 Relative expression levels of ACS3 in cortical, cerebellar astrocytes and neurons. A. Western
Blot Analysis of ACS3 using ACS3 specific antibody. Beta-Actin was used as a marker for equal protein
loading.

54

4. GLUT-l EXPRESSION

1 8o 8: as to as as
‘ g «I» a. .5-“ .55.-.. GLUT-l
~ Beta-Actin
2.

- - hp _ in “I, GLUT-1

Beta-Actin

-_---'-

Fig c.6 FFA induced GLUT-l downregulation in cortical but not in cerebellar astrocytes. Astrocytes from
the cortical and cerebellum were treated with 0.2 mM Palmitic Acid (PA) or 0.5 mM PA or 5% BSA
(Control). 1,2. Western Blot Analysis of GLUTl expression using GLUTl specific antibody in cortical
and cerebellar astrocytes. Beta-Actin was used as a marker for equal protein loading.

55

APPENDIX D

CAVEATS IN MEASUREMENT OF REACTIVE OXYGEN SPECIES

In the study undertaken we have employed two different techniques to visualize the
release of reactive oxygen species (ROS) namely Fluorescence Microscopy and
Quantification of the fluorescence using a plate reader. Both the methods mentioned
above employs a similar principle in visualizing the ROS released by the neurons on
treatment with the media conditioned by the palmitic acid treated astrocytes for a given
period of time. Detection of the intracellular ROS were done by staining with a dye 5-(6)-
chloromethyl-2’,7’ dichlorodihydrofluorescein diacetate (CM-H2DCFDA, from
Molecular Probes). Intracellular esterases cleave the ester group to form H2DCFDA and
is further converted to a membrane impermeable H2DCF. The ROS released oxidizes the
H2DCF resulting in formation of 2,7-dichlorofluorescein (DCF) which is a fluorescent
compound. The intensity of the fluorescence correlates with the ROS produced by the

neurons.

We have used the 24-well plate for quantification purposes since it is easier to use one
plate at a time for fluorescence readings and thereby eliminating errors due to cell count
and conditions. At the same time, we had the problem of cell clustering especially in the
case of cerebellar neurons when kept in the media for more than 24 hours, which is
probably due to density differences. This also the reason as to why we do not observe any
fluorescence in the microscopy images since they were taken using the cells plated on 6-
well culture plates. Further imaging using Confocal Microscopy would have yielded

much clearer and better images than the ones taken on the Fluorescence Microscope.

56

 

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