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This is to certify that the
dissertation entitled

INVOLVEMENT OF SATURATED FATTY ACIDS IN
CAUSING PATHOPHYSIOLOGICAL AND METABOLIC
CHANGES ASSOCIATED WITH
ALZHEIMER’S DISEASE

presented by
SACHIN PATIL

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Chemiml EngineerinL

Major Professor‘s Signature
9/24 o7

Date

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INVOLVEMENT 0F SATURATED FATTY ACIDS
IN CAUSING PATHOPHYSIOLOGICAL AND METABOLIC CHANGES
ASSOCIATED WITH ALZHEIMER’S DISEASE
By

SACHIN PATIL

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Chemical Engineering and Materials Science

2007

ABSTRACT
INVOLVEMENT OF SATURATED FATTY ACIDS IN CAUSING
PATHOPHYSIOLOGICAL AND METABOLIC CHANGES
ASSOCIATED WITH ALZHEIMER’S DISEASE
By

SACHIN PATIL

Alzheimer’s disease (AD) is a very complex, age-related neurodegenerative disorder.
Pathologically, AD brain is characterized by extracellular deposits of amyloid beta (AB)
protein and intracellular accumulation of neurofibrillary tangles (NFTS) composed of
hyperphosphorylated tau protein. The present studies were undertaken to investigate the
possible causal role of saturated free fatty acids (FFAS) in the pathogenesis of AD with
the primary focus on establishing the underlying mechanism, which may prove vital in

developing novel therapeutic strategies for AD.

We found that saturated FFAS had no direct effect on primary rat cortical neurons in
terms of causing both the pathophysiological (AB production and tau
hyperphosphorylation) as well as metabolic (glucose metabolism) abnormalities. In
contrast, saturated FFAS significantly increased AB production and tau
hyperphosphorylation in neurons through astroglial mediation. The conditioned media
from FFA-treated astroglia induced increased production of reactive oxygen species
(ROS) in neurons and co-treatment of neurons with N-acetyl cysteine, an anti-oxidant,
inhibited FFA-astroglia-induced AB production and tau hyperphosphorylation, suggesting

a central role of astroglia-mediated oxidative stress in the FFA-induced

pathophysiological abnormalities in neurons. Furthermore, saturated FFAS significantly
decreased the level of astroglial glucose transporter (GLUTl) and downregulated glucose
uptake and lactate release by astroglia. By using a powerful mathematical technique,
metabolic flux analysis (MFA), we found that de novo synthesis of ceramide in PA-
treated astroglia was significantly higher as compared to controls. The treatment of
astroglia with L-cycloserine inhibited FFA-induced de novo synthesis of ceramide in
astroglia and in turn, ROS production, AB production and tau hyperphosphorylation in
neurons. The data suggest that astroglial ceramide may play a central role in FFA-

induced, AD-associated pathophysiological changes in neurons.

To conclude, our results establish an underlying mechanism by which saturated FFAS
induce AD-associated pathophysiological as well as metabolic changes, placing
“astroglial FFA metabolism” at the center of the pathological cascade of AD. Further
understanding of astroglial FFA metabolism, both in vitro and in vivo, may help in
uncovering new aspects of AD pathogenesis that may be translated into potential targets

for therapeutic intervention in AD.

© COPYRIGHT

SACHIN PATIL

2007

 

DEDICATED TO MY FAMILY

ACKNOWLEDGMENTS

No Matter How Hard You Work, You have Only One Brain and Two Handr,

Great Work Demandr Sac/z Numerou: Once, W/ziclt You Find In Your Friends:

At this juncture of successful completion of my dissertation, I wish to convey my deepest
sense of gratitude towards those colleagues and friends who have helped me to achieve
my goal. First and foremost, I would like to thank my advisor Dr. Christina Chan, for her
guidance, constant inspiration and complete intellectual freedom throughout my research
tenure. I am also thankful to all the members of my Ph.D. advisory committee-Dr. Bill
Atchison, Dr. Worden and Dr. Walton. Their support and interest in my work, together
with the critical evaluation of the work, helped me tremendously to improve the quality
of the present dissertation. Words fall short to express my heartfelt feelings and gratitude

towards these great personalities. I am proud that I got a chance to work with them.

I would also like to thank my M.S. advisor, my GURU, Professor G. D. Yadav (UICT,
Mumbai, India). His inspiring guidance has carved a niche in my heart. He imbibed in me
the qualities of confidence, perseverance and unwavering commitment towards one’s

work, which I am sure would definitely help me throughout my life.

If “a friend in need is a true friend indeed”, then all my colleagues are my friends, in true
sense. I wouldn’t be able to justify my thankful feelings by mere words towards my
labmates (from Drs. Chan and Walton labs) who have always been there whenever I

needed their help. My special thanks my senior colleague, Dr. Shireesh Srivastava who

vi

helped me throughout my tenure in the lab with constant encouragement and valuable
suggestions. The mathematical work (MFA) that I performed for dissertation wouldn’t
have been possible without his kind help. I am also thankful to my friends for their
constant, unwavering encouragement- Dr. Mehul (Netherlands) and his wife, Dr. Sameer
(Netherlands), Dr. Lek (Scotland) and his wife Heena, Dr. Ambareesh (Texas), Dr. Navin
(MSU), Dr. Zheng Li (Boston), Dr. Srivatsan (Harvard), Sachin Injal (Lawyer), Dhanu,
Adam, Nandu, Priti, Lufang, Chisa, Tanmay, Susan, Katie, Dana, Tao, Alison, Bahareh,
John, Ketan, Hemant, Joe, Mike, Shengnan, Linxia, Deebika and Xureui. My special
thanks also to Shirley Owens, for her help in terms of confocal microscopy. I was also
very fortunate to have hard-working, sincere undergraduates assisting me- Alexis (Shell),

Robert (U of M) and Joe. Their youthful exuberance was pleasantly encouraging.

I would also like to thank the Department of Chemical Engineering and Materials
Science, the College of Engineering, Quantitative and Biological Modeling Initiative
(QBMI) at MSU, the office of international student and scholars, council of graduate
studies (COGS) and the graduate school for the financial support in the form of Food,
Nutrition and Chronic Disease Fellowship and travel fellowships. Their contributions

helped me present my research at the national and international scientific meetings.

Also, my apartment at Student living Center (SLC) was my home-away-from-home and
my roomies made it that way with whom I will always share the deepest friendship-
Sarfaraz (India), Arda (Turkey), Jorge (Peru), Kensuke (Japan), Qiang (China) and Paul

(USA). It was truly an international experience. I have learned so many things from them

vii

 

that I cannot repay them in any way. Here, I would also like to express my gratitude
towards all my ward mates at SLC for their true friendship- Stephanie, Tiffany, Kelly,
Kerry, Emily, Conrad, Justin, Don, Dan, Ben, Josh, Gary. My special thanks to Cindy,
Vern, Nicole and President Hinkle. Also thanks to the Elders who taught me how to tie a

double knot.

My sincere thanks to Amit and his wife Deboleena, for inviting me over for countless
lunches and dinners. But, obviously it’s much more than food that is and will be keeping

us in the bond of friendship!

Last but not the least, it was the confidence laid in me by my Mom, Dad, my sisters and
brother-in-laws, which enabled me to reach this pinnacle of success. I owe a debt of
gratitude for their constant encouragement for higher education and unflinching, altruistic

help during this work.

viii

-“fi‘fi'va- , . .- . *- 7

TABLE OF CONTENTS

 

 

 

 

 

LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF ABBREVIATIONS xviii
CHAPTER 1. INTRODUCTION 1
1.1 ALZHEIMER’S DISEASE .................................................................................... 1
1.2 THE CURRENT HYPOTHESES OF AD ................................................. 4
1.2.1 The Amyloid Cascade Hypothesis ................................................................... 4
1.2.2 The Cholesterol Metabolism and AD .............................................................. 6
1.2.3 Oxidative Stress and AD ................................................................................. 7
1.2.4 The Cholnergic Hypothesis of AD .................................................................. 8
1.2.5 Homocysteine and AD .................................................................................... 9
1.2.6 The Pathogen Hypothesis of AD ................................................................... 10
1.3 POTENTIAL INVOLVEMENT OF SATURATED FATTY ACIDS IN THE
PATHOGENESIS OF AD ......................................................................................... 11
1.4 GOALS OF THE PRESENT STUDY .................................................................. 12
1.5 THESIS OUTLINE .............................................................................................. 12
CHAPTER 2. SATURATED FATTY ACID-INDUCED
HYPERPHOSPHORYLATION OF TAU IN PRIMARY RAT CORTICAL
NEURONS--- - - - 14
2.1 INTRODUCTION ............................................................................................... 14
2.2 MATERIALS AND METHODS ......................................................................... 18
2.2.1 Isolation and Culture of Primary Rat Cortical Neurons and Astroglia ............ 18
2.2.2 Lactate Dehydrogenase (LDH) Assay ........................................................... 18
2.2.3 Western Blot Analysis ................................................................................... 19
2.2.4 Immunofluorescence Analysis of Neurons and Astroglia ............................... 20
2.2.5 Immunostaining of Reactive oxygen Species ................................................. 20
2.2.6 Data Analyses ............................................................................................... 21
2.3 RESULTS AND DISCUSSION ........................................................................... 21
2.3.] Direct Treatment of Neurons with Saturated Fatty Acids ............................... 21
2.3.2 Involvement of Saturated F FAS in Tau Hyperphosphorylation Through
Astroglial Mediation .............................................................................................. 22
2.3.3 Involvement of Oxidative Stress in FFA-Astroglia-Induced Tau
Hyperphopshorylation in Neurons .......................................................................... 26
2.4 CONCLUSIONS ................................................................................................. 27

ix

CHAPTER 3. SATURATED FATTY ACID-INDUCED AMYLOIDOGENIC

PROCESSING OF APP IN PRIMARY RAT CORTICAL N EURONS ................... 28
3.1 INTRODUCTION ............................................................................................... 28
3.2 MATERIALS AND METHODS ......................................................................... 33

3.2.1 Isolation and Culture of Primary Rat Cortical Neurons and Astroglia ............ 33
3.2.2 Western Blot Analysis ................................................................................... 34
3.2.3 Immunofluorescence Analysis of BACEl in Neurons ................................... 34
3.2.4 Data Analyses ............................................................................................... 35
3.3 RESULTS ........................................................................................................... 35
3.3.1 Direct Treatment of Neurons with Saturated Fatty acid (Palmitic Acid) ......... 35
3.3.2 Involvement of Saturated FF As in BACEl Up-regulation and Amyloidogenic
Processing of APP Through Astroglial Mediation .................................................. 36
3.3.3 Involvement of Oxidative Stress in FFA-Astroglia-Induced BACEl Up-
regulation and Amyloidogenic Processing of APP in Primary Neurons .................. 39
3.4 CONCLUSIONS ................................................................................................. 41

CHAPTER 4. SATURATED FATTY ACID-INDUCED ABNORMAL

METABOLIC CHANGES ASSOCIATED WITH ALZHEIMER'S DISEASE ....... 42
4.1 INTRODUCTION ............................................................................................... 42
4.2 MATERIALS AND METHODS ......................................................................... 45

4.2.1 Isolation and Culture of Primary Rat Cortical Neurons and Astroglia ............ 45
4.2.2 Western Blot Analysis ................................................................................... 46
4.2.3 Biochemical Measurements of Cellular Metabolites ...................................... 47
4.2.4 Metabolic Flux Analysis (MFA) .................................................................... 48
4.2.5 Measurement of Intracellular ATP in Astroglia ............................................. 54
4.2.6 Measurement of Intracellular Ceramide in Astroglia ..................................... 55
4.2.7 Data Analyses ............................................................................................... 56
4.3 RESULTS AND DISCUSSION ........................................................................... 56
4.3.1 FFA-Induced Abnormal Glucose Metabolism ............................................... 56
4.3.2 Cellular Mechanism of FF A-Induced Abnormal Glucose Metabolism in
Astroglia ................................................................................................................ 59
4.3.3 FFA-Induced Global Metabolic Changes in Astroglia ................................... 61
4.3.3.1 Verification of the psueudo-steady state assumption and linearity of
fluxes over 24 hours ...................................................................................... 62
4.3.3.2 MFA Analysis ................................................................................... 64
4.4 CONCLUSIONS ................................................................................................. 69

CHAPTER 5. INVOLVEMENT OF CERAMIDE IN FFA-ASTROGLIA-
INDUCED PATHOPHYSIOLOGICAL ABNORMALITIES ASSOCIATED WITH
AD -- 70

 

 

 

 

5.1 INTRODUCTION ............................................................................................... 70
5.1 MATERIALS AND METHODS ......................................................................... 72
5.2.1 Isolation and Culture of Primary Neurons and Astroglia from Rat Cortex and
Cerebellum ............................................................................................................ 72
5.2.21mmunostaining of Reactive Oxygen Species (ROS) ..................................... 74
5.2.3 Western Blot Analysis ................................................................................... 74
5.2.4 ELISA measurements of AB4O and AB42 ..................................................... 76
5.2.5 Data Analyses ............................................................................................... 76
5.3 RESULTS AND DISCUSSION ........................................................................... 76
5.3.1. Involvement of Astroglial Ceramide in F FA-Astroglia-Induced ROS
production in Neurons ........................................................................................... 76
5.3.2 Involvement of astroglial ceramide in FFA-astroglia-induced amyloidogenesis
and tau hyperphoshorylation in neurons ................................................................. 78
5.3.3 A possible explanation for the region-specific and cell type—specific damage
observed in AD ...................................................................................................... 84
5.4 CONCLUSIONS ................................................................................................. 87
CHAPTER 6 CONCLUSIONS--- -- - ------- - 88
APPENDIX- - - 96
1. Brain cells from older animals ............................................................................... 96
2. Trans-well experiments .......................................................................................... 96
3. Exogenous addition of ceramide as a positive control ............................................ 97
LIST OF PUBLICATIONS -- - - - -- - 99
BIBLIOGRAPHY -- ----- - -------------- - ------------- 100

 

xi

 

LIST OF TABLES

Table 4.1 List of the cellular metabolic reactions ............................................... 43
Table 4.2 List of intracellular metabolites ....................................................... 46
Table 4.3 List of measured fluxes ................................................................. 47
Table 4.4 Ratio of intracellular to extracellular lactate levels ................................. 57
Table 4.5 Metabolic flux values calculated by MFA ........................................... 58

xii

Images in this dissertation are presented colored.

LIST OF FIGURES

Figure 1.1. The shrinkage of brain in Alzheimer’s disease. The brain regions involved
in cognitive functions (frontal and temporal lobes, left and lower part of brain,
respectively) show significant shrinkage in AD brain as compared to normal brain ........ 1

Figure 1.2. The pathophysiological lesions in AD brain. (A) Amyloid beta (AB) plaque
and (B) Neurofibrillary tangle (NFT) .............................................................. 2

Figure 1.3. The decreased glucose metabolism in Alzheimer’s disease. The positron
emission tomography (PET) images Show significant decline in cerebral glucose
metabolism in AD brain as compared to normal brain; red and yellow-high metabolism,
blue-low metabolism ................................................................................. 2

Figure 2.1. The physiology and pathology of tau. (A) In healthy neurons, tau stabilizes
microtubules. (B) In AD, tau hyperphosphorylates and detaches from microtubules
leading to tangle formation .......................................................................... 8

Figure 2.2. Direct treatment of neurons with saturated FFAS. Primary rat cortical
neurons were treated for 24 hours with 0.2mM of either palmitic acid (PA) or stearic acid
(SA) or with 5% bovine serum albumin (BSA), vehicle for FFAS (control). Detergent cell
lysates from fatty acid-treated and control cells were immunoblotted with PHF-l and
AT8 antibodies, which recognize phosphorylated tau. B-actin is shown as a marker for
protein loading. ..................................................................................... 15

Figure 2.3. MAP-2, GFAP and AT8 immunostaining. (A) Neurons treated directly
with FFAS for 24 hours. (B) Astrocytes treated for 12 hours with 0.2mM of either
palmitic acid (PA) or stearic acid (SA) or 5% BSA (control). (C) Neurons treated for 24
hours with conditioned media from fatty acid-treated or control astrocytes. (D)
Immunofluorescence labeling of phosphorylated tau with AT8 antibody in neurons
treated with conditioned media from fatty acid-treated or untreated astrocytes (control).
Images were obtained with confocal fluorescence microscopy. (Objective lens
magnification- 40X for MAP-2 and GFAP, 63X for AT8) .................................... 16

Figure 2.4. Measurement of LDH release from astroglia treated with PA. 24h
treatment with 0.2mM PA failed to liberate LDH from astroglia as compared to controls.
In contrast, 1hr treatment of astroglia with 300mM H202 (positive control) induced
significant LDH liberation after 24h as compared to both control and PA-treated cells.
Data are taken from 3 different experiments and are expressed as mean :1: SD. One-way

xiii

 

 

ANOVA with Tukey’s post hoc method was used for analyzing the differences between
treatment groups. *, p<0.05 compared with control ............................................ 17

Figure 2.5. Astroglia-mediated, fatty acid-induced hyperphosphorylation of tau in
neurons. (A) Western blot analysis of hyperphosphorylated tau was performed using
phospho-specific antibodies PHF-l and AT8. (B) Histograms corresponding to PHF-l
and AT8 blots represent quantitative determinations of intensities of the relevant bands.
Data represent mean :1: SD. of 3 independent experiments. One-way ANOVA with
Tukey’s post hoc method was used for analyzing the differences between treatment
groups. *, p<0.05 compared with control; #, p<0.05 compared with fatty acid
treatment ............................................................................................. 18

Figure 2.6. Intracellular accumulation of ROS in neurons. (A) Astroglia and (B)
neurons were stained with CM-H2DCFDA for intracellular ROS detection and examined
with confocal fluorescence microscopy (Zeiss LSM 5 Pascal). (Objective lens
magnification, 63X) ................................................................................. 19

Figure 3.1. Processing of amyloid precursor protein (APP). (A) The non-
amyloidogenic pathway catalyzed by a- and y-secretase. (B) The amyloidogenic pathway
catalyzed by B- and y-secretase ................................................................... 23

Figure 3.2. Direct treatment of neurons with palmitic acid. Primary rat cortical
neurons were treated for 24 hours with 0.2mM of palmitic acid (PA) or with 4% bovine
serum albumin (BSA), vehicle for PA (control). Detergent cell lysates from PA-treated
and control cells were immunoblotted with BACEl antibody. B-actin is shown as a
marker for protein loading. The blots are representative of 3 independent experiments..29

Figure 3.3. BACEl immunostaining. Immunofluorescence labelling of BACEl in
neurons treated for 24 hours with conditioned media from PA-treated (for 24 hours) or
untreated astrocytes (control). Images were obtained with confocal fluorescence
microscopy (objective lens magnification- 40X) ................................................ 30

Figure 3.4. Astroglia-mediated, PA-induced BACEI upregulation in neurons.
Astrocytes were treated for 12 and 24 hours with 0.2mM of PA or 4% BSA (control),
followed by transfer of the astrocytes-conditioned media to neurons (24 hours treatment).
(A) Western blot analysis of BACEl protein levels in neurons. B-actin is shown as a
marker for protein loading. (B) Histograms represent quantitative determinations of
intensities of the relative bands. Data represent mean 1 SD. of three independent
experiments. Student’s t-test was used for analyzing differences between different
treatment groups. *, p<0.05 compared with respective control ............................... 31

Figure 3.5. Immunoblot analysis of presenilin-l (PSI) levels in neurons treated with
astrocytes-conditioned media. Astrocytes were treated for 24 hours with 0.2mM PA or
5% BSA (control), followed by transfer of the astrocytes-conditioned media to neurons
(24 hours treatment), with or without lOmM DMU. Detergent cell lysates from PA—

xiv

 

treated and control cells were immunoblotted for PS] and PSl-CTF levels. The
immunoblot is representative of 3 independent experiments .................................. 32

Figure 3.6. Oxidative stress involved in astroglia-mediated, PA-induced elevations in
BACEl and C99 levels in neurons. Astrocytes were treated for 24 hours with 0.2mM
PA or 5% BSA (control), followed by transfer of the astrocytes-conditioned media to
neurons (24 hours treatment), with or without lOmM DMU. (A) Western blot analysis of
BACEl, APP and C99 protein levels in neurons. (B) Histograms represent quantitative
determinations of intensities of the relative bands. Data represent mean at SE. of three
independent experiments. One-way ANOVA with Tukey’s post hoc method was used for
analyzing the differences between treatment groups. *, p<0.05 compared with control; **
p<0.05 compared with PA treatment ............................................................. 33

Figure 4.1. Conventional view of cerebral glucose metabolism ........................... 37

Figure 4.2. Cerebral glucose metabolism based on the novel astrocyte-neuron lactate
shuttle hypothesis. ................................................................................ 38

Figure 4.3. Astroglial metabolic network. Boxes represent extracellular metabolites,
while ovals represent intracellular metabolites. The direction of reaction assumed in the
model is indicated by arrows. ..................................................................... 45

Figure 4.4. PA downregulates glucose uptake and lactate release by astroglia. The
cortical neurons and astroglia were treated for 24 hours with 0.2mM of PA or 4% BSA.
(A) In neurons, PA treatment did not change glucose uptake and lactate production. (B)
PA treatment Significantly decreased glucose uptake and lactate production by astroglia.
AMPK-activator AICAR did not inhibit PA-induced downregulation in glucose
metabolism. Data represent mean :i: SD. of Six experiments. Student’s t-test was used for
analyzing the differences between the two treatment groups. *, p<0.05 compared with
respective control. ................................................................................... 50

Figure 4.5. Measurement of intracellular ATP in astroglia. The cortical astroglia were
treated for 24 hours with 0.2mM of PA or 4% BSA. PA treatment increased cellular ATP
production in astroglia. Data represent mean :1: SD. of 3 experiments. Student’s t—test was
used for analyzing the differences between the two treatment groups. *, p<0.05 compared
with respective control. ............................................................................. 51

Figure 4.6. PA downregulates GLUTI level in astroglia. Astroglia were treated for 24
hours with 0.2mM of PA or 4% BSA. The immunoblot analysis shows that PA-treatment
significantly decreased the levels of GLUTl as compared to the untreated ones. B-actin is
shown as a marker for protein loading. Histogram represents quantitative determinations
of intensities of the relative bands normalized with actin. Data represent mean :t SD. of
three independent experiments. Student’s t-test was used for analyzing the differences
between the two treatment groups. *, p<0.05 compared with respective control ........... 53

Figure 4.7. FFA—metabolizing pathways involved in cellular ROS production ......... 54

XV

Figure 4.8A. Time-dependent measurements of intracellular lactate levels. Astroglia
were treated for 6, 12 and 24 hours with 0.2mM of PA or 4% BSA. The cells were
trypsinized, washed with TBS and lyzed by using 0.7% perchloric acid. The lactate
levels in cell lysate were measured by enzymatic assay. Data represent mean 3: SD. of
three independent experiments .................................................................. 56

Figure 4.8B. Time-dependent measurements of extracellular lactate levels. Astroglia
were treated for 6, 12 and 24 hours with 0.2mM of PA or 4% BSA. The conditioned
media were collected and the lactate levels in the media were measured by enzymatic
assay. Data represent mean :t SD. of three independent experiments ....................... 57

Figure 4.9. PA-induced, de novo synthesis of ceramide in astroglia. Astroglia were
treated for 24h with 0.2mM PA or 4% BSA (control), afier which cellular lipids were
extracted for ceramide determination by HPLC. PA significantly increased ceramide
synthesis in astroglia, which was completely inhibited by treatment of astroglia with
2mM L-CS, an inhibitor of de novo synthesis of ceramide. Data are taken from 3
different experiments and are expressed as mean i S.D. One-way ANOVA with Tukey’s
post hoc method was used for analyzing the differences between treatment groups. *,
p<0.05 compared with control; #, p<0.05 compared with PA treatment ..................... 61

Figure 5.1. Cellular ceramide production. Ceramide is produced in cells by de novo
synthesis from serine and palmitoyl-COA or by breakdown of membrane
sphingomyelins ...................................................................................... 64

Figure 5.2. Involvement of astroglial ceramide in PA-astroglia-induced ROS
production in neurons. Co-treatment of astroglia with 2mM L-CS inhibited PA-
astroglia-induced ROS production in neurons. The neurons were stained with CM-
H2DCFDA for intracellular ROS detection and examined with confocal fluorescence
microscopy (Zeiss LSM 5 Pascal). (Objective lens magnification, 40X) .................... 70

Figure 5.3. The expression of iNOS and IL-6 in astroglia. The expression of iNOS, but
not IL-6 was increased by PA treatment in astroglia. Co-treatment of astroglia with 2mM
L-CS inhibited PA-induced iNOS expression in astroglia. B-actin is shown as a marker
for protein loading ................................................................................... 71

Figure 5.4. Involvement of astroglial ceramide in PA-astroglia—induced elevations
in BACEl and amyloidogenic processing of APP in neurons. In neurons treated with
conditioned media from PA-treated astroglia, the levels of (A) BACEl, (B) C99 and (C)
AB40 and AB42 were found elevated. These PA-astroglia-induced tau abnormalities were
blocked by inhibiting astroglial ceramide synthesis with 2mM L-CS. Data represent mean
:1: SD. of three independent experiments. One-way ANOVA with Tukey’s post hoc
method was used for analyzing the differences between treatment groups. *, p<0.05
compared with control; #, p<0.05 compared with PA treatment .............................. 72

Figure 5.5. Involvement of astroglial ceramide in PA-astroglia-induced tau
hyperphosphorylation in neurons. In neurons treated with conditioned media from PA-

xvi

treated astroglia, tau was found pathologically hyperphosphorylated as shown by
irnmunoblotting with PHF -1 and AT8 antibodies. Tau-l detects dephosphorylated tau,
thus Showing decreased levels in PA-astroglia-treated neurons. These PA-astroglia-
induced tau abnormalities were blocked by inhibiting astroglial ceramide synthesis with
2mM L-CS. Histograms corresponding to PHF-l and AT8 blots represent quantitative
determinations of intensities of the relevant bands normalized with actin. Data represent
mean i SD. of three independent experiments. One-way ANOVA with Tukey’s post hoc
method was used for analyzing the differences between treatment groups. *, p<0.05
compared with control; #, p<0.05 compared with PA treatment .............................. 73

Figure 5.6. PA-astroglia-induced activation of AD-specific kinases in neurons is
mediated by astroglial ceramide. Conditioned media from PA-treated astroglia
activated (A) GSK-3 (increased levels of phosphorylated GSK-3) and (B) cdkS
(increased cleavage of p35 to p25) but not (C) MAP- Erkl/2 (no change in the levels of
phosphorylated MAP- Erkl/2). The treatment of astroglia with 2mM L-CS inhibited PA-
astroglia-induced activation of both GSK-3 and cdk5. Data are representative of 3
different experiments ............................................................................... 75

Figure 5.7. GSK—3 is involved in PA-astroglia-induced tau hyperphosphorylation in
neurons. Astroglia-conditioned media were transferred to neurons, with or without
various kinase inhibitors, viz., lOmM LiC12 (GSK-3 inhibitor), lOmM Roscovitine (cdk-
5 inhibitor) and 30mM PD98059 (MAPK inhibitor). Immunoblot analysis with PHF-l
and AT8 antibodies Show that only LiC12 inhibited the observed, PA-induced tau
hyperphosphorylation in neurons. Histogram data represent mean :1: SD. of three
independent experiments. One-way ANOVA and Tukey’s post hoc method was used for
analyzing the differences between treatment groups. *, p<0.05 compared with control; #,
p<0.05 compared with PA treatment ............................................................. 77

Figure 5.8. Differential effects of cortical and cerebellar astroglia on cortical
neurons. Cortical neurons (CTN) were treated with conditioned media from (A) cortical
astroglia (CTA) and (B) cerebellar astroglia (CBA). Western blot analysis of
hyperphosphorylated tau was performed using AT8 antibody. Cortical astroglia but not
cerebellar astroglia were involved in PA-induced tau hyperphosphorylation in neurons.
Data represent mean :l: SD. of three independent experiments. Student’s t-test was used
for analyzing differences between different treatment groups. *, p<0.05 compared with
control ................................................................................................ 79

Figure 6.1. The “FFA-AD” hypothesis. Proposed cellular mechanism by which
astroglial FFA metabolism may play a central role in causing pathophysiological and
metabolic changes associated with AD .......................................................... 83

xvii

LIST OF ABBREVIATIONS

AB: Amyloid beta

AChE: Acetylcholine esterase

AD: Alzheimer’s disease

ADDLs: AB-derived diffusible ligands
AMPK: AMP-activated protein kinase
ApoE4: Apolipoprotein E4

APP: Amyloid precursor protein
BACE: B-site APP cleaving enzyme
BBB: Blood-brain barrier

BSA: Bovine serim albumin

cdk: cycline-dependent kinase

CBA: Cerebellar astroglia

CTA: Cortical astroglia

CTN: Cortical neurons

DAG: Diacyl glycerol

DMU: 1,3-dimethyl urea

ELISA: Enzyme-linked immunosorbent assay
FACS: Fatty acyl-CoA synthetase
FAD: Familial Alzheimer’s disease
FFA: Free fatty acid

GSK: Glycogen synthase kinase

4-HNE: 4-hydroxynonenal

xviii

‘_ fl“..—

m”...—

 

HPLC: High performance liquid chromatography
IL-6: Interleukin-6

iNOS: inducible nitric oxide synthase
L-CS: L-cycloserine

LDH: Lactate dehydrogenase
MAPK: Mitogen-activated protein kinase
MCI: Mild cognitive impairment
MFA: Metabolic flux analysis

NAC: N-acetyl cysteine

NFTs: Neurofibrillary tangles

N MDA: N-methyl D-aspartate

NO: Nitric oxide

PA: Palmitic acid

PET: Positron emission tomography
PHF: Paired helical filaments

PKA: Protein kinase A

PKC: Protein kinase C

PS: Presenilin

ROS: Reactive oxygen species

SA: Stearic acid

SAD: Sporadic Alzheimer’s disease
SPT-l: Serine palmitoyltransferase-l

TBARS: Thiobarbituric acid reactive substances

xix

CHAPTER 1. INTRODUCTION

1.1 ALZHEIMER’S DISEASE

Dementia is one of the most complex and heterogeneous age-related disorders and the
most common cause of dementia is Alzheimer’s disease (AD) '. AD is a progressive
neurodegenerative disease clinically characterized by severe memory loss and cognitive
impairment 2. As shown in Figure 1.1, AD brain exhibits significant shrinkage as
compared to healthy controls 3. Pathologically, AD is characterized by extracellular
deposits of amyloid beta (AB) protein and intracellular accumulation of neurofibrillary
tangles (NFTS) composed of hyperphosphorylated tau protein (Figure 1.2) 3. In addition
to these two classical neuropathological hallmarks, AD brain is also characterized by
abnormal metabolic changes; abnormal cerebral glucose metabolism is one of the distinct
characteristics of AD brain (Figure 1.3) 3'5.

Healthy Brain AD Brain

   

Figure 1.1. The shrinkage of brain in Alzheimer’s disease. The brain regions involved in wgnitive
functions (frontal and temporal lobes, lefi and lower part of brain, respectively) show signifith shrinkage
in AD brain as compared to normal brain (Figure taken from Ref. 3, with permission from Nature
Publishing Group).

 

Figure 1.2. The pathophysiological lesions in AD brain. (A) Amyloid beta (AB) plaque and (B)
Neurofibrillary tangle (N FT) (Figures taken from www.alzheimers.org, 08-09-2007).

 

Healthy Brain AD Brain

Figure 1.3. The decreased glucose metabolism in Alzheimer's disease. The positron emission
tomography (PET) images show significant decline in cereme glucose metabolism in AD brain as
compared to normal brain; red and yellow-high metabolism, blue-low metabolism (Figure taken from Ref.
3, with permission from Nature Publishing Group).

AD is classified into two categories- familial Alzheimer’s disease (FAD) and sporadic

Alzheimer’s disease (SAD). FAD has been shown to be associated with the mutations in

2

 

APP, presenilin 1 and 2 (PSI and P82) genes on chromosome 21, 14 and 1, respectively
6‘9. Of all the AD cases only 5-10% are due to FAD mutations and the mutations in P8]
are the most frequent of the FAD causes '0’ ”. Furthermore, apolipoprotein E4 (ApoE4)
gene has been shown to cause slight predisposition to AD '2. On the other hand, SAD is
the major form of AD and comprises 90-95% of all the cases 10. Unlike FAD, the etiology
of SAD is not well understood and several possible risk factors in the development of
SAD have been identified. Age is the most significant risk factor for the development of
AD. Additional risk factors based upon the epidemiological studies are high fat diet,
gender, head trauma and vascular risk factors such as diabetes, ischemia, hypertension,

etc. ‘3.

AD was first described in 1906 by the German psychiatrist Alois Alzheimer '4. Today,
AD affects approximately 5-10% of the population over 65 years of age and more than
20% over the age of 80 years ’5. It represents 40-70% of all dementia cases ’5' 16. There
are approximately 5 million AD patients in the US. alone and the total direct/indirect
cost associated with the disease is estimated to be more than $140 billion annually
[www.alz.org]. With the increasing aging population in the western world, AD has
become one of the leading socio-economical challenges today. At the rate of 1 new
patient every 72 seconds, the number of AD patients is expected to grow to 13 million by
year 2050 and the associated cost will be more than the total US. budget [www.alz.org].
Currently, there is no cure for AD ’7. The available treatments include the use of
acetylcholinesterase (AChE) inhibitors (donezepil, rivastigmine and galanthamine) and

the N-methyl D-aspartate (NMDA) antagonist, memantine, which have beneficial, but

short-lived effects on the symptoms of AD. This emphasizes a significant need for

identifying novel targets for therapeutic intervention in AD.

1.2 THE CURRENT HYPOTHESES OF AD

AD etiology is very complex and a large number of factors are hypothesized to play key
roles in the pathogenesis of AD. Based on the extensive literature search, we present
variety of such major scientific hypotheses about the pathogenesis of AD below. In case
of each Of these hypotheses, we present supporting data in terms of epidemiological
studies as well as various cell culture and animal models. In addition, the limitations

associated with these hypotheses are also explained.

1.2.1 The Amyloid Cascade Hypothesis

The amyloid hypothesis is the most widely studied hypothesis in AD research field.
According to this hypothesis, the different gene defects can lead to altered expression or
proteolytic processing of amyloid precursor protein (APP) leading to chronic imbalance
between AB production and clearance. This results in the gradual accumulation of AB
plaques, which in turn initiates a pathological cascade leading to the gliosis,
inflammatory changes, synaptic change, neurofibrillary tangles and neurotransmitter
loss”. Many studies support the amyloid cascade hypothesis. The brains of AD patients
are characterized by the presence of AB plaques and their number far exceeds that found
in the brains of age-matched healthy controls 19. Furthermore, the amount of AB plaques
is highly correlated with the degree of cognitive impairment 20. In addition, all four genes

associated with FAD have been shown to be involved in increased production of AB

(APP, PSI and PS2) 2"28 and its aggregation (ApoE4) 29’ 3° 3'. ApoE4 leads to excessive
deposition of AB in the brain long before AD symptoms occur 32. Down's syndrome
patients who produce significantly higher amount of AB from birth and deposit AB
plaques in their brains as early as age 12, inevitably develop AD by age the of 50 33. This
further emphasizes the central role of AB in the pathogensis of AD. In addition, in various
cell culture models AB fibrils have been shown to induce neuronal damage and activate
inflammatory cells (microglia and astroglia) 34’ 35 36. Also transgenic animal models
expressing human APP gene have been shown to develop AB plaques leading to the
neuronal and microglial damage. These animal models reproduce most of the major

features associated with the AD pathology ”'3’.

Despite these data, the amyloid cascade hypothesis falls short in comprehensively
interpreting AD pathology. In addition to the increased AB deposition, AD brain is also
characterized by the formation of NFTS. In this context, although AB has been Shown to
induce hyperphosphorylation of tau and its tangle formation in cell cultures, the APP
transgenic mice do not show formation of NFTS in their brain 4°. Also, NFTS have been
shown to occur independent of AB plaques“. In addition, it is not clear whether the
behavioral deficits observed in APP transgenic animals are related exclusively to the
increased AB production and deposition in these animals. Furthermore, the amyloid
cascade hypothesis does not provide an explanation for the region-specific damage
observed in AD. Finally, suspension of the AB vaccination clinical trial due to the
development of encephalitis in a small percentage of individuals further undermined the

amyloid cascade hypothesis 42.

1.2.2 The Cholesterol Metabolism and AD

Recently, it has been hypothesized that the abnormalities in cholesterol homeostasis may
play a central role in causing synaptic impairment, neuronal degeneration and other
hallmarks associated with AD 43. The hypothesis is based on the observations that statins
which inhibit cholesterol synthesis protect against age-associated dementia and AD 44’ 45.
Also, the dietary hypercholesterolemia in APP transgenic mice and rabbits has been
shown to accelerate AB deposition in their brains 46' 47. Various cell culture studies further
support the involvement of abnormal cholesterol metabolism in AB production “8‘50. In
addition to increased AB production, abnormal cholesterol metabolism may lead to
neurite degeneration, neuronal cell death, cholinergic dysfunction, oxidative stress and

behavioral impairment, thus suggesting its central role in AD pathogenesis 43.

The main limitation of the cholesterol hypothesis is that the causal factor behind the
abnormal brain cholesterol metabolism is not well established. Furthermore, It is not clear
how dietary hypercholesterolemia affects brain cholesterol metabolism as it has been
shown that the cholesterol pools in the plasma and the brain are independent of each
other; in rats where hypercholesterolemia was induced by diet, all the brain cholesterol
was synthesized in situ and did not come from circulation 5'. Furthermore, although
cholesterol levels enhance AB production, tau hyperphosphorylation is caused by cellular
cholesterol deficiency 52’ 53. Thus, how abnormal cholesterol metabolism may lead to the
formation of both the pathophysiological changes associated with AD (AB production

and tau hyperphosphorylation) is not clear.

1.2.3 Oxidative Stress and AD

Age is the most important risk factor for AD and one of the most accepted theories of
aging is increased oxidative stress 54. In this context, it has been hypothesized that the
age-associated increase in oxidative stress may play a key role in the pathogenesis of AD.
The increased oxidative stress in AD brain can also be attributed to various other factors
such as increased levels of metal ions in the brain, inflammatory response from activated
microglia and astroglia and increased levels of advanced glycation endproducts (AGES)
associated with AD. Iron has been shown to be increased in NFTS as well as in AB
plaques and involved in reactive oxygen species (ROS) production 55’ 56. Iron catalyzes
the formation of hydroxyl radicals from H202 and thus may lead to lipid peroxidation and
oxidation of cellular DNA and proteins. The iron-induced lipid peroxidation is further
potentiated by aluminum 57, which also accumulates in neurofibn'llary tangle-containing
neurons 58. The activated microglia surround the AB plaques 59 and are a source of NO
and oxygen radicals 60, which can react to form peroxynitrite 6'. Furthermore, AGES in
the presence of transition metals can undergo redox cycling with consequent ROS
production 62‘“. The role of oxidative stress in AD pathogenesis is supported by various
studies where free radicals were shown to be involved in increased AB production 65'7" as

well as hyperphosphorylation of tau "’75, two hallmarks of AD.

Here it is interesting to note that Amyloid-B itself has been directly implicated in ROS
formation through peptidyl radicals 76'78. Furthermore, increased oxidative stress has been
associated with many other neurodegenerative diseases such as Parkinson's disease and

Huntington's disease, which have clinical and pathological features different than AD.

Therefore, it is not clear whether oxidative stress plays a causal role in AD pathogenesis

or merely a part of the complex AD etiology.

1.2.4 The Cholinergic Hypothesis of AD

The cholinergic system in the brain is involved in controlling cerebral blood flow,
cortical activity and sleep-wake cycle as well as in modulating cognitive function 79. The
severe deficiency in the brain cholinergic system which is associated with the cognitive
impairment has been proposed to be a central aspect of AD pathology 80. The strong
correlation of clinical dementia ratings with the reductions in the cerebral cholinergic
markers such as choline acetyltransferase and levels of acetylcholine support the
association of cholinergic dysfunction with AD pathology 3" 82. The direct correlation of
dysfunctional cholinergic system and AD pathology is supported by various cell culture
and animal model studies that showed a central role of cholinergic system in regulating
amyloidogenic processing of APP and hyperphopshorylation of tau, two main
characteristics of AD. The activation of muscarinic acetylcholine receptor (M2-mAChR)
in SH-SY5Y neuroblastoma cells significantly downregulated level of BACEl, which is
involved in amyloidogenic processing of APP 83. Furthermore, selective lesion of basal
forebrain cholinergic neurons in rat brain significantly increased AB production and
deposition in cortical areas 84’ 85. Also, activation of nicotinic receptors (nAChR) has been

86. 87

shown to decrease amyloidogenic of APP both in cell culture and in vivo 88. In

addition, the activation of mAChR has been shown to prevent tau phosphorylation 89‘ 9°.

Despite these supporting data, the cholinergic hypothesis has some limitations. It is
unclear if the cholinergic dysfunction leads to the AB production or AB leads to the death
of cholinergic neurons associated with AD. Furthermore, basal forebrain cholinergic
neuronal loss is not specific to AD and is also associated with many diseases e.g.
Parkinson’s disease, Parkinsonism with dementing complex of Guam, Pick’s disease,

Jakob-Creutzfeld disease etc. 79.

1.2.5 Homocysteine and AD

The elevated plasma level of homcysteine (homocysteinuria) has been proposed to be an
independent risk factor for the development of AD 9’. The level of total homocysteine is
significantly higher in serum of AD patients compared to healthy subjects 92‘9” . The role
of homocysteine in AD pathology is further emphasized by the cell culture studies where
homocysteine caused oxidative stress, tau hyperphosphorylation and apoptosis in neurons
99' 100. There is a significant positive correlation between homocysteine and 4-
hydroxynonenal (4-HNE), a lipid peroxidation product, in AD ’0'. Furthermore, BACEl
and PS], two important enzymes involved in AB production have been shown to be
regulated by methylation. In context to this, increased homocysteine levels (caused by the
reduction of folate and vitamin BIZ in culture medium) cause a reduction of s-
adenosylrnethionine, the universal methyl-group donor, thus consequently increasing PSI

and BACEl levels '02. In this context, a positive correlation between elevated levels of

homocysteine and AB40 has been established in AD '03.

 

 

In addition to these supportive data, there are many studies that oppose any key role of
homocysteine in AD. It has been observed that although plasma homocysteine levels
were higher in AD cases than controls, this difference was not significant and

homocysteine levels were not related to cognitive status ’04

. This was further supported
by another study, which showed that high homocysteine levels were not associated with
AD and were not related to a decrease in memory scores over time '05. Furthermore,
homocysteine has been shown to potentiate copper-induced oxidative stress in primary
mouse neurons, but homocysteine alone had no effect '“.Thus, it is clear that the area of
homocysteine and AD needs to be further studied. We need additional, long-term studies

using a variety of populations to determine if elevated homocysteine level is a significant

and consistent risk factor for AD.

1.2.6 The Pathogen Hypothesis of AD

The pathogen hypothesis proposes a potential role of microbes such as herpes simplex
virus 1 (HSVl) and Chlamydophila pneumoniae (Cp) in the pathogenesis of AD. The
pathogens have been accepted to play central role in causing many diseases which earlier
had been thought to be non-infectious; Helicobacter pylori has been shown to cause
duodenal ulcers and gastric cancers ’07, which previously were thought of as the result of
stress, chemical irritants and genetic mutations. Also, human papillomavirus (HPV) virus
has been accepted to cause virtually all cases of cervical cancer '08. Furthermore, C.
pneumoniae has been recently suggested to play a role in atherosclerosis 109' ”0. In light
of these data, proponents of the pathogen hypothesis suggest its serious consideration in

the case of AD, given the fact that the dominant hypothesis in the AD field (the amyloid

10

 

cascade hypothesis) remains open to many questions. Interestingly, although various

studies showed the presence of HSVl and Cp in brains of AD patients “H”

, others
failed to confirm these data “6"”. These contradictory results may be attributed to the
fact that both HSVl and Cp are highly challenging to detect. Recently, an infection-based
animal model showed that intranasal inoculation of mice with Cp results in the formation

of amyloid plaques in their brain 120, thus further supporting the pathogen hypothesis of

AD. However, these data await further independent replication.

1.3 POTENTIAL INVOLVEMENT OF STURATED FATTY ACIDS

IN THE PATHOGENESIS OF AD

Epidemiological studies suggest that high fat diets significantly increase the risk of AD
and the degree of saturation of fatty acids is critical in determining the risk for AD ’2'.
This notion is further supported by various in vivo studies where mice fed a western, high
fat (21-40%)-high cholesterol (0.15-1%) diet developed AD—like pathophysiological

changes in their brain ”242"

. Diets rich in saturated fats may increase brain uptake of
intact free fatty acids (FF As) from the plasma through the blood brain barrier (BBB) 125;
the BBB is not a barrier for fatty acids ’26. Here, it is interesting to note that diabetes
mellitus, which is a significant risk factor for AD 127 is characterized by elevated plasma
levels of saturated FFAS '28. Due to the interaction between the FFA pools in the plasma
and the brain 125’ ’26’ ’29, diabetes-associated increases in plasma FFAS may affect the
level of F FAs in the brain and in turn increase the risk for AD. Likewise, traumatic brain

injury, which has been established as an independent risk factor for AD '30 is associated

with elevated levels of pahnitic, stearic and arachidonic acids in the brain '3’. Following

11

traumatic brain injury, palmitic acid increases from ~ 60 to 180 M and stearic acid from
~ 50 to 350 M ’32. In addition, the fatty acid profile of NFTS in AD brain has been
shown to be rich in palmitic and stearic fatty acids ’33. Similarly, the white matter in AD
brain is characterized by high fatty acid content ’34. Finally, apolipoprotein E4 (ApoE4) is
an important genetic risk factor for AD and its risk may be further increased by a
hyperlipidemic life-style 135. Despite these accumulating data, the basic mechanism of

how elevated levels of fatty acids are involved in the pathogenesis of AD is unclear.

1.4 GOALS OF THE PRESENT STUDY

The present study was undertaken with the following aims: I) investigate the possible
involvement of saturated fatty acids in causing the pathophysiological and metabolic
changes associated with AD; 2) establish the basic mechanism behind these potential,
fatty acid-induced abnormalities; and 3) understand the causal interrelation between the
neuropathological and metabolic changes. These data may be useful in identifying the
possible causal role of elevated levels of saturated fatty acids in the pathogenesis of AD

and may further help in identifying novel therapeutic targets.

1.5 THESIS OUTLINE

In line with the main focus of the present work to investigate a possible causal link
between saturated FFAS and AD-associated abnormalities, this dissertation is subdivided
as follows: Chapter 2 presents results describing the involvement of saturated FFAS in
causing oxidative stress and hyperphosphorylation of tan in neurons through astroglial

mediation. Chapter 3 describes the role of saturated FFAS in causing amyloidogenic

12

 

 

processing of amyloid precursor protein (APP) through astroglia-mediated oxidative
stress. In Chapter 4, FFA-induced abnormal glucose metabolism is studied. Metabolic
flux analysis (MFA) is applied to obtain a comprehensive picture of the global metabolic
changes caused by FFA treatment. This led to the identification of abnormal astroglial
sphingolipid metabolism as a pathway of interest in relation to the FFA-induced
pathophysiological changes observed in neurons. Chapter 5 further establishes the causal
role of astroglial ceramide in the FFA-induced, AD-associated pathophysiological
abnormalities in neurons. Chapter 6 presents the conclusions based on the present study

and future research directions.

13

CHAPTER 2. SATURATED FATTY ACID-INDUCED
HYPERPHOSPHORYLATION OF TAU IN PRIMARY RAT
CORTICAL NEURONS

2.1 INTRODUCTION

Neurofibrillary tangles (NFTS) are one of the classical neuropathological hallmarks of
AD brain 3. NFTS have been suggested to play a central role in AD pathogenesis since
they have been shown to correlate with the severity of dementia. According to a proposed
model of pathological evolution of AD, NFTS appear first in the entorhinal cortex during
the preclinical phase, spreading to the hippocampus in the middle phase and, eventually,
to the neocortex during the late stage of AD ’36. This strong correlation between the
number of NFTS and the disease severity was further supported by many other studies 137’
’38. NFTS are composed of paired helical filaments of tau protein, which is
hyperphosphorylated in AD '39. Physiologically, tan is one of the major microtubule-
associated proteins that stabilize the microtubules, which play important structural and

functional roles in the neurons (Figure 2.1A). Tau is involved in signal transduction 140’

14', anchoring various protein kinases and phosphatases 142' ”3

and interacting with the
actin cytoskeleton ”4. Six isoforrns of tau exist in the central nervous system, which are
derived from a single gene and vary between having 3 or 4 microtubule-binding repeat
domains and in the number and size of N-terminal inserts 145. Interactions between tau
and microtubules are regulated by the length and more importantly, phosphorylation of
the microtubule-binding repeat domains 146. Tau has about 30 possible phosphorylation

sites '47 and in its phosphorylated form cannot stabilize microtubules 148. In this context,

tan is phosphorylated to a degree of ~8 Pi/mol in AD as compared to ~2 Pi/mol for

14

normal tau and this hyperphosphorylation of tau in AD leads to the disruption of the
cytoskeleton of neurons, which in turn leads to their degeneration, thereby playing an

important role in AD pathology (Figure 2.13) 3.

 

(A) Control

 

 

 

    
   
 

  

Disintegrating
Mierotubule

 
 

:‘7 Diseased
“i Neuron

 

(B) AD

    

Disintegrating
Microtubules‘

 

‘ .4

Figure 2.1. The physiology and pathology of tau. (A) In healthy neurons, tau silzes mcrules.
(B) In AD, tau hyperphosphorylates and detaches from microtubules leading to tangle formation
(figure taken from www.alz.org).

The hyperphosphorylation of tau has been proposed to be critical in promoting
aggregation of tau in NFTS 149. The hyperphosphorylation of tau has been shown to
precede the formation of NFTS in degenerating neurons 150’ ’5'. The importance of
hyperphosphorylation of tau in its aggregation is further emphasized by additional studies
that showed that the aggregation of tau depends on the degree of its phosphorylation and
in vitro dephosphorylation of phosphorylated-tau from AD brain prevents its aggregation

’52. Furthermore, in human neuroblastoma cells phosphorylated but not the

unphosphorylated tau has been Shown to form tau filaments '53.

The hyperphosphorylation of tau has been suggested to be due to the imbalance between
the activities of protein kinases and phosphatases although the actual mechanism is still
not well understood. The kinases involved in tau phosphorylation are divided into two
groups, proline-directed protein kinases (PDPKs) and non-PDPKs 15". The PDPKs
include glycogen synthase kinase 3 (GSK—3), cycline-dependent kinase 5 (cdk5) and
mitogen activated protein kinases (MAPKS). The non-PDPKS include protein kinase C
(PKC), protein kinase A (PKA) and Ca2+/calrnodulin-dependent kinase II (CaMKII).
Different kinases may phosphorylate tau at different amino acid residues with some
overlapping of sites. The phosphatases involved in dephosphorylation of tau are divided
into two major groups, S or T site protein phosphatases and protein tyrosine
phosphatases, of which PP-l, PP-2A and PP-2B dephosphorylate tau with a certain
degree of overlap in sites ’5 5 '157. AD brain has been characterized by decreased activity of

158

phosphatases and increased activity of various kinases resulting in

hyperphoshorylation of tau "’8’ ”9"“. Increased activity of these tau phosphorylating

l6

kinases in AD has been suggested to be a direct result of increased oxidative stress '62.
Increased oxidative stress, manifested by increased lipid peroxidation and protein

163,164

oxidation is one of the major and earliest characteristics of AD brain 73' ’65. Lipid

peroxidation is an important aspect of AD brain and has been shown to be elevated in the

regions of brain affected in AD '66' ‘67

. There is a strong correlation between
thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, and
the presence of NFTS in AD brain ’66. Similarly, the levels of 4-hydroxynonenal (4-HNE)
and acrolein have been shown to be elevated in AD brain "’8' ’69. Furthermore, protein
oxidation is an inevitable aspect of aging and age-related neurodegenerative diseases 76'
'70. Protein oxidation is Significantly increased in the cortex and hippocampus of AD

brain as compared to age-matched healthy controls ”"172.

In addition to increasing the activity of various stress-dependent kinases, which are
involved in hyperphosphorylating tau thus leading to its aggregation, oxidative stress in
itself has been shown to be involved in forming tau dimers, which are the building blocks
of paired helical filaments (PHFs) in NFTS. The aggregation of tau is increased when tau
molecules crosslink into dimers by an oxidized disulfide bond at Cys322 173’ ’74.
Furthermore, 4-HNE, which has been shown to co-localize with NFTS in AD brain “’5’ ”’9'
’75, can act as an adduct to phosphorylated tau and enhance tau filament formation '53. In
summary, oxidative stress and hyperphosphorylation are important pathological events in
the formation of tau aggregates, which are one of the characteristics signatures of AD

brain. Therefore, the aim of the current study was to investigate possible involvement of

saturated fatty acids in causing oxidative stress and hyperphosphorylation of tau.

l7

2.2 MATERIALS AND METHODS

2.2.1 Isolation and Culture of Primary Rat Cortical Neurons and Astroglia

Primary cortical neurons were isolated from one-day-old Sprague-Dawley rat pups and
cultured according to the published methods as described in Chandler et al. ’76. The cells
were plated on poly-D-Lysine coated, 6-well plates at the concentration of 2 x 106 cells
per well in fresh cortical medium [Dulbecco's Modified Eagle's Medium (DMEM and all
other media are from Invitrogen, CA) supplemented with 10% horse serum (Sigma, MO),
25 mM glucose, 10 mM HEPES (Sigma), 2 mM glutamine (BioSource International,
CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycine]. Three days after incubation
(37°C, 5% C02), the medium was subsequently replaced with 2 ml of cortical medium
supplemented with 5 uM cytosine-B-arabinofuranoside (Arac, from Calbiochem, CA).
After 2 days, the neuronal culture was switched back to cortical medium without Arac.
The experiments were performed on 6-7 day old culture. To obtain primary cultures of
astroglial cells, the cortical cells from one-day-old Sprague-Dawley rat pups were
cultured in DMEM/Ham’s F12 medium (1:1), 10% fetal bovine serum (Biomeda, CA),
100 IU/ml penicillin, and 0.1 mg/ml streptomycin 177. The cells were plated on poly-D-
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% C02) and culture medium was changed every 2 days. 24
hours prior to treatment with fatty acids, the medium was changed to neuronal cell
culture medium.

2.2.2 Lactate Dehydrogenase (LDH) Assay

The secreted and intracellular levels of LDH were measured to determine the level of cell

toxicity in astroglial cells by using cytotoxicity detection kit (Roche, IN, USA). The

18

cytotoxicity was determined as the fraction of LDH released into the medium, normalized

to the total LDH (released + intracellular), as shown in the equation below-.

LDH (medium)
%LDH release = X 100
LDH (medium+intracellular)

2.3 Western Blot Analysis

 

For western blot analysis, cells were washed three times with ice-cold TBS (25 mM Tris,
pH 8.0, 140 mM NaCl, and 5 mM KCl) and lysed for 20 minutes by scraping into ice-cold
radioimmunoprecipitation assay (RIPA) buffer [1% (v/v) Triton, 0.1% (w/v) SDS, 0.5%
(w/v) deoxycholate, 20 mM Tris, pH 7.4, 150 mM NaCl, 100 mM NaF, 1 mM Na3VO4,
1 mM EDTA, 1 mM EGTA, and 1 mM PMSF, all chemicals from Sigma] ’7’. The total
cell lysate was obtained by centrifugation at 12,000 rpm for 15 minutes at 4 0C. The total
protein concentration was measured by BCA protein assay kit from Pierce (Rockford,
IL). Equal amounts of total protein from each condition were run at 200 V on 10% SDS-
PAGE gels (BioRad, CA) for phosphorylated tau and actin. The separated proteins were
transferred to nitrocellulose membranes for 1 hour at 100 V and incubated at 4 0C
overnight with the appropriate primary antibodies [1:200 PHF-l (from Dr. P. Davies,
Albert Einstein, NY), 1:200 AT8 (Pierce Biotechnology, IL), 122000 actin (Sigma, MO)].
Blots were washed three times in PBS-Tween (PBS-T) and incubated with appropriate
HRP-linked secondary antibodies (Pierce Biotechnology, IL) diluted in PBS-T for 1 hr.
After an additional three washes in PBS-T, blots were developed with the Pierce
SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) and
imaged with the BioRad ChemiDoc. Quantity One software from Bio-Rad was used to

quantify the signal intensity of the protein bands.

19

2.2.4 Immunofluorescence Analysis of Neurons and Astroglia

To perform confocal immunofluorescence microscopy study, neurons and astroglia
cultures were fixed for 20 minutes in 4% paraforrnaldehyde and permeabilized with 0.1%
Triton X-100 and 5% goat serum (Invitrogen) in PBS. Cells were then labeled overnight
at 4 0C with appropriate primary antibodies [1:50 MAP-2 (Santa Cruz Biotechnology,
CA) for neurons, 1:1000 GFAP (Dako, CA) for astroglia and 1:200 AT8 for
hyperphosphorylated tan] in 5% goat serum in PBS. After three PBS washes, primary
antibodies were detected with rhodamine conjugated (Chemicon, CA) or Alexa Flour 594
conjugated (Molecular Probes, OR) secondary antibodies. The cells were visualized with
the confocal microscope Zeiss LSM 5 Pascal (Carl Zeiss, Jena, Germany) using a 40X

(for MAP-2 and GFAP) or 63x (for AT8) oil-immersion objective lens.

2.2.5 Immunostaining of Reactive Oxygen Species (ROS)

Intracellular reactive oxygen species (ROS) were detected by staining with the oxidant-
sensitive dye 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-
HzDCFDA, from Molecular Probes, OR). HzDCFDA is cleaved of its ester groups by
intracellular esterases and converted into membrane impermeable, nonfluorescent
derivative HzDCF. Oxidation of HzDCF by ROS results in highly fluorescent 2,7-
dichlorofluorescein (DCF) '79. The cells were incubated for 30 minutes at 37 0C with 2
uM CM-HzDCFDA in Hanks’ Balanced Salt Solution without phenol red (Invitrogen).
The cells were then washed three times with PBS and analyzed with confocal

microscopy. A 63X oil-immersion objective lens was used for data acquisition.

20

2.2.6 Data Analyses

Data are shown as means : SD. for indicated number of experiments. Student’s t-test
and one-way ANOVA with Tukey’s post hoc method were used to evaluate statistical

significances between different treatment groups. Statistical significance was set at

p<0.05.

2.3 RESULTS AND DISCUSSION

2.3.11 Direct Treatment of Neurons with Saturated Fatty Acids

Although tan is present in astroglia and oligodendrocytes in the brain and in some
peripheral tissues, it is mainly synthesized by neurons "’0’ ’8'. Tau is mainly located in
neuronal axons and hyperphosphorylated tau is deposited in dystrophic neurites and
neuronal bodies in the form of NFTS. Therefore, to examine the possible involvement of
saturated FFAS in the hyperphosphorylation of tau, primary rat cortical neurons were left
untreated or treated with 0.2 mM of palmitic acid (PA) or stearic acid (SA) for 24 hours.
After 24 hours, the cells were lysed and western blot analysis was performed to
determine the cellular levels of hyperphosphorylated tau. There was no change in the
levels of phosphorylated tan in rat cortical neurons treated directly with pahnitic or
stearic acid, as compared to controls (Figure 2.2). Furthermore, the morphology of the
neurons was not affected by the FFA treatment, as shown by MAP-2 immunostaining
(Figure 2.3A). The observed lack of F FA effect on neurons may be attributed to the low

capacity of primary neurons to take up and metabolize saturated fatty acids ’82.

21

Previously, primary rat cortical neurons have been shown to possess a very low capacity

to take up PA and incorporate it into glycerolipids and sphingolipids '82.

Control PA SA

PHF-1 -~-
AT8 it” “he—a In...

Is-actin m

Figure 2.2. Direct treatment of neurons with saturated FFAs. Primary rat cortical neurons were treated
for 24 hours with 0.2 mM of palmitic acid (PA) or stearic acid (SA) or with 5% bovine serum albumin
(BSA), vehicle for FFAS (control). Detergent cell lysates from fatty acid-treated and control cells were
immunoblotted with PHF-l and AT8 antibodies, which recognize phosphorylated tau. B-actin is shown as a
marker for protein loading.

2.3.2 Involvement of Saturated FFAS in Tau Hyperphosphorylation Through

Astroglial Mediation

Compared to primary neurons, primary astroglia possess a significantly higher capacity to

182

utilize saturated fatty acids . The uptake and incorporation of PA into glycerolipids and

sphingolipids have been Shown to be more than 3 times higher in primary rat cortical

astroglia as compared to primary rat cortical neurons '82.

Therefore, we treated cortical
astroglia with 0.2 mM of PA or SA for 12 hours and transferred the conditioned media to
treat the cortical neurons for 24 hours. The morphologies of the fatty acid-treated
astroglia and neurons are shown using GFAP and MAP-2 immunostaining, respectively
(Figures 2.38 and C). No significant change in the cell morphologies was observed,

except, the characteristic dotted MAP-2 labeling of the neurites was less significant in the

22

neurons treated with conditioned media from FFA-treated astroglia as compared to

controls.

 

(A) MAP-2

Figure 2.3. MAP-2, GFAP and AT8 immunostaining. (A) Neurons treated directly with FFAS l'or 24
hours (B) Astrocytes treated for 12 hours with 0.2 mM of either palmitic acid (PA) or stearic acid (SA) or
5% BSA (control). (C) Neurons treated for 24 hours with conditioned media from fatty acid-treated or
control astrocytes. (D) Immunot‘luorcsccncc labeling of phosphorylated tau with AT8 antibody in neurons
treated with conditioned media from fully acid-treated or untreated astrocytes (control). Images were
obtained with confocal fluorescence microscopy. (Objective lens magnification- 40X l‘or MAP-2 and
GFAP, 63X for AT8)..

Furthermore, treatment of astroglia with 0.2mM of PA did not affect the astroglial cell
viability compared to controls as measured by % LDH release (Figure 2.4). 300 IIM

H202 treatment was used as a positive control and significantly increased LDH release

23

from astroglia (Figure 2.4), which is in accordance with a previous study in the literature

183

50 *
451
40-
35-
30-
’75..
20-
151
10-
5..
0+

%LDH released

   

 

Control PA 300uM H202

Figure 2.4. Measurement of LDH release from astroglia treated with PA. 24hr treatment with 0.2mM
PA failed to liberate LDH from astroglia as compared to controls. In contrast, 1hr treatment of astroglia
with 300 uM H202 (positive control) induced significant LDH liberation after 24h as compared to both
control and PA-treated cells. Data are taken from 3 different experiments and are expressed as mean i S.D.
One-way ANOVA with T‘ukey’s post hoc method was used for analyzing the differences between treatment
groups. *, p<0.05 compared with control.

The conditioned media from FFA-treated astroglia caused significant
hyperphosphorylation of tan in cortical neurons, as observed by immunostaining with
AT8 (Figure 2.3D) and immunoblotting with AT8 and PHF-l antibodies Gilgure 2.5).
AT8 and PHF-l antibodies recognize tau protein hyperphosphorylated at AD-specific

184

phospho-epitopes; AT8 recognizes tau phosphorylated at Ser202 and Thr205 , while

PHF-l is specific for tau phosphorylated at Ser396 and Ser404 ”‘5. Ser202, Ser396 and

24

Ser404 are three of the nine abnormal phosphorylation sites of the hyperphosphorylated

tau associated with NFTS in AD '36.

(A)
C PA SA PA+NAC SA+NAC
PHF-l - u ,- -
AT8 “"“ ~ - — -1

Actin

(B)

Pbospho-Tau
(% of control)

  

 

PA+NAC SA+NAC

400‘ AT8
350. _ .
m.

Phospho—Tau 250 i

(% of control) 200 1 # #
150 ‘
100 .
50 J

0
C PA SA PA+NAC SA+NAC

 

Figure 2.5. Astroglia-mediated, fatty acid-induced hyperphosphorylation of tau in neurons. (A)
Western blot analysis of hyperphosphorylated tau was performed using phospho—specific antibodies PHF-l
and AT8. (B) Histograms corresponding to PHF-l and AT8 blots represent quantitative determinations of
intensities of the relevant bands. Data represent mean in SD. of 3 independent experiments. One-way
ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment groups.
‘, p<0.05 compared with control; #, p<0.05 compared with fatty acid treatment.

25

2.3.3 Involvement of Oxidative Stress in FFA-Astroglia-Induced Tau
Hyperphopshorylation in Neurons

As shown in Figure 2.6A, treatment of astroglia with 0.2mM of PA or SA did not cause
any ROS production in cortical astroglia. On the other hand, we found that intracellular
levels of ROS were elevated in the neurons treated with conditioned media from FFA-

treated astroglia as compared to controls (Figure 2.68).

(A)

Control

 

(3)

control

PA+NAC

     

Figure 2.6. Intracellular accumulation of ROS in neurons. (A) Astroglia and (B) neurons were stained
with CM-HZDCFDA for intracellular ROS detection and examined with confocal fluorescence microscopy.
(Objective lens magnification, 63X).

26

Previously, various in vitro and in vivo studies have implicated increased oxidative stress
in the hyperphosphorylation of tau 7” 72. 4-HNE and acrolein, the lipid peroxidation
products found elevated in AD brain 73, have been shown to induce hyperphosphorylation

74, 75

of tan in neurons . Furthermore, although acute administration of a high

concentration of H202 (lmM for 1 hr) decreases tau phosphorylation '87

; chronic
exposure of a low concentration of H202 (10 [AM for 24, 48 or 72 hrs), more relevant to
AD, increases tau phosphorylation in primary rat cortical neurons (Mark Smith and
Xiongwei Zhu, personal communication). Therefore, we treated neurons with lOmM N-
acetyl cysteine (NAC), an anti-oxidant. The co-treatment of neurons with NAC inhibited

the observed FFA-astroglia-induced increase in ROS levels (Figure 2.68) as well as tau

hyperphosphorylation in neurons (Figure 2.5).

2.4 CONCLUSIONS

In conclusion, saturated F F As had no direct effect on the neuronal morphology and the
level of phosphorylated tau in neurons. In addition, saturated FFAS had no effect on
astroglial morphology and viability. However, the conditioned media from FFA-treated
astroglia affected the classical dotted MAP-2 labeling of the neuronal axons and also
increased the levels of phosphorylated tan in neurons. Furthermore, there was a
significant increase in the ROS production in neurons treated with conditioned media
from FFA-treated astroglia, without any increase in astroglial ROS levels. The elevated
levels of ROS in the neurons were found to be involved in the observed, FFA-astroglia-
induced hyperphosphorylation of tan in the neurons. Thus, the present results establish a
central role of saturated FFAS in causing hyperphosphorylation of tau in neurons through

astroglia-mediated oxidative stress.

27

CHAPTER 3. SATURATED FATTY ACID-INDUCED
AMYLOIDOGENIC PROCESSING OF APP IN PRIMARY
RAT CORTICAL NEURONS

3.1 INTRODUCTION

The “amyloid cascade hypothesis”, which suggests the accumulation of aggregated
amyloid beta (AB) in the brain as a main trigger for AD, has been extensively studied
since the first characterization of AB deposits in 1984 “’8. According to this hypothesis, a
chronic imbalance between the production and clearance of AB results in the formation of
AB plaques, which leads to a multi-step cascade including reactive gliosis, inflammatory
changes, synaptic change and transmitter loss 2" ”9"”. In AD brain, two major types of
AB plaques are observed, diffuse plaques and neuritic plaques. Diffuse plaques mainly
consist of nonfibrillar AB, while neuritic plaques are more developed consisting of dense
AB fibrils together with degenerating dendrites and axons, serum amyloid P, (11-
antichymotrypsin, al-antitrypsin, sulphated glycosaminoglycans, apolipoproteins E and
D, and the neurotrophic factor midkine ”3"”. Recently, the soluble AB intermediates
have been shown to play a more important role in AD pathogenesis as compared to the
mature neuritic plaques '90' '96' '97. These soluble AB oligomers, AB protofibrils and AB-
derived diffusible ligands (ADDLS) cause synaptic dysfunction, which have been

suggested to be an early event in AD-associated memory loss '98.

AB is generated from the proteolytic processing of amyloid precursor protein (APP). APP
is an integral type I membrane glycoprotein of 110-120kDa in size and contains a large

amino terminal extracellular domain and a small COOH-terrninal intracellular domain "’9‘

28

20°. APP has three major isoforms containing 695, 751 or 770 amino acids. The APP 69,
isoform is mostly present in neurons, while the others are present in peripheral and glial
cells 20'. APP m and APP 770 have serine protease inhibitor domain called Kunitz protease
inhibitor domain, while APP ,9, lacks this domain. The physiological functions of APP
include neuronal survival, cell adhesion, axonal adhesion, neuritic outgrowth, synaptic

plasticity and signaling "202.

The proteolytic processing of APP takes place by sequential cleavage by proteases named
01-, B- and y-secretase (Figure 3.1A). The a-secretase is a member of the ADAM (a
disintegrin and metalloprotease) family such as ADAM17 or TACE (tumor necrosis
factor-a converting enzyme), ADAM 9, ADAMIO, MDC9 and an aspartyl protease
BACE2 20’. The ct—secretase cleavage of APP may occur at the cell surface, within
calveolae or in the trans-Golgi compartment "’0' 20". The a-secretase has been shown
mainly to be active in non-raft regions of the membrane 2°" 20". The a-secretase cleaves
APP within AB domain (shown as red) between residues Lysl6 and Leul7, thus avoiding
the generation of intact AB peptides. It leads to the formation of a soluble domain
(SAPPa), which is released into extracellular space and a lO-kDa C-tenninal fragment

(C83), which remains within the cellular membrane 207.

The B-secretase, also called BACE (B-site of APP cleaving enzyme), Asp-2 or
memapsin-2 is a trans-membrane protein and an aspartic-acid protease 20“”. BACE

contains aspartate residues in its extracellular protein domain which are involved in

BACE activity 20”.

29

 

AMA MR c...

/ K

APPSB

  
  

M

if" tit

i Liideaft

(A) (B)

Flgu3re ”lProcmingo ofa amylororidpecurs pro i (APP)()Th n-aomylioesdge cawpth ay
catalyz edbyu -andy- steecreas .e(B)Th eoioenamyldg icpatwh ayc atazely zedbyBoa an-sedy setacre

 

30

BACE] is a major B-secreatse involved in the amyloidogenic processing of APP in
neurons 2”. Cleavage of APP by B-secretase may occur in endosomes and trans-Golgi

compartments 212-214

, which provide an acidic environment that has been shown to be
critical for maximal activity of BACE 2". The B-secretase has been shown mainly to be
active in lipid raft regions of the membrane 206' 2'6' 2”. The B-secretase cleaves APP at the

Asp+1 residue of the AB region and leads to the generation of a secreted soluble fragment

(SAPPB) and a membrane-bound C-terminal fragment (C99).

Both the a-secretase product of APP (C83) and the B-secretase product of APP (C99) act
as immediate substrates for y-secretase. The y-secretase is a membrane-bound complex of
at least four enzymes including components such as Presenilin 1 and 2 (PSI and PS2),
Nicastiin (th), anterior pharynx-defective phenotype (APH-l) and Presenilin enhancer
(PEN-2) 2". The PS1 C-terminal tail (PSl-CTF) is critical for y-secretase activity “9‘ 22°.
The y —secretase activity resides in various cellular compartments such as the ER, late-
Golgi/trans-Golgi, endosomes and the plasma membrane ”"22". Similar to the B-
secretase, the y-secretase has been associated with lipid raft microdomains of the
membrane 22’. The y-secretase cleavage of ‘C83 is a non-amyloidogenic pathway, which
leads to the generation of a short peptide (p3) containing the C-terminal domain of the
AB peptide. The physiological or pathological significance of p3 remains unclear. On the
other hand, the y-secretase cleavage of C99 is an amyloidogenic pathway, which leads to
the generation of a spectrum of AB peptides. The AB peptides containing 40 or 42 amino
acids (AB40/42) are the two most common amyloidogenic AB peptides. Both AB40 and

AB42 are produced during normal cellular metabolism but the production of AB42 is

31

considered to be elevated in AD 226' 227. AB42 is more prone to aggregation as compared
to AB40 228‘ 229. AB42 initially forms non-filamentous, diffuse plaques onto which AB40

starts to aggregate, which leads to the mature, neuritic plaques.

The importance of amyloidogenic processing of APP in AD is emphasized by the
involvement of mutations in the APP, presenilin-l (PSI) and presenilin-2 (P82) genes,
localized on chromosome 21, 14 and 1, respectively, in FAD ”0'23". These APP and PS
mutations alter APP processing leading to the pathological increase in the production of
total AB or AB42 which is highly fibrilogenic “'2" “’9. In APP mutations linked to FAD,
clinical and pathological symptoms are identical to those of the late-onset sporadic AD,
which consists of more than 95% of total AD cases. This strongly suggests that
amyloidogenic processing of APP plays a central role not only in FAD but also in
sporadic AD. Furthermore, ApoE4, a genetic risk factor for AD, has been shown to play
an important role in the production and clearance of AB 2’7. This further suggests that
amyloidogenic processing of APP is a central pathological event in AD pathology. In this
context, the aim of the current study was to investigate possible involvement of saturated
fatty acids in causing amyloidogenic processing of APP, potentially by affecting the

levels and/or activities of B- and y-secretases.

32

3.2 MATERIALS AND METHODS

3.2.1 Isolation and Culture of Primary Rat Cortical Neurons and Astroglia

Primary neurons and astroglia were isolated from the cortex of one-day-old Sprague—
Dawley rat pups and cultured according to the methods as described in chapter 2. The
cells were plated on poly-L-Lysine coated, 6-well plates at the concentration of 2 x 10'5
cells per well in fresh cortical medium [Dulbecco's Modified Eagle's Medium (DMEM
and all other media are from Invitrogen, CA) supplemented with 10% horse serum
(Sigma, MO), 25 mM glucose, 10 mM HEPES (Sigma), 2 mM glutamine (BioSource
International, CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin] ’76. To obtain pure
neuronal cell cultures, after 3 days of incubation (37°C, 5% C02) the medium was
replaced with the cortical medium supplemented with 5 uM cytosine-B-arabinofuranoside
(Arac, from Calbiochem, CA). After 2 days of Arac treatment, the neuronal culture was
switched back to cortical medium without Arac. The neuronal cell culture of more than
95% was obtained by this procedure. The experiments were performed on 6-7 day old
neuronal culture. To obtain primary cultures of astroglial cells, the cortical cells from
one-day-old Sprague-Dawley rat pups were cultured in DMEM/Ham’s F12 medium
(1:1), 10% fetal bovine serum (Biomeda, CA), 100 IU/ml penicillin, and 0.1 mg/ml
streptomycine '77. Cells were grown for 8-10 days (37°C, 5% C02) and culture medium
was changed every 2 days. The astroglial cell culture of more than 95% was obtained by
this procedure. 24 hours prior to treatment with fatty acids, the medium was changed to

neuronal cell culture medium.

33

3.2.2 Western Blot Analysis

For western blot analysis, cells were washed three times with ice-cold TBS (25 mM Tris,
pH 8.0, 140 mM NaCl, and 5 mM KCl) and lysed for 20 minutes by scraping into scraping
into ice-cold radioimmunoprecipitation assay (RIPA) buffer [1% (v/v) Nonidet P-40,
0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 50 mM Tris, pH 7.2, 150 mM NaCl, 1 mM
Na3VO4 and lmM PMSF, all chemicals from Sigma] 238. The total cell lysate was
obtained by centrifugation at 12,000 rpm for 15 minutes at 4 0C. The total protein
concentration was measured by BCA protein assay kit from Pierce (Rockford, IL). Equal
amounts of total protein from each condition were run at 200 V on 10% Tris-HCl gels
(for BACEl , actin), 12% Tris-HCl gels (for PS1) and 10-20% Tris-Tricine gels (for APP,
C99). The separated proteins were transferred to nitrocellulose membranes for 1 hour at
100 V and incubated at 4 0C overnight with the appropriate primary antibodies [1:1000
BACE] (chemicon, CA), 1:1000 PS1 (Calbiochem, CA), 1:2000 actin (Sigma, MO),
1:1000 APP/C99 (QED Bioscience Inc, CA)]. Blots were washed three times in PBS-
Tween (PBS-T) and incubated with appropriate HRP-linked secondary antibodies (Pierce
Biotechnology, IL) diluted in PBS-T for 1 hr. After an additional three washes in PBS-T,
blots were developed with the Pierce SuperSignal West Femto Maximum Sensitivity
Substrate (Pierce Biotechnology) and imaged with the BioRad ChemiDoc. Quantity One

software from Bio-Rad was used to quantify the signal intensity of the protein bands.

3.2.3 Immunofluorescence Analysis of BACEl in Neurons

To perform confocal immunofluorescence microscopy study, neuronal cultures were

fixed for 20 min in 4% paraforrnaldehyde and permeabilized with O. 1% Triton X-100 and

34

5% goat serum (Invitrogen) in PBS. Cells were then labeled overnight at 4 0C with the
primary antibody [1:100, BACEl] in 5% goat serum in PBS. After three PBS washes,
primary antibody was detected with Alexa Flour 594 conjugated (Molecular Probes, OR)
secondary antibody. The cells were visualized with confocal microscope Zeiss LSM 5

Pascal (Carl Zeiss, Jena, Germany) using a 40x oil-immersion objective lens.

3.2.4 Data Analyses
Data are shown as means :9: SD. for indicated number of experiments. Student’s t-test
and one-way ANOVA with Tukey’s post hoc method were used to evaluate statistical

significances between different treatment groups. Statistical significance was set at

p<0.05. ‘

3.3 RESULTS

3.3.1 Direct Treatment of Neurons with Saturated Fatty acid (Palmitic Acid)

The BACEl enzyme involved in the first step of amyloidogenic processing of APP has
been localized in the brain, mainly in the neurons, thus suggesting that neurons are the
prominent source of AB peptides in the brain 239. The BACEl levels have been shown to

240. Therefore, to

be significantly elevated in AD brain as compared to healthy controls
examine the possible effect of saturated FFAS on the BACE] levels, primary rat cortical
neurons were left untreated or treated with 0.2mM of palmitic acid (PA) for 24 hours. In
this and all subsequent studies, we treated cells with only PA, since both PA and SA had

similar effects as demonstrated in chapter 2. Also, in typical high-fat American diets,

35

more than 60% of the saturated fat is PA, while SA contributes only 25% of the dietary
energy derived from saturated fats 2‘“. After 24 hrs of direct treatment with PA, the cells
were lysed and western blot analysis was performed to determine the cellular levels of
BACE]. There was no change in the BACEl levels in primary rat cortical neurons
treated directly with PA as compared to controls (Figure 3.2). This lack of FFA effect on
neurons may be attributed to the low capacity of primary neurons to take up and
metabolize saturated fatty acids '82. This effect is similar to that shown in Chapter 2,
where saturated FFAS had no direct effect on neurons; there was no change in the levels

of phosphorylated tan in rat cortical neurons treated directly with both PA and SA.

BPCEI “'""‘"" M —70Koa

Actin ‘- ~—42 KDa
Control PA

Figure 3.2. Direct treatment of neurons with palmitic acid. Primary rat cortical neurons were treated for
24 hours with 0.2mM of palmitic acid (PA) or with 4% bovine serum albumin (BSA), vehicle for PA
(control). Detergent cell lysates from PA-treated and control cells were immunoblotted with BACE]
antibody. B—actin is shown as a marker for protein loading. The blots are representative of 3 independent
experiments.

3.3.2 Involvement of Saturated FFAS in BACE] Up-regulation and

Amyloidogenic Processing of APP Through Astroglial Mediation

As mentioned earlier in Chapter 2, primary astroglia possess a significantly higher

182

capacity (>3 times) to take up and utilize saturated fatty acids and the conditioned

media from FFA-treated astroglia significantly increased phosphorylation of tau in

36

neurons. Therefore, in this study, we first cultured the rat cortical astroglia with 0.2mM
PA for 12 or 24 hrs and transferred the conditioned media to treat the cortical neurons for
24 hours. The conditioned media from PA-treated astroglia induced BACEl upregulation
in cortical neurons, as observed by immunofluorescence imaging (Figure 3.3) and

immunoblotting (Figure 3.4).

   

Control PA

Figure 3.3. BACEl immunostaining. lmmunofluorescence labelling of BACEI in neurons treated for 24
hours with conditioned media from PA-treated (for 24 hours) or untreated astrocytes (control). Images were
obtained with confocal fluorescence microscopy (objective lens magnification- 40X).

The PA-induced BACEl upregulation was observed to be dependent on the length of
time that the astroglia were treated with PA, which might be attributed to the time-
dependent increase in PA metabolism by the astroglia '82. BACEl cleaves APP at the
major Asp+l site and minor Glu+l I site to generate C99 and C89 fragments, respectively
209. Accordingly, we found increased C99 levels in the PA-astroglia-treated cortical

neurons as compared to controls (Figure 3.6). C-tenninal fragments of APP are found to

37

(A)
BACEl a” “I“? w 1'

Actin "1“"

C PA C PA
12 hrs 24 hrs

 

(B)

l 80

l 60

140

BACEI l 20
(% Control) 1 00
80

60

40

20

 

   

C PA C PA
12 hrs 24 hrs

Figure 3.4. Astroglia-mediated, PA-lnduced BACE] upregulation in neurons. Astrocytes were treated
for 12 and 24 hours with 0.2mM of PA or 4% BSA (control), followed by transfer of the astrocytes-
conditioned media to neurons (24 hours treatment). (A) Western blot analysis of BACE] protein levels in
neurons. B-actin is shown as a marker for protein loading. (B) Histograms represent quantitative
determinations of intensities of the relative bands. Data represent mean at SD. of three independent
experiments. Student’s t-test was used for analyzing differences between different treatment groups. ‘,
p<0.05 compared with respective control.

 

be elevated in AD brain and are more toxic to neurons than AB, which is obtained by
cleavage of C99 by y-secretase 242. The presenilin (PS) complex, including PS, nicastrin,
APH-l and PEN-2, forms a central core of the y-secretase enzyme and the PSI C-

218

terminal tail (PSl-CTF) is critical for y-secretase activity . We found no change in the

38

levels of PSl—CTF suggesting the y-secretase activity is unchanged in the PA-astrocytes-
treated neurons as compared to controls (Figure 3.5). It is noteworthy that a slight
increase in BACEl levels, without any change in y-secretase activity, has been shown to
increase AB production significantly 243. Thus, the increased BACE] levels in the PA-
astrocyte-treated cortical neurons resulting in elevated C99 levels, may be followed by

increased AB production, despite the lack of change in y-secretase activity.

P51 ‘ “ ' --- —98 KDa

PSl-CTF F—_‘
—21 KDa

Control PA PA+DMU

Figure 3.5. Immunoblot analysis of presenilin-l (PSI) levels in neurons treated with astrocytes-
conditioned media. Astrocytes were treated for 24 hours with 0.2mM PA or 5% BSA (control), followed
by transfer of the astrocytes-conditioned media to neurons (24 hours treatment), with or without 10 mM
DMU. Detergent cell lysates from PA-treated and control cells were immunoblotted for PS] and PSl-CTF
levels. The immunoblot is representative of 3 independent experiments.

3.3.3 Involvement of Oxidative Stress in FFA-Astroglia-Induced BACEl Up-

regulation and Amyloidogenic processing of APP in Primary Neurons
As shown in Chapter 2, intracellular levels of reactive oxygen species (ROS) were
significantly elevated in the neurons cultured with the conditioned media from FFA-

treated astroglia as compared to controls. To investigate the possible involvement of

39

oxidative stress in FFA-induced BACEl up-regulation and amyloidogenic processing of
APP, we treated neurons with 1,3-dimethyl urea (DMU), an antioxidant. The co-
treatment of neurons with lOmM DMU inhibited PA-astroglia-induced BACE]

upregulation and increased C99 production in neurons (Figure 3.6).

   

(A) (B)
203
183
183
._ ._ ,..-. m
BACE] 1:
Actin - ~ ~ . 2
C PA PA+DMU 2: ..
PA+DMU

ppp _I--« b-‘N ‘33

3k

10
. *Ilt
' C99/ 1m
APP an
E)
- 0
C99 ‘0‘” k ‘. 23
0 A ._ __7 V
C PA

C PA PA+DMU PA+DMU

Figure 3.6. Oxidative stress Involved in astroglia-mediated, PA-induced elevations in BACE] and
C99 levels in neurons. Astrocytes were treated for 24 hours with 0.2mM PA or 5% BSA (control),
followed by transfer of the astrocyteSoconditioned media to neurons (24 hours treatment), with or without
lOmM DMU. (A) Western blot analysis of BACE], APP and C99 protein levels in neurons. (B)
Histograms represent quantitative determinations of intensities of the relative bands. Data represent mean :l:
8.5. of three independent experiments. One-way ANOVA with Tukey’s post hoc method was used for
analyzing the differences between treatment groups. ‘, p<0.05 compared with control; '”' p<0.05 compared
with PA treatment.

This suggests a central role of astroglia-mediated oxidative stress in PA-induced
upregulation of BACEl and increased production of C99 in neurons. The role of
oxidative stress in causing increased amyloidogenic processing of APP is further

supported by various in vitro and in vivo studies 244. Oxidative stress has been shown to

40

increase the expression and activity of BACEl in NT2 neurons, which was accompanied
by a proportional elevation of the c-terminal fragments of APP 65’ 66. H202 and UV

irradiation have been shown to increase production of AB peptides “'70

, and antioxidants
Trolox and dirnethyl sulfoxide blocked stress-induced AB production 70. It is noteworthy

that AB itself can cause oxidative stress and increase its own production 69, thus

sustaining a vicious cycle 68.

3.4 CONCLUSIONS

In conclusion, PA had no direct effect on BACEl levels and the amyloidogenic
processing of APP in neurons. On the other hand, the conditioned media from PA-treated
astroglia significantly increased BACEl levels in neurons, which was dependent on the
length of time that the astroglia were treated with PA. This emphasizes a central role of
PA metabolism by astroglia in the observed pathological effects in neurons. Furthermore,
elevated BACEl increased amyloidogenic processing of APP, as evident by increased
levels of C99 in PA-astroglia-treated neurons as compared to controls. Treatment of
neurons with anti-oxidant blocked PA-induced abnormalities in neurons. Thus, the
present study illustrates that elevated levels of saturated fatty acids play an important role
in the up-regulation of BACE] and consequent amyloidogenic processing of APP

through astroglia-mediated oxidative stress.

41

CHAPTER 4. SATURATED FATTY ACID-INDUCED
ABNORMAL METABOLIC CHANGES ASSOCIATED
WITH ALZHEIMER’S DISEASE

4.1 INTRODUCTION

In addition to the two major pathophysiological lesions mentioned earlier (AB plaques
and NFTS), AD pathology is also characterized by abnormal metabolic changes.
Decreased cerebral glucose metabolism is a distinct characteristic of AD 4’ 5. In AD, brain
glucose utilization and ATP formation are decreased significantly (approximately 46%
and 19% respectively) as compared to healthy controls 245. In vivo imaging of AD brains
using positron emission tomography (PET) with 2-[F-l 8]-fluoro-2-deoxy-D- glucose as a
label shows progressive reduction in brain glucose metabolism, which is fiirther
correlated with disease severity 246. In accordance with this, glucose hypometabolism has
been suggested to be an important marker for early diagnosis of AD 4. The metabolic
abnormalities observed in AD are widespread in that even the peripheral cells
(fibroblasts) from AD patients show decreased metabolic activities 247’ 248. The activities
of various metabolic enzymes, mainly pyruvate dehydrogenase, a-ketoglutarate
dehydrogenase, glutamine synthetase, creatine kinase, aconitase and cytochrome oxidase
have been shown to be decreased in AD 249'253. Interestingly, decreased cytochrome
oxidase activity in post-mortem brain tissue from AD has been shown to be particularly
located in NFT-bearing neurons 254’ 255. The decreased activities of these enzymes may be
attributed to the increased oxidative stress in AD, as these enzymes are highly vulnerable
to oxidative modification 256. Also, patients with mild cognitive impairment (MCI),

which is characterized by reduced glucose metabolism, often develop AD 257. Finally, in

42

patients that are genetically predisposed to AD, cerebral metabolic changes occur well

before any pathophysiological signs of the disease manifest 258.

Glucose is a very important substrate in the efficient physiological functioning of the
brain. Glucose, through cellular glycolysis, produces pyruvate that is further oxidized to
acetyl-CoA. Acetyl-CoA is utilized by cells to produce cholesterol, acetylcholine and
ATP 259. Thus, decreased glucose metabolism in AD may result in decreased production
of these important cellular metabolites. Cholesterol is a primary sterol in cellular
membranes and is important in the production of various neurosteroids 26°. Acetyl choline
is a very important neurotransmitter involved in various cognitive functions, which have
been shown to be decreased in AD 26'. In AD, the activity of choline cetyl transferase has
been shown to be decreased in the presynaptic cholinergic neurons which might be
attributed to decreased availability of acetylcholine 262. Muscarinic M1/M3 acetylcholine
receptors have been shown to be involved in regulating APP processing and decreased
acetylcholine levels may induce increased amyloidogenic processing of APP 263. In this
context, degeneration of cholinergic system in AD has been correlated with disease
severity 264. Finally, ATP is a cellular energy currency required for various cellular
functions such as synthesis, folding, transport and degradation of proteins, maintenance
of ion homeostasis and synaptic transmission among others 265. Experimental evidence
suggests that decreased glucose metabolism and energy production may lead to increased
amyloidogenesis and hyperphosphorylation of tau 266'270. Taken together, these data
suggest that abnormal cerebral metabolism is central to AD pathology and may precede

the neuropathological changes associated with the disease '0 .

43

Traditionally, it is considered that glucose in the brain is mainly metabolized by neurons
and it is the substrate of choice for most activity-associated neuronal metabolism (Figure
4.1) 27‘. However, recent data suggest that astroglia take-up and metabolize glucose and
produce lactate, the latter may be used by neurons as a fuel for metabolic activities and
energy production (Figure 4.2) 27"273. In this context, the aim of the current study was to
investigate the possible involvement of saturated fatty acids in causing abnormal glucose
metabolism in both neurons and astroglia. Furthermore, we also focus on FFA-induced
global metabolic changes in astroglia and their possible involvement in observed FFA-
astroglia-induced ROS production in neurons, which in turn is involved in tau

hyperphosphorylation and amyloidogenic processing of APP as discussed in chapters 2

 

and 3.
NI! «rm- Juana-{n
Gl..cn.~.r: ‘
:‘nmr-u- ‘ (L ..l 3 lil l.’ t ‘ Hut-nan a
Cl .17 I
(in
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. z) p ' p p In
t Eh I U)
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Pyramtn Pin-.116

‘0 1
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l 3H 5:
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,
'CA J4 '18!”-' .1! "IRA'P TCI
. l v v '
(yelp - Iv'fl . A
05: , 3 a my- GP
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ADP - P

 

 

 

 

Figure 4.1. Conventional view of cerebral glucose metabolism.

44

 

 

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Figure 4.2. Cerebral glucose metabolism based on the novel astrocyte-neuron lactate shuttle
hypothesis.

4.2 MATERIALS AND METHODS

4.2.1 Isolation and Culture of Primary Rat Cortical Neurons and Astroglia

Primary neurons and astroglia were isolated from the cortex of one-day-old Sprague-
Dawley rat pups and cultured according to the methods as described in chapter 2. The
cells were plated on poly-L-Lysine coated, 6-well plates at the concentration of 2 x 106
cells per well in fresh cortical medium [Dulbecco's Modified Eagle's Medium (DMEM
and all other media are from Invitrogen, CA) supplemented with 10% horse serum
(Sigma, MO), 25 mM glucose, 10 mM HEPES (Sigma), 2 mM glutamine (BioSource

lntemational, CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycine] '76. To obtain

45

pure neuronal cell cultures, after 3 days of incubation (37°C, 5% C02) the medium was
replaced with the cortical medium supplemented with 5 uM cytosine-B—arabinofiiranoside
(Arac, from Calbiochem, CA). After 2 more days, the neuronal culture was switched
back to cortical medium without Arac. The neuronal cell culture of more than 95% was
obtained by this procedure. The experiments were performed on 6-7 day old neuronal
culture. To obtain primary cultures of astroglial cells, the cortical cells from one-day-old
Sprague-Dawley rat pups were cultured in DMEM/Ham’s F12 medium (1:1), 10% fetal
bovine serum (Biomeda, CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycine ‘77.
Cells were grown for 8-10 days (37°C, 5% C02) and culture medium was changed every
2 days. The astroglial cell culture of more than 95% was obtained by this procedure. 24

hours prior to treatment with fatty acids, the medium was changed to neuronal cell

culture medium.

4.2.2 Western Blot Analysis

For western blot analysis, cells were washed three times with ice-cold TBS (25 mM Tris,
pH 8.0, 140 mM NaCl, and 5 mM KCl) and lysed for 20 minutes by scraping into scraping
into ice-cold radioimmunoprecipitation assay (RIPA) buffer [1% (v/v) Nonidet P40,
0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 50 mM Tris, pH 7.2, lSOmM NaCl, 1 mM
Na3VO4 and lmM PMSF, all chemicals from Sigma] 238. The total cell lysate was
obtained by centrifugation at 12,000 rpm for 15 minutes at 4 0C. The total protein
concentration was measured by BCA protein assay kit from Pierce (Rockford, IL). Equal
amounts of total protein from each condition were run at 200 V on 10% Tris-HCl gels for
detection of GLUTl and actin. The separated proteins were transferred to nitrocellulose

membranes for 1 hour at 100 V and incubated at 4 0C overnight with the appropriate

46

primary antibodies [1:500 GLUTl, 1:2000 actin]. Blots were washed three times in PBS-
Tween (PBS-T) and incubated with appropriate HRP-linked secondary antibodies (Pierce
Biotechnology, IL) diluted in PBS-T for 1 hr. After an additional three washes in PBS-T,
blots were developed with the Pierce SuperSignal West Femto Maximum Sensitivity
Substrate (Pierce Biotechnology) and imaged with the BioRad ChemiDoc. Quantity One

software from Bio-Rad was used to quantify the signal intensity of the protein bands.

4.2.3 Biochemical Measurements of Cellular Metabolites

For measurement of various cellular metabolites the conditioned media were collected
immediately after experimental treatment and centrifuged for 10 minutes at 3000 rpm to
remove any cell debris. To calculate the glucose uptake and lactate production in
particlular, their concentrations in the media were measured by using enzymatic glucose
(Stanbio Laboratories, TX, USA) and lactate (Trinity Biotech, MO, USA) assays. The
glucose uptake and lactate production were calculated by using the differences between
the metabolite concentrations in the media before and after the treatment. The data were
normalized by using total intracellular protein levels. Similarly, the concentrations of
FFA (Assay kit from Roche Biochemicals), beta-hydroxybutyrate and acetoacetate (both
assays from Stanbio Laboratories) in the extracellular media were measured according to
manufacturer’s instructions. High-performance liquid chromatography (HPLC) method
(Waters AccQTag amino acid analysis with fluorescence detector) was used to measure
concentrations of various amino acids such as Asp, Glu, Gly, Arg, Thr, Ala, Pro, Tyr,
Val, Met, Om, Lys, Ile, Leu and Phe. The concentrations of Ser, Asn, Gin and His were
measured by a slight modification of the AccQTag method. The extracellular fluxes of

these metabolites were measured for metabolic flux analysis by calculating the changes in

47

the levels of the metabolites in the cell culture media after 24 hours of treatment. The
linearity of some of these fluxes over this interval was verified.

4.2.4 Metabolic Flux Analysis (MFA)

MFA is a powerful mathematical technique that can provide a comprehensive snapshot of
the metabolic profile of cells as a function of their environment. The basis of this method
is that metabolic pathways have a well-defined stoichiometry relating reactants to
products 274. In MFA, a mass-balance on various intracellular metabolites is performed
and under the assumption of pseudo-steady state the differential equations representing
the changes in the concentrations of the individual metabolites may be written as
algebraic summation of the fluxes associated with that metabolite 275. These pseudo-
steady state balances for all the intracellular metabolites under consideration can be
written in matrix form as:

S *v = 0

where, S is the stoichiometric matrix and v is the vector of metabolic fluxes. If m numbers
of fluxes are measured, then it is possible to divide the matrix S into two submatn'ces, S,,,
and S“, corresponding to the measured and unknown fluxes, respectively. This leads to-
S..."‘vm + S..*v.. = 0

v. = (S.)"* (-s..*v..)

Thus, with the knowledge of the stoichiometry and measured fluxes v,,,, the vector of the
unknown fluxes vu can be calculated.

Assumptions

The assumptions pertaining to the MFA model employed in the current study are as

follows:

48

ii.

iii.

iv.

Pseudo-steady state assumption. The intracellular metabolites are assumed to be
under the condition of pseudo-steady state in that there is no significant intracellular
accumulation of any metabolite. Experiments were conducted to verify the pseudo-
steady state assumption.

Linearity of metabolic fluxes over 24 hour period Fluxes of the uptake or release of
metabolites were assumed to be linear over the 24 hour period, that is the net
change in the extracellular concentration of a given metabolite after 24 hours
duration represents the flux of that metabolite.

The metabolites are distributed uniformly inside the cell. Based on this assumption,
a single MFA model could be applied to perform metabolite balances for the entire
cell and separate models were not needed for the individual cellular compartments
or organelles. This assumption has been successfully employed previously in
various MFA studies 276 275.
Urea cycle is not active in brain tissue. Urea cycle, present in liver, is critical in
efficiently clearing up ammonia. However, the urea cycle is not present in the brain.
The brain depends on the amidation reaction (glutamate to glutamine) catalyzed by
glutamine synthetase, to clear ammonia 277. Therefore, the present model consisted
of the glutamine synthetase reaction and the urea cycle reactions were not included.

Glycerol is converted by the cells to glycerol-3-phosphate, which leads to
dicylglycerol (DAG) formation or to glyceraldehyde-3-phosphate, the latter enters
glycolysis leading to the production of pyruvate. However, the enzyme involved in

the conversion of glycerol-3-phosphate to glyceraldehyde-3-phosphate, glycerol-3-

49

phosphate dehydrogenase, is not expressed in astroglia 278. Therefore, only the

DAG formation reaction is included in the present model.
Based on these assumptions, a MFA model consisting of 71 cellular metabolic reactions
involved in the metabolism of glucose, amino acids and lipids was constructed (Table 4.1
and Figure 4.3). The model consisted of 54 intracellular metabolites as shown in Table
4.2. A total of 23 metabolic fluxes were measured (Table 4.3), yielding an over-
determined system of equations, which was solved by least-squares fit using the Moore-
Penrose pseudo-inverse calculation. This method has been successfully used in our
275

laboratory to study F F A-induced abnormalities in HepGZ cells

Table 4.1. List of the cellular metabolic reactions

 

 

Flux
# Equation
Glycolysis
1 G+ATP -> G-6-P
2 G-6-P -) F-6-P
3 F-G-P +ATP -) Glyceraldehyde-B-P + DHAP
4 DHAP -> Glyceraldehyde-B-P
5 Glyceraldehyde-B-P -) 3-PGA
6 3PGA -) PEP + NADH (+ ZATP)
7 PEP -) Pyr
8 Pyruvate + NADH -) (Lactate)
9 Pyruvate -) Acetyl-CoA + NADH +C02
TCA cycle

10 Acetyl-CoA + 0AA -) Citrate

1 1 Citrate -> a-KetoGlutarate + NADPH +C02

12 a-Ketoglutarate -> Succinyi-CoA + NADH + C02
13 Succinyl-CoA -) Fumavate + FADHZ + ATP

14 Fumarate -) 0AA + NADH

Pentose-phosphate pathway
15 G-6-P -) 12 NADPH + 6 C02
16 C02 out

Ketone Body production

17 2 Acetyl-COA -) Acetoacetyl-CoA
18 Acetoacetyl-CoA —) Acetoacetate

50

19
20

Table 4.1 Continued

Acac out
Acetoacetate + NADH -) (B-OH butyrate)

Oxygen uptake and Oxidative Phosphorylation

21
22
23

02 (In)
NADH + 0.5 02 -> 2.5 ATP
FADHZ + 0.5 02 -) 2 ATP

FFA synthesis and oxidation

25
69
68
26
24

Amino
27
28
29
30
31
32
33
34
35
36
37
38
49
4O
41
42
43
44
45
46
47
48
59
SO
51
52

53
S4
54

8 Acetyl-CoA + 14 NADH -> FA-CoA
Glycerol + ATP -) Glycerol-3-P
ZFA-CoA + Gcherol-3-P -) DAG
FA-CoA + DAG -) TG

FA-CoA (In)

acid metabolism

Ser (In)

3-PGA + Glu --> Ser + a-KG + NADH

Gln In

Asp (In)

Glu (In)

Gly (In)

Gly 9 2 C02 + NH3 + NADH + THF + ATP

NH4 (In)

Arg (In)

Thr In

Ala (In)

Glu + Pyr -) Ala + aKG

Pro In

Tyr (In)

Tyr + aKG + 2 02 -) Glu + C02 + Acetoacetate + Fumarate
Val (In)

Orn IN

Lys IN

Ile IN

Lue In

Phe In

Gln -) Glu + NH4

0m + a-KG + 0.5 NADPH + 0.5 NADH --> Pro

Asp + NH4 -) Asn

Thr -) Pyr + C02 + NH4 + 2 NADH + FADHZ

Val + aKG -) Glu + C02 + 2NADH + FADHZ + Succ-CoA

Lys + 2 aKG + NADPH -) ZGIu + Acetoacetyl-CoA + 2C02 + 4 NADH +
FADHZ

Ile + aKG -) Glu + Succ-CoA + Acetyl-CoA + NADH + FADHZ
Leu + aKG -) Glu + HMG-CoA + NADH + FADHZ

51

Table 4.1 Continued
56 Phe + 02 9 Tyr
59 a-KG + NH4 + NADPH --> Glu
70 Cys + 02 + a-KG --> Glu + Pyr + $04
71 Cystine --> 2 Cys
Synthesis of Cholesterol and Cholesteryl ester
60 AcetoacetyI-CoA + Acetyl-CoA 9 HMG-CoA
61 HMG-CoA + 2 NADPH (+ 3ATP) 9 IPP
62 2 IPP 9 GeranyI-PP
63 Geranyl-PP + IPP 9 Farnesyl-PP
64 2 FarnesyI-PP + 0.5 NADPH + 0.5 NADH 9 Squalene
65 Squalene + 02 + NADPH --> Lanosterol
66 Lanosterol + 10.5 NADPH + 4.5 NADH + 10 02 9 Chol + 3 C02
67 Chol + FA-CoA --> Cholesterol Ester
Sphingolipid Metabolism
57 Ser + I Palm-CoA + 1 FA-CoA + NADPH 9 Ceramide + C02 + FADHZ
58 Ceramide + Phosphatidleholine 9 Sphingomyelin

illumn I I Quanta 15’16 p< PPP) 69 I glymrol I /E|0WM°>
26
2,3,4 Shawls?-
phosvhab 68 Diacayl
<1praldehydo glprol
SphingomyelD

-3-ph/oaphab Palmihb
3U 58 /
Palm CoA 57

[Puruwb56 /\-CAC c A) - 17 “Comm“;
CPU/v 51 70 W” C“ 18.19.20
’ \ 22:22: I
0AA)“ K 56 henylalanin) C Val >

14 Co"- I] (”X41 5, 55/...)
(min!) (”'3‘ 53 /

c223 G“'°°'")+\/_/-@m.>.4/ .448 6......)

(when!) /12 38 w
vmb (I. 54 CM)

13

Figure 4.3. Astroglial metabolic network. Boxes represent extracellular metabolites. while ovals represent
intracellular metabolites. The direction of reaction assumed in the model is indicated by arrows.

52

 

Table 4.2. List of intracellular metabolites

 

Metabolite

 

_a
ommeU'l-th—i

DhAb-hwwwwwwwwwwNNNNNNNNNN-'-|-'-'did—Dd
AwN-‘O‘Dmflmm-bdeOtOmeUl-bwml—Iommem-th-i

 

glucose-6-P
fructose—6-P
glyceraldehyde-3—P
3-PGA

DHAP
Phosphoenolpyruvate
Pyruvate

C02
acetyl-CoA
Oxaloacetate
Citrate
a-ketoglutarate
succinyI-CoA
Fumarte
Acetoacetate
acetoacetyl-CoA
B-OH

02
GcheroI-3-P
Palm-CoA
NADH

NADPH
FADHZ

Ser

NH4

Glu

Gln

Val

Ile

Leu

Arg

Orn

Ala

Gly

Tyr

Thr

Lys

Phe

Pro

Asp

Asn

Cys

Ceramide
Sphingomyelin

 

53

Table 4.2 Continued
45 HMG-CoA

46 lsopentenyI-PP
47 GeranyI-PP

48 FarnesyI-PP

49 Squalene

50 Lanosterol

51 Cholesterol

52 Cholesterol Ester
53 DAG

54 Sulfate

 

 

 

Table 4.3. List of measured fluxes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Flux # Metabolite
1 Glucose

8 Lactate

19 Acetoacetate
20 Beta-hydroxybugrate
21 02 In

24 Palm

27 Ser

29 Gln

30 Asp

31 Glu

32 Gly

34 NH4

35 Arg

36 Thr

37 Ala

39 Pro

40 Tyr

42 Val

43 Orn

44 Lys

45 lie

46 Leu

47 Phe

 

 

 

 

4.2.5 Measurement of Intracellular ATP in Astroglia

To measure the intracellular ATP levels, astroglia were washed with ice-cold PBS and

then lysed with 0.7% perchloric acid (PCA). The PCA was neutralized by using 0.7N

54

NaOH. The neutralized samples were used to measure intracellular ATP levels with a

luciferase-based chemiluminescent assay (Molecular Probes, CA).

4.2.5 Measurement of Intracellular Ceramide in Astroglia

Astroglia were washed with ice-cold PBS and then lipids were extracted by using a
chloroform/methanol method as described by Bligh and Dyer 279. The organic phase was
dried under N2 and the ceramide was measured after its deacylation to sphingolipid base
and derivitization with o-phthaldehyde (OPA) as described earlier 28°. Briefly, the dried
lipids from the organic phase were re-suspended in 500 pl of 1N KOH in methanol and
incubated at 100°C for 1 hour to deacylate ceramide to free sphingolipid bases. The lipids
were then dissolved in 50 ul of methanol and 50 pl of OPA reagent is added to this. The
CPA reagent was prepared by mixing 99 ml of boric acid (3% w/v in water, pH 10.5),
lml of ethanol containing 50 mg OPA (Sigma) and 50 ul of B-mercaptoethanol (Sigma).
The derivatized sample aliquots (20 ul) were quantified by high performance liquid
chromatography (HPLC) using Nova Pak C18 column (60 A0, 4 pm, 3.9 mm x 150 mm;
from Waters, MA, USA). Fluorescent-labeled lipids were eluted isocratically by using
methanol:5mM potassium phosphate (pH 7.0) (90:10, v/v) at a flow rate of 0.6 ml/min
and detected with a fluorescence detector (excitation wavelength 340 nm, emission
wavelength 455 nm). A standard curve obtained by running known amounts of ceramide
(type 111, from bovine brain Sphingomyelin; from Sigma) was used as a comparison to

determine the ceramide levels in the experimental samples.

55

4.2.6 Data Analyses
Data are shown as means : SD. for indicated number of experiments. Student’s t-test
and one-way ANOVA with Tukey’s post hoc method were used to evaluate statistical

significances between different treatment groups. Statistical significance was set at

p<0.05.

4.3 RESULTS AND DISCUSSION

4.3.1 FFA-Induced Abnormal Glucose Metabolism

To study the effects of PA on cellular glucose metabolism, neurons and astroglia were
treated with 0.2mM of PA for 24 hours. As shown in Figure 4.4A, there was no change
in the glucose uptake and lactate production in neurons treated with PA as compared to
the untreated ones. This is in line with the observed, lack of direct effect of PA on
neurons in terms of AD-associated pathophysiological changes as discussed earlier
(Chapters 2 and 3) and may similarly be attributed to the low affinity of primary neurons
towards PA '82. However, as mentioned earlier, astroglia have a higher capacity to take
up and metabolize PA '82. Thus, in the case of astroglia, PA may compete with glucose
for cellular uptake. Previously, PA has been shown to inhibit glycolysis in primary
hepatocytes 28" 282. Furthermore, high fat diet has been shown to increase palrnitate
oxidation and decrease fructose oxidation in isolated primary hepatocytes 283. We found
that PA treatment significantly decreased basal glucose uptake and lactate release from

astroglia (Figure 4.4B).

56

Glucose 35 Lactate

Jumoleslmg protein

     

60 . Glucose

umoleslmg protein
to h in
O O O

N
O

1 FL“
,:

 

o
.
flu. <- —4r_-;E‘

0;:

PA

80 Lactate

 

umoleslmg protein
.h
0

    

C PA PA+AICAR

Figure 4.4. PA downregulates glucose uptake and lactate release by astroglia. The cortical neurons and
astroglia were treated for 24 hours with 0.2mM of PA or 4% BSA. (A) In neurons, PA treatment did not
change glucose uptake and lactate production. (8) PA treatment significantly decreased glucose uptake and
lactate production by astroglia. AMPK-activator AICAR did not inhibit PA-induced downregulation in
glucose metabolism. Data represent mean 1- SD. of six experiments. Student’s t-test was used for analyzing
the differences between the two treatment groups. *, p<0.05 compared with respective control.

57

Here it is imperative to note that although PA did not directly affect glucose metabolism
in neurons, PA-astroglia-induced ROS production in neurons (as discussed in chapter 2)
may affect glucose uptake and metabolism in neurons; oxidative stress has been shown to
affect the activities of glucose transporter and glycolytic enzymes and in turn affect
glucose uptake and metabolism in neurons 28”“. To investigate perturbed astroglial
metabolism due to PA treatment, we measured cellular ATP production in astroglia. We
expected PA-treatment to decrease astroglial ATP production in light of our observation
that PA decreases glucose uptake. We, however, found that there was an increase in ATP

production in the PA-treated astroglia as compared to the untreated ones (Figure 4.5).

140

it
120
100 '
80
60
40
20
0 V .
C PA

Figure 4.5. Measurement of intracellular ATP in astroglia. The cortical astroglia were treated for 24
hours with 0.2mM of PA or 4% BSA. PA treatment increased cellular ATP production in astroglia. Data
represent mean i SD. of 3 experiments. Student's t-test was used for analyzing the differences between the
two treatment groups. ', p<0.05 compared with respective control.

ATP (°/o of control)

increased ATP production in PA-treated astroglia may be due to the uptake and increased
oxidization of PA by astroglia. In support of this, previously it has been shown that
although PA inhibits glucose oxidation, PA-oxidation itself makes up for this and thereby

prevents ATP loss, which otherwise would be expected due to the reduced glucose

58

oxidation 287’ 288. Furthermore, it has been shown that although glucose utilization is
reduced in AD brain, oxygen consumption and C02 production are unchanged or even

increased as compared to healthy controls 289' 29°

. Thus, it has been hypothesized that
substrates other than glucose (e.g. FF As and endogenous amino acids) may be oxidized
in AD brain 245, which may partially compensate for the energy deficit observed in AD

brain due to decreased glucose metabolism 29'.

4.3.2 Cellular Mechanism of FFA-Induced Abnormal Glucose Metabolism in

Astroglia

We hypothesized that the observed, PA-induced abnormal glucose metabolism may be
due to the potential effect of PA on the level of astroglial glucose transporter (GLUTl) or
due to possible involvement of PA in perturbing the signaling mechanism involved in the
cellular glucose metabolism, e.g. AMP-activated protein kinase (AMPK). Along those
lines, it has been previously shown that polyunsaturated fatty acids (arachidonic acid)
deficiency results in down-regulation of the glucose transporter in astroglia 292. However,
the effects of elevated levels of saturated fatty acids on astroglial glucose transporters
have not been studied. AMPK is a serine-threonine kinase, which is involved in
regulating cellular metabolism 293. It was first isolated from liver and is also expressed in
many other tissues including lung, kidney, heart, skeletal muscle, and brain 294. At the
sub-cellular level in the brain, AMPK is expressed in both neurons and astroglia;
however, its activity is 3X higher in astroglia as compared to neurons 295. The importance
of AMPK in AD research is emphasized by the fact that inflammation is central to the

AD pathology and AMPK activation has been shown to inhibit the production of

59

296

inflammatory cytokines in astrocytes AMPK activation has also been shown to

protect cortical and hippocampal neurons from oxygen-glucose deprivation 297.

Therefore, we treated astroglia for 24 hours with a cell-permeable pharmacological

 

activator of All/IPK, 5 ' ' 'J ‘ 4 L " ribonucleoside (AICAR). Co-
treatment of PA-treated astroglia with 0.25mM AICAR did not increase glucose uptake
and lactate production as compared to astroglia treated with PA (Figure 4.4B). This
suggests that AMPK may not be involved in the observed PA-induced abnormal glucose
metabolism in astroglia. On the other hand, we found that the level of astroglial glucose
transporter (GLUTl) was significantly downregulated in PA-treated astroglia as
compared to untreated cells (Figure 4.6). Thus, the observed downregulation in glucose
uptake by astroglia in the presence of PA may be attributed to the PA-induced
downregulation of GLUT] levels in astroglia. Interestingly, AD brain is characterized by

298

significant reductions in GLUTl levels , and disease severity is associated with

progressive decline in GLUTl gene expression 299.
120

l

100 -

80 -

GLUTl «I. -—..

60-

Actin w- ‘0 ‘
C PA 20'

   

 

C PA

Figure 4.6. PA downregulates GLUTI level in astroglia. Astroglia were treated for 24 hours with 0.2mM
of PA or 4% BSA. The immunoblot analysis shows that PA-treatment significantly decreased the levels of
GLUTl as compared to the untreated ones. B-actin is shown as a marker for protein loading. Histogram
represents quantitative determinations of intensities of the relative bands normalized with actin. Data
represent mean at SD. of three independent experiments. Student’s t-test was used for analyzing the
differences between the two treatment groups. ‘, p<0.05 compared wim respective control.

60

4.3.3 FFA-Induced Global Metabolic Changes in Astroglia

Abnormal metabolism precedes the cascade of neuropathological changes in AD
pathology 1°. Decreased glucose metabolism and energy production have been shown to
be involved in increased amyloidogenesis and hyperphosphorylation of tau in neurons 2“
27°. However, as we showed here, PA treatment did not affect the glucose metabolism in
neurons but only in astroglia. Therefore, we hypothesized that metabolic changes, other
than the abnormal glucose metabolism induced by PA in astroglia may be involved in
causing the observed, PA-astroglia-induced pathophyiological changes in neurons
(chapters 2 and 3). The main focus was to investigate various FFA-metabolizing
pathways in astroglia that may potentially be involved in causing cellular ROS
production, specifically in neurons, as is observed in our studies. Based on extensive
literature review, we propose 3 major pathways by which PA may induce cellular ROS

production (Figure 4.7).

 

 

     
 

FFA metabolism

fi-oxidation Diacylglycerol Sphingolipid
(DAlG) metabolism ®
Ha/(c ramifie)——~—’/vh’*

Ros NAD(P) /7/'

 

 

 

 

 

 

oxidase iNOS (NO)
ROS Cytokines
Astrocyte Neuron

Figure 4.7. FFA-metabolizing pathways involved in cellular ROS production.

61

Elevated levels of PA may lead to its increased cycling through B-oxidation pathways in
mitochondria and other organelles such as peroxisomes and glyoxysomes. The increased
oxidation of PA may lead to enhanced cellular ROS production 300. PA may also increase
diacylglyecrol (DAG) production, which in turn may activate NAD(P)H oxidase and
increase cellular ROS 300’ 30'. However, as shown earlier in Chapter 2, PA did not induce
ROS production in astroglia (Figure 2.6), which may suggest the lack of or minimal
activation of these two pathways (ti-oxidation and DAG production) in PA-treated
astroglia. Finally, PA may also be metabolized by the cells to synthesize ceramide 302.
Ceramides are also potent intracellular activators of NAD(P)H oxidase 303 and thus may
increase cellular ROS production. More importantly, ceramide secreted from astrocytes
may directly act on neurons and mediate oxidative stress-induced effects in neurons 304.
Furthermore, PA-induced increase in ceramide levels may induce secretion of cytokines
305 or other signaling molecules, e.g., NO 306 by astrocytes, which in turn may elevate
production of ROS in the neurons 307’ 308. Thus, taken together, these data may suggest the
sphingolipid pathway (ceramide) to be predominantly activated in PA-treated astroglia
and ceramide to be a possible mediator of the FFA-induced pathological damage in

neurons as shown in our present studies. We sought to support this hypothesis by using

mathematical modeling (MFA) and additional experiments.

4.3.3.1 Verification of the pseudo-steady state assumption and linearity of fluxes
over 24 hours
To verify whether the pseudo-steady state assumption for MFA is valid, intracellular and

extracellular lactate concentrations were measured after 6, 12 and 24 hour treatments.

62

Lactate was the metabolite of choice due to its highest flux, which offered two
advantages 275-(1) due to its highest molar synthesis, it is likely the most concentrated
metabolite, simplifying accurate detection, and (2) its highest molar change provides the
most stringent test of the pseudo-steady state assumption. As shown in Figures 4.8A and
B, both the intracellular and extracellular concentrations of lactate increased with time in
response to PA treatment. The extracellular lactate release was approximately linear over
the 24 hour period for both the control and PA treatments. Furthermore, as shown in
Table 4.4, the changes in the intracellular lactate levels were about a thousand-fold
smaller as compared to changes in the extracellular lactate levels. This indicates that the
intracellular accumulation of lactate is negligible and the pseudo-steady state hypothesis

is valid.

Lactate (Intracellular)

0.09

 

+C
0.08 H + pA

0.07

 

 

 

tein

0.06

E 0.05
\

3 0.04
0.03

mlcromol

0.02
0.01 -

6 h 12 h 24 h
Time

 

Figure 4.8A. Time-dependent measurements of intracellular lactate levels. Astroglia were treated for 6,
12 and 24 hours with 0.2mM of PA or 4% BSA. The cells were trypsinized, washed with TBS and lyzed by
using 0.7% perchloric acid. The lactate levels in cell lysate were measured by enzymatic assay. Data
represent mean :1: SD. of three independent experiments.

63

Lactate (Extracellular)

IO
0

 

+C
+PA

m
0
l

 

 

 

micromoles/ mg protein
H N U A 01 Ch \I
O O o O o o o
. i . i 1
Hi.

0

6 n 12 h 24 h
Time

Figure 4.88. Time-dependent measurements of extracellular lactate levels. Astroglia were treated for 6,
12 and 24 hours with 0.2mM of PA or 4% BSA. The conditioned media were collected and the lactate
levels in the media were measured by enzymatic assay. Data represent mean :1: SD. of three independent
experiments

 

 

 

| I24h lntra I24h Extra IRatio (lntralExtra) |
IControl | 0.07303I 73.35371l 9.32E-04I
IPA | 0.0777eel 61.93194] 1.26503

 

Table 4.4. Ratio of intracellular to extracellular lactate levels.

4.3.3.2 MFA analysis

Table 4.5 shows a complete list of fluxes calculated from MFA analysis for both the
control and PA-treated astroglia. Firstly, in both the control and PA-treated cells fatty
acid synthesis but not the fatty acid oxidation, was more prominent (flux 25, Table 4.5).

In PA-treated astroglia fatty acid synthesis was lower as compared to controls, which

64

might be attributed to the exogenously added PA. In this context, there is evidence that
astrocytes do synthesize fatty acids 309 and exogenous addition of fatty acids decrease the
endogenous fatty acid synthesis 3'0. Secondly, in PA-treated cells, glycerol uptake was
significantly decreased (flux 69, Table 4.5). Previously, PA treatment has been shown to
decrease glycerol uptake inHepGZ cells 275. As glycerol is involved in DAG production,
PA-induced decrease in glycerol uptake may further contribute to decreased synthesis of
DAG in the PA—treated astroglia (flux 68, Table 4.5). Finally, flux through de novo
synthesis of ceramide was significantly elevated in PA-treated astroglia as compared to
controls (flux 57, Table 4.5). Experimental measurements also showed higher
intracellular ceramide levels in PA-treated astroglia as compared to controls and co-
treatment of astroglia with 2mM L-cycloserine (L-CS), an inhibitor of de novo synthesis
of ceramide, inhibited PA-induced ceramide increase (Figure 4.9), thus further validating

the MFA findings.

Table 4.5. Metabolic flux values calculated by MFA

 

Flux Control Palmitate
# Equation Average Error Average Error

 

 

52.8850 5.8490 37.4540 3.8160
1 G+ATP -> G-6-P

52.7431 5.8491 37.2262 3.8160
2 G-G-P -) F-6-P
F-G-P +ATP —) Gcheraldehyde-3-P + 52.7431 5.8491 37.2262 3.8160
3 DHAP
52.7431 5.8491 37.2262 3.8160

4 DHAP —) Glyceraldehyde-3-P

105.4862 11.6982 74.4523 7.6320
5 Glyceraldehyde-3-P -) 3-PGA

104.1620 11.7016 72.4859 7.6465
6 3PGA -> PEP + NADH (+ ZATP)

104.1620 11.7016 72.4859 7.6465
7 PEP -) Pyr

78.3500 1.2500 61.9300 3.1000
8 Pyruvate + NADH -) (Lactate)
9 Pyruvate 9 Acetyl-CoA + NADH +C02 24-4819 “-7797 94574 82970

65

10
11
12
13
14
15
16
17
18
19
20
21
22
23

24

25
26
27
28
29
30
31
32
33

34

Table 4.5 Continued.

Acetyl-CoA + 0AA 9 Citrate

Citrate 9 a-KetoGIutarate + NADPH
+C02

a-Ketoglutarate 9 SuccinyI-CoA +
NADH + C02

Succinyl-CoA 9 Fumarate + FADHZ +
ATP

Fumarate 9 0AA + NADH

G-6-P 9 12 NADPH + 6 C02

C02 out

2 Acetyl-COA -) AcetoacetyI-CoA
AcetoacetyI-CoA 9 Acetoacetate

Acac out
Acetoacetate + NADH 9 (B-OH
butyrate)

02 (In)
NADH + 0.5 02 9 2.5 ATP
FADHZ + 0.5 02 9 2 ATP

FA-CoA (In)
8 Acetyl-CoA + 14 NADH -)
FA-CoA

FA-CoA + DAG 9 TG

Ser (In)

3-PGA + Glu --> Ser + a-KG + NADH
Gln In

Asp (In)

Glu (In)

Gly (In)

Gly 9 2 C02 + NH3 + NADH + THF +
ATP

NH4 (In)

66

5.8041
6.1589

0.7998

4.7440

5.4492
0.1419
39.0347
1.2702
1.9907
2.3390
0.0020
24.5230
33.2840
13.5899
0.1510

3.3083

0.8802
0.7100
1.3241
4.7620
0.2140
-0.9460
1.7060

1.5013

3.5020

1.0401
1.035

1.1461

1.0824

1.0467
0.0359
10.9023
0.9366
0.8355
0.8040
0
2.1000
3.7417
1.1080
0.0210

1.5521

0.5339
0.0380
0.2431
0.7520
0.0030
0.1830
0.1180

0.1373

0.1760

5.0068
5.4583

0.1808

3.8455

4.5552
0.2278
25.7209
0.9077
2.1769
2.4320
0.0030
23.2030
32.5936
12.5787
1 .0600

1 .2357

0.2139
0.7260
1 .9665
4.8990
0.2050
-0.5090
2.9720

2.5585

4.0760

0.9997
1 .0044

1 .5399

1.1778

1.0048
0.0047
7.8214
1.2612
0.9631
0.8280
0.0010
2.1000
4.4499
1.1972
0.1450

1.1552

0.4917
0.0790
0.4702
1.8360
0.0010
0.4850
0.9830

0.9404

0.0220

35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51

52

S3
54

55

56

57

58
59

Table 4.5 Continued.
Arg (In)

Thr In

Ala (In)

Glu + Pyr 9 Ala + aKG
Pro In

Tyr (In)
Tyr+aKG+2029Glu+C02+
Acetoacetate + Fumarate

Val (In)
Orn IN
Lys IN
Ile IN
Lue In
Phe In

Gln 9 Glu + NH4
0m + a-KG + 0.5 NADPH + 0.5 NADH
--> Pro

Asp + NH4 9 Asn

Thr 9 Pyr + C02 + NH4 + 2 NADH +
FADHZ

Val + aKG 9 Glu + C02 + 2NADH +
FADHZ + Succ-CoA

Lys + 2 aKG + NADPH 9 ZGIu +
Acetoacetyl-CoA + ZCOZ + 4 NADH +
FADHZ

Ile + aKG 9 Glu + Succ-CoA + Acetyl-
CoA + NADH + FADHZ

Leu + aKG 9 Glu + HMG-CoA + NADH
+ FADHZ

Phe + 02 9 Tyr

Ser 4» 1 Palm-CoA + 1 FA-CoA
+ NADPH 9 Ceramide + C02
+ FADl-IZ

Ceramide + PhosphatidyIChoIine 9
Sphingomyelin

a-KG + NH4 + NADPH --> Glu

67

1.5340
1.7910

-2.5070
2.7117

-0.6020
-0.3900

0.3503

0.1580
-0.0440
1.7030
3.1310
2.1720
0.4400
-4.8166
0.1016
0.2094
1.5863
0.3082

1.2936

3.2812

1.9673

0.5902

0.4094

0.2047

1.3589

0.1240
0.4230

0.2100

0.2138

0.0360

0.0980

0.2274

0.0350

0.0060

0.4310

0.2010

0.1600

0.1230

0.6129

0.0309

0.0402

0.4071

0.1005

0.3607

0.2044

0.1710

0.1461

0.1607

0.0804

0.5890

1 .4980
2.0220

-2.1700
2.5835

-0.4570
-0.2940

0.2581

0.4430
-0.0380
2.7340
2.6940
1.5910
0.4760
-5.2744
-0.0163
0.3092
1 .6085
0.481 1

1.9070

2.7321

1.1775

0.5141

0.8270

0.4135

2.2459

0.2810
0.5570

0.1290
0.1973

0.0530

0.2420

0.4920

0.3480
0.01 1 0
0.0560
0.2630
0.3230
0.0500
1 .4865

0.0557

0.0776

0.5480

0.3861

0.3133

0.3272

0.3420

0.2304

0.31 05

0.1552

1.3018

Table 4.5 Continued.

Acetoacetyl-CoA + Acetyl-CoA 9 4.9673 0.1710 -1-1775 0.3420
60 HMG-CoA
e1 HMG-c°A + 2 NADPH (+ 3ATP) .9 |PP -o.oooo 0.0000 -0.0000 0.0000
-0.0000 0.0000 ~0.0000 0.0000
62 2 lPP 9 Geranyl-PP
-0.0000 0.0000 -0.0000 0.0000
63 Geranyl-PP + IPP 9 FarnesyI-PP
2 Farnesyl-PP + 0.5 NADPH + 0.5 00000 (1000‘) 0-0000 00000
64 NADH 9 Squalene
Squalene + 02 + NADPH --> -0.0000 0.0000 -0.0000 0.0000
65 Lanosterol
Lanosterol + 10.5 NADPH + 4.5 NADH 00000 00000 00000 00000
66 +10 02 9 Chol+ 3 C02
-0.0000 0.0000 -0.0000 0.0000

67 Chol + FA-CoA -—> Cholesterol Ester
ZFA—CoA + GcheroI-3-P -) 0.8802 0.5339 0.2139 0.4917

68 DAG

0.8802 0.5339 0.2139 0.4917
69 Glycerol + ATP 9 Glycerol-3-P
-0.2047 0.0804 -O.4135 0.1552
70 Cys + 02 + a-KG --> Glu + Pyr + 504
-0.1024 0.0402 -0.2067 0.0776
71 Cystine --> 2 Cys
200 ~ *
180
1130 -
140 4 #
Ceramide 12° ‘

(% Control) 100 .

 

C PA PA+L-CS

Figure 4.9. PA-lnduced, de novo syntnests or ceramtne in astroglia. Astroglia were treated tor 24h with
0.2mM PA or 4% BSA (control), after which cellular lipids were extracted for ceramide determination by
HPLC. PA significantly increased ceramide synthesis in astroglia, which was completely inhibited by
treatment of astroglia with 2mM L-CS, an inhibitor of de novo synthesis of ceramide. Data are taken from
3 different experiments and are expressed as mean :1: SD. One-way ANOVA with Tukey’s post hoc
method was used for analyzing the differences between treatment groups. ‘, p<0.05 compared with control;
#, p<0.05 compared with PA treatment.

68

4.4 CONCLUSIONS

In conclusion, PA had no direct effect on either glucose uptake and lactate production in
neurons. On the other hand, PA significantly decreased both glucose uptake and lactate
release in astroglia. The observed downregulation in glucose uptake by astroglia in the
presence of PA may be attributed to PA-induced downregulation of astroglial glucose
transporter (GLUTI) levels. In addition to these PA-induced abnormalities in astroglial
glucose metabolism, we also showed by using MFA that flux through de novo synthesis
of ceramide is elevated in PA-treated astroglia as compared to control. This MFA finding
was further validated experimentally; PA-treated astroglia had elevated intracellular
levels of ceramide as compared to controls, which were decreased by the astroglial
treatement with L-CS, which inhibits serine palmitoyltransferase-l (SPT-l), an enzyme
that catalyzes the first committed step of de novo synthesis of ceramide. Thus, the present
data establish a central role of saturated FFAS in causing abnormalities in astroglial
glucose metabolism and further warrant investigating a possible causal role of increased

astroglial ceramide levels in FFA-astroglia-induced pathological changes in neurons.

69

CHAPTER 5. INVOLVEMENT OF CERAMIDE IN FFA-
ASTROGLIA-INDUCED PATHOPHYSIOLOGICAL
ABNORMALITIES ASSOCIATED WITH AD

5.1 INTRODUCTION

Ceramides are one of the most important sphingolipids and are composed of sphingosine
and fatty acid, which are joined in an amide bond. Ceramides are involved in the
synthesis of Sphingomyelin, one of the major components of the cellular lipid bilayer,
thus playing a critical role in structural integrity of cell membranes. In addition,
ceramides also act as signaling molecules and are involved in many important signaling

31 1. Ceramides are

functions such as cell growth, differentiation, cell death etc.
synthesized in cells through two pathways- (1) the sphingomyelinase (Smase) pathway
and (2) the de novo synthesis pathway (Figure 5.1). Smase is regulated by the cellular
redox state; increased oxidative stress activates Smase 3”. Currently, there are five
different enzymes characterized as Smases based on their pH dependence, cation
dependence and cellular localization 312. Smases break down membrane sphingomyelins
into ceramide and fiee fatty acids. On the other hand, the de novo synthesis of ceramide
uses serine and palmitoyl-CoA to synthesize ceramide and is initiated by serine
palmitoyltransferase (SPT-l), which is the rate-limiting step of ceramide synthesis.
Ketosphinganine formed in this first reaction is then converted to sphinganine by
ketosphinganine reductase. A double bond is introduced to sphinganine by

dihydroceramide synthase leading to the production of dihydroceramide, which is

converted to ceramide by dihydroceramide desaturase.

70

 

serine 8pm

 

, , ketosphinganine . '
——* ketosphmganlne ; sphinganine
PalmitoyI-COA reductase
dihydroceramide
synthase

dihydroceramide

dihydroceramide
desaturase
Smase

Sphingomyelin .____' ceramide

 

 

 

Figure 5.1. Cellular ceramide production. Ceramide is produced in cells by de novo synthesis from
serine and palmitoyl-CoA or by breakdown of membrane sphingomyelins.

Abnormal ceramide metabolism has been implicated in many diseases such as diabetes,
AIDS and neurodegenerative disorders 3 11_ Ceramides may induce insulin resistance
associated with diabetes and inhibition of ceramide synthesis has been shown to
ameliorate glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance 313'
Ceramide level has been also shown to be elevated in the cerebrospinal fluid of AIDS
patients; elevated cerebral ceramides may be involved in neuronal cell death thus leading
to the dementia often associated with AIDS 3 '4. In AD brain, ceramide levels were found
elevated as compared to those of healthy controls, and the levels were higher in the
affected regions (cortex and hippocampus) as compared to the unaffected regions
(cerebellum) of the AD brain 3'5' 3'6, thus suggesting a central role of elevated ceramide
levels in AD pathology. On the sub-cellular level, immunohistochemical analysis of AD

brain showed abnormal expression of ceramides in the astroglia as compared to neurons

3 '7. In addition, ceramides have also been shown to be elevated in the white matter of AD

7l

brain 318. The involvement of increased ceramide levels in AD pathogenesis is further
emphasized by a recent study that showed that the gene expression of the enzymes
involved in the de nova synthesis of ceramides is significantly upregulated in AD brain
3 '9. In addition, increased ceramide synthesis has been implicated in Aft-induced death of

neurons 320' 32'

and oligodendrocytes 322 in AD. In chapters 2 and 3, we showed that
saturated fatty acids induced tau hyperphosphorylation and amyloidogenic processing of
APP in neurons through astroglia-mediated oxidative stress. Furthermore, in chapter 4 we
showed that astroglia treatment with PA significantly increased de nova synthesis of
ceramide. Therefore, the aim of the current study was to investigate the possible

involvement of PA-induced, increased astroglial ceramide levels in causing AD-

associated, pathophysiological changes observed in neurons.

5.2 MATERIALS AND METHODS

5.2.1 Isolation and Culture of Primary Neurons and Astroglia from Rat

Cortex and Cerebellum

Primary cortical neurons and astroglia were isolated from the brains of one-day-old
Sprague-Dawley rat pups and cultured according to the methods as described in chapter
2. The cells were plated on poly-L-Lysine coated, 6-well plates at the concentration of 2
x 106 cells per well in fresh cortical medium [Dulbecco's Modified Eagle's Medium
(DMEM and all other media are from Invitrogen, CA) supplemented with 10% horse
serum (Sigma, MO), 25 mM glucose, 10 mM HEPES (Sigma), 2 mM glutamine

(BioSource lntemational, CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycine] '76.

72

To obtain pure neuronal cell cultures, the medium was replaced with the cortical medium
supplemented with 5 11M cytosine-B-arabinofuranoside (Arac, from Calbiochem, CA)
after 3 days of incubation (37°C, 5% C02). After 2 more days, the neuronal culture was
switched back to cortical medium without Arac. The neuronal cell culture of more than
95% was obtained by this procedure. The experiments were performed on 6-7 day old
neuronal culture. Primary cerebellar neurons were isolated from 7-day-old rat pups,
according to enzyme digestion and trituration techniques described previously 323.
Briefly, dissected cerebellar tissue was placed in a cerebellar buffer solution containing
136.89mM NaCl, 5.36mM KCl, 0.34mM NazHPO4, 0.44mM KH2P04, 5.55mM dextrose,
20.02mM Hepes, and 4.17mM NaHCO3, PH 7.4. Cerebellar tissue was minced,
transferred to 0.025% trypsin solution in cerebellar buffer, and incubated in water bath
for 15 minutes at 37°C. 0.04% DNase I solution in cerebellar medium (DMEM
supplemented with 10% horse serum, 25mM KC], 5 mg/ml insulin, 50 M GABA,
lOOIU/ml penicillin, and 0.1 mg/ml streptomycine) was then added to inactivate trypsin.
After the supernatant was collected from the trituration steps, cerebellar neurons were
separated from the debris into 4% BSA solution in cerebellar buffer supplemented with
0.03% MgSO4. Finally, the purified neurons were plated onto poly-D-Lysine coated six-
well culture dishes at a density of 2.0><105 cells/ml in 2ml of fresh cerebellar medium.
One day after incubation (37°C, 10% C02/95% air), half the medium was subsequently
replaced with fresh cerebellar medium supplemented with 20uM cytosine-[3-
arabinofaranoside. Total medium was replaced with fresh cerebellar medium afier three
days. Afterwards, half of the fresh cerebellar medium was replaced every third day. The

experiments were performed on 10-12 day old neuronal culture. To obtain primary

73

cultures of astroglial cells, the cortical and cerebellar cells from one-day-old and seven-
day-old Sprague-Dawley rat pups, respectively were cultured in DMEM/Ham’s F 12
medium (1:1), 10% fetal bovine serum (Biomeda, CA), 100 IU/ml penicillin, and 0.1
mg/ml streptomycine 177. Cells were grown for 8-10 days (37°C, 5% C02) and culture
medium was changed every 2 days. The astroglial cell culture of more than 95% was
obtained by this procedure. 24 hours prior to treatment with fatty acids, the medium was

changed to neuronal cell culture medium.

5.2.2 Immunostaining of Reactive Oxygen Species (ROS)

Intracellular reactive oxygen species (ROS) were detected by staining with the oxidant-
sensitive dye 5-(6)-chloromethyl-Z',7'-dichlorodihydrofluorescein diacetate (CM-
HzDCFDA, from Molecular Probes, OR). HzDCFDA is cleaved of the ester groups by
intracellular esterases and converted into membrane impermeable, nonfluorescent
derivative H2DCF. Oxidation of I-IzDCF by ROS results in highly fluorescent 2,7-
dichlorofluorescein (DCF) '79. The cells were incubated for 30 minutes at 37 0C with
2M CM-HzDCFDA in Hanks’ Balanced Salt Solution without phenol red (Invitrogen).
The cells were then washed three times with PBS and analyzed with confocal microscopy

(Zeiss LSM 5 Pascal). A 63X oil-immersion objective lens was used for data acquisition.

5.2.3 Western Blot Analysis

For Western blotting, the following antibodies were used: BACE] (Chemicon, CA,
USA), APP/C99 (QED Bioscience Inc., CA, USA), AT8 (Pierce Biotechnology, IL,
USA), PHF-l (from Dr. P. Davies, Albert Einstein, NY, USA), Tau-l (Chemicon), GSK-

3a/B (Chemicon), Phospho GSK-3a/8 (Sigma), MAP Erkl/2 (Cell Signaling), Phospho

74

MAP Erkl/2 (Cell Signaling Technology, MA, USA), cdk5 (Santa Cruz Biotechnology,
CA, USA), p35/p25 (Santa Cruz) and actin (Sigma). To extract membrane proteins
(BACEI and APP/C99), cells were washed three times with ice-cold TBS (25 mM Tris,
pH 8.0, 140 mM NaCl, and 5 mM KCl) and lysed for 20 minutes by scraping into ice-cold
radioimmunoprecipitation assay (RIPA) buffer [1% (v/v) Nonidet P40, 0.1% (w/v) SDS,
0.5% (w/v) deoxycholate, 50 mM Tris, pH 7.2, 150 mM NaCl, 1 mM Na3VO4 and 1 mM

PMSF, all chemicals from Sigma] 238

. To extract all other proteins, RIPA buffer
containing 1% (v/v) Triton, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 20 mM Tris, pH
7.4, 150 mM NaCl, 100 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, and 1 mM
PMSF [all chemicals from Sigma] was used '78. The total cell lysate was obtained by
centrifugation at 12,000 rpm for 15 minutes at 4 0C. The total protein concentration was
measured by BCA protein assay kit from Pierce (Rockford, IL, USA). Equal amounts of
total protein from each condition were run at 200 V on SDS-PAGE gels (BioRad, CA,
USA). The separated proteins were transferred to nitrocellulose membranes for 1 hour at
100 V and incubated at 4 0C overnight with the appropriate primary antibodies [1:1000
BACE], 1:1000 APP/C99, 1:200 AT8, 1:200 PHF-l, 1:2000 Tau-l, 1:1000 GSK-301/8,
1:1000 Phospho GSK-3a/B, 1:1000 MAP Erk1/2, 1:1000 Phospho MAP Erk1/2, 1:1000
cdk5, 1:1000 P35/p25, 1:500 GLUTl, 1:2000 actin]. Blots were washed three times in
PBS-Tween (PBS-T) and incubated with appropriate HRP-linked secondary antibodies
(Pierce) diluted in PBS-T for 1 hr at room temperature. After washing three times in
PBS-T, blots were developed with the Pierce SuperSignal West Femto Maximum

Sensitivity Substrate (Pierce) and imaged with the BioRad ChemiDoc. Quantity One

software from Bio-Rad was used to quantify the signal intensity of the protein bands.

75

5.2.4 ELISA measurements of A840 and A842

For AB measurements, the media and neuronal cells were collected after 24 hours of
treatment. The media were treated with protease inhibitor cocktail (Sigma, MO, USA)
and cleared by brief centrifugation (3000 rpm, 5 minutes, 40C). A840 and A842 in the
media were measured by using colorimetric sandwich ELISA according to the
manufacturer's instructions (Wako Chemicals, VA, USA). The cells were lysed and the
intracellular protein was measured by using the BCA protein assay kit from Pierce

(Rockford, IL, USA), which was used to normalize the A040 and A842 values.

5.2.5 Data Analyses

Data are shown as means :1: SD. for indicated number of experiments. Student’s t-test
and one-way ANOVA with Tukey’s past hac method were used to evaluate statistical
significances between different treatment groups. Statistical significance was set at

p<0.05.

5.3 RESULTS AND DISCUSSION

5.3.1. Involvement of Astroglial Ceramide in FFA-Astroglia-Induced ROS
Production in Neurons

In chapter 2, we showed that treatment of neurons with the conditioned media from F FA-
treated astroglia increased ROS production in neurons. This suggests a central role of

astroglial FFA metabolism in the observed FFA-astroglia-induced ROS production in

neurons. Furthermore, our literature-based hypothesis followed by mathematical and

76

experimental studies suggested astroglial ceramide as a possible mediator of ROS
production in neurons (as discussed in chapter 4). In this context, here we found that
inhibition of de nova synthesis of ceramide in PA-treated astroglia by using 2 mM L-CS,
significantly inhibited PA-astroglia—induced ROS production in neurons (Figure 5.2).
The treatment of neurons directly with L-CS did not inhibit the PA-astroglia-induced
ROS production observed in neurons. This further emphasizes the role of astroglial

ceramide in causing ROS production in neurons.

Control PA PA+L-CS

   

Figure 5.2. Involvement of astroglial ceramide in PA-astroglia-induced ROS production in neurons.
Co-treatment of astroglia with 2mM L-CS inhibited PA-astroglia-induced ROS production in neurons. The
neurons were stained with C M-HZDCFDA for intracellular ROS detection and examined with confocal
fluorescence microscopy. (Objective lens magnification, 40X).

As mentioned earlier, ceramide secreted from astroglia can act directly on neurons and
mediate the oxidative stress-induced effects in the neurons 304. In addition, PA-induced

305

increase in ceramide levels can induce the secretion of cytokines (e.g. IL-6) or other

signaling molecules, e.g. NO 306 by astrocytes, which in turn may elevate the production

307‘ 308. In this context, we found that PA treatment did not induce

of ROS in the neurons
IL-6 expression in astroglia, however it increased the level of inducible nitric oxide
synthase (iNOS) in astroglia, which was blocked by the co-treatment of astroglia with
2mM L-CS, thus suggesting an involvement of ceramide in astroglial iNOS expression

(Figure 5.3).

77

iNOS .............. ——' "“""

 

lL-6

 

 

actin -— “I

C PA PA+L-CS
Figure 5.3. The expression of iNOS and IL-6 in astroglia. The expression of iNOS, but not IL-6 was
increased by PA treatment in astroglia. Co-treatment of astroglia with 2mM L-CS inhibited PA-induced
iNOS expression in astroglia. B-actin is shown as a marker for protein loading.

5.3.2 Involvement of astroglial ceramide in FFA-astroglia-induced

amyloidogenesis and tau hyperphoshorylation in neurons

We treated cortical astroglia with 0.2mM PA for 24 hours and then used the astroglia-
conditioned media to treat cortical neurons for 24 hours. After 24 hours of treatment, the
media were collected for measurement of secreted A6 levels and the neurons were
washed, lysed and the total cellular protein was used for western blot analysis of a
number of proteins. As shown in Figure 5.4, treatment of neurons with the conditioned
media from PA-treated astroglia significantly increased BACE] levels and consequent
amyloidogenic processing of APP leading to the formation of c-terrninal fragments of
APP (C99) (Figure 5.4). C99 is processed by y-secretase to form A8. BACEI is a rate-
limiting enzyme in the amyloidogenic processing of APP and a slight increase in BACE]
levels leads to a dramatic increase in the production of AB40/42 243. Indeed, we found
that the PA-astroglia-treated neurons significantly increased secretion of A840 and A842

as compared to controls (Figure 5.4).

78

(A) 180 9c

BACEl—> ' .1 5...,

Actin 7* -"

C PA PA+L-CS

BACE1 (‘5 of cancel)

   

 

 

   

C PA PA+L-CS
183
:1:
140
g 120 #
5
g 100 -
.3 so-
I
a .1
8 “I
C PA PA+L-CS 2‘“
D 4
PA PA+L-CS
(C) A 42
3... AB40 '3
* 350 a
23m 2300
c 9
g 250 # g 250 #
g 200 a; 200
39 150 g: 150
O 100 N 100
v wr
21 50 it 50
g 0
C PA PA+L-CS C PA PA+L-CS

Figure 5.4. Involvement of astroglial ceramide in PA-astroglla—induced elevations in BACEl and
amyloidogenic processing of APP in neurons. In neurons treated with conditioned media from PA-treated
astroglia, the levels of (A) BACE], (B) C99 and (C) A840 and A842 were found elevated. These PA-
astroglia—induced tau abnormalities were blocked by inhibiting astroglial ceramide synthesis with 2mM L-
CS. Data represent mean :t SD. of three independent experiments. One-way ANOVA with Tukey’s post
hoc method was used for analyzing the differences between treatment groups. ‘, p<0.05 compared with
control; it, p<0.05 compared with PA Ireatrnent.

79

In addition, phosphorylation of tau was significantly increased in neurons treated with the
conditioned media from PA-treated astroglia as observed by immunoblotting with two
different antibodies, PHF-l and AT8 (Figure 5.5). As mentioned earlier, PHF—l and AT8
antibodies recognize tau protein hyperphosphorylated at two different, AD-specific
phospho-epitopes. In addition to PHF-l and AT8 antibodies, we also carried out
immunoblotting with Tau-1 antibody, which detects all the isoforms of dephosphorylated
tau, thus acting as a negative control. As expected, dephosphorylated Tau-1 was
significantly decreased in PA-astroglia-treated neurons as compared to control-astroglia-

treated neurons (Figure 5.5).

160 *

120
100

AT8 as-“ :3

20
-_-~ 0

   

PHF'l PA PA+L-CS

140 ‘ *

Tau-1 “one. 120 #

Actin ‘1 I . 10°
80

C PA PA+L-CS 60

40

20

0

 

c PA+L-CS

Figure 5.5. Involvement of astroglial ceramide in PA-astroglia-induced tau hyperphosphorylation in
neurons. In neurons treated with conditioned media from PA-treated astroglia, tau was found
pathologically hyperphosphorylated as shown by immunoblotting with PHF-l and AT8 antibodies. Tau-l
detects dephosphorylated tau, thus showing decreased levels in PA-astroglia-treated neurons. These PA-
astroglia—induced tau abnormalities were blocked by inhibiting astroglial ceramide synthesis with 2mM L-
CS. Histograms corresponding to PHF-l and AT8 blots represent quantitative determinations of intensities
of the relevant bands normalized with actin. Data represent mean i SD. of three independent experiments.
One-way ANOVA with Tukey’s post hoc method was used for analyzing the differences between treatment
groups. *, p<0.05 compared with control; #, p<0.05 compared with PA treatment.

80

As PA treatment was found to elevate de nova synthesis of ceramide in astroglia, we
wanted to investigate a possible involvement of astroglial ceramide in the observed, PA-
astroglia-induced amyloidogenesis and tau hyperphosphorylation in neurons. For this, we
treated astroglia with 2mM L-CS together with 0.2mM PA for 24 hours and then
transferred the astroglia—conditioned medium to the neurons (24 hours treatment). The
inhibition of astroglial ceramide synthesis by L-CS treatment blocked PA-astroglia-
induced BACEl upregulation, AB production and hyperphosphorylation of tau (Figures
5.4 and 5.5), strongly suggesting a central role of astroglial ceramide in causing AD-
associated pathophysiological changes in neurons. Previously, exogenous addition of

24 -
3 . Furthermore, increased

ceramide to neurons has been shown to induce A8 production
tau phosphorylation has been reported in cholesterol-deficient neurons, which had a
significant increase in ceramide levels 325. Our findings reported here, however, are the
first to demonstrate a direct causal role of astroglial PA metabolism and endogenously

synthesized ceramide in astroglia, in causing A8 production and tau

hyperphosphorylation in neurons, two characteristic signatures of AD pathology.

We further studied the possible activation of various AD-related kinases (GSK-3a/8,
cdk5 and MAPK Erkl/Z) 75, one or more of which may be responsible for the observed
PA-astroglia-induced tau hyperphosphorylation, and potentially activated by ceramide.
An increase in the phosphorylation of these enzymes was used as an indicator of their
activation. As shown in Figure 5.6A, levels of phosphorylated GSK-3ot/[3
(Tyr279/Tyr216) were significantly increased in PA-astroglia-treated neurons as

compared to controls, without any significant change in the levels of total GSK-3a/8.

81

GSK—3a phosphorylated at Tyr279 and GSK3-B phosphorylated at Tyr216 suggest
increased activity 326. In addition to activating GSK-3a/l3, PA-treatment also increased
the cleavage of cdk5 activator p35 to p25 (Figure 5.6B). p25 accumulates in the brains of
AD patients and the conversion of p35 to p25 suggests augmented activity of cdk5 327' 328.
Finally, there was no change in the levels of both total and phosphorylated MAPK Erkl/Z
in the PA-astroglia-treated neurons as compared to control-astroglia-treated ones (Figure
5.6C), suggesting no change in its activity. The treatment of astroglia with 2mM L-CS

inhibited both the PA-astroglia-induced increase in the level of phosphorylated GSK-

301/[3 and the cleavage of p35 to p25 (Figure 5.6A and B).

(A) GSK-3a/B-
nosrzsa — "3.... fl ..
P-GSK-Bp —
Us? 011 ’
6:8 113 - — H u
c PA PA+L-CS
(B) cdk5-
p35 —'
p25—e W

0 PA PA+L-CS
(C) h'IAP-Er'kl/Z-

P-MAP-Erkr-a—II- ~ .5...
P-MAP-ErkZ-F ? P

INLAP-E kl --—- _—- 'h ..
MAP—Eikfd -- . =
C PA PA+L-CS

Figure 5.6. PA-astroglia-induced activation of AD-speciflc kinases in neurons is mediated by
astroglial ceramide. Conditioned media from PA-treated astroglia activated (A) GSK-3 (increased levels
of phosphorylated GSK-3) and (B) cdk5 (increased cleavage of p35 to p25) but not (C) MAP- Erkl/Z (no
change in the levels of phosphorylated MAP- Erkl/Z). The treatment of astroglia with 2mM L-CS inhibited
PA-astroglia-induced activation of both GSK-3 and cdk5. Data are representative of 3 different
experiments.

82

To further evaluate the possible involvement of these enzymes in PA-astroglia-induced
tau hyperphosphorylation in neurons, we treated neurons with pharmacological inhibitors
of these enzymes; lOmM LiCl (for GSK-Ba/B), lOuM Roscovitine (for cdk5) and 3011M
PD98059 (for MAPK Erkl/2). Treatment with PD98059 did not inhibit PA-astroglia-
induced tau hyperphosphorylation in neurons (Figure 5.7), which is in agreement with
the lack of activation of MAP Erk1/2 (Figure 5.6C). Furthermore, although cdk5 was
activated in neurons (Figure 5.6B), co-treatment of neurons with a potent cdk5 inhibitor,
roscovitine, did not inhibit the observed PA-astroglia-induced hyperphosphorylation of
tau (Figure 5.7). This suggests that PA-astroglia-induced hyperphosphorylation of tau in
primary rat cortical neurons is independent of the observed activation of the cdk5 .
pathway. Our results agree with a previous report that showed that cleavage of p35 to p25
and subsequent activation of cdk5 were not involved in the hyperphosphorylation of tau
in primary rat hippocampal neurons 329. On the other hand, GSK-3 inhibitor (LiCl)
inhibited the PA-astroglia-induced hyperphosphorylation of tau in neurons (Figure 5.7).
Previously, GSK-3 has been shown to be involved in causing both tau
hyperphosphorylation and A8 production and thus, is central and essential in the
development of AD. GSK-3B is involved in hyperphosphorylation of tau and its other
isoforrn, GSK-3a, is involved in AB production by regulating the activity of y-secretase
330' 33'. These studies together with our present data further emphasize the central role of
PA-induced, abnormal astroglial ceramide metabolism in causing AD-associated

pathophysiological characteristics.

83

AT8 - Hub“ SE

PA PA PA

PHF-l wfififl" C M . .

+
LiCl rosco PD98

 

150
140
Ta“ ~‘_-— 120
100
. so
Actm m..— 5°
40
C PA 20
Pf PA PA 0
. + +
LICI rosco PD98 C PA Pf P: +
.1.
v1 me 059 LiCl rosco PD98
vitine 059

Figure 5.7. GSK-3 is involved in PA-astroglia-indueed tau hyperphosphorylation in neurons.
Astroglia-conditioned media were transferred to neurons, with or without various kinase inhibitors, viz.,
lOmM LiC12 (GSK-3 inhibitor), IOmM Roscovitine (cdk-S inhibitor) and 30mM PD98059 (MAPK
inhibitor). Immunoblot analysis with PHF-l and AT8 antibodies show. that only LiCl inhibited the
observed, PA-induced tau hyperphosphorylation in neurons. Histogram data represent mean i SD. of three
independent experiments. One-way ANOVA and I‘ukey’s post hoc method was used for analyzing the
differences between treatment groups. ‘, p<0.05 compared with control; #, p<0.05 compared with PA
treatment.

5.3.3 A possible explanation for the region-specific and cell type-specific
damage observed in AD

AD is a peculiar neurodegenerative disease in that the observed pathophysiological and
metabolic damages are found to be region-specific and cell type-specific Basal forebrain,
cortex and hippocampus are affected while cerebellum is relatively spared. Also,
cholinergic neurons are most affected 332’ 333. Here, it is interesting to note that different
brain regions show differential FFA metabolic activities; the activity of fatty acyl—CoA
synthetase (FACS) is 10X lower in cerebellum as compared to the other affected brain

regions 334. FACS is the first enzyme involved in cellular FFA metabolism, which

84

converts fatty acid to fatty acyl-CoA, which is then utilized by the cells in catabolic (e. g.
8-oxidation) and anabolic (e. g. ceramide synthesis) pathways 335. ceramide production in
cerebellum is 2X and 4X lower as compared to cortex and hippocampus, respectively 336.
These studies together with our present data may suggest that under pathologically
elevated levels of saturated FF As, cerebellum may be less likely to be affected by
abnormal F FA metabolism due to its lower activity level of F ACS and consequent, lower
levels of ceramide, as compared to cortex and hippocampus. This may explain in part the
region-specific damage observed in AD brain. To further investigate this hypothesis, we
carried out experiments, whereby we treated cortical neurons (CTN) with the conditioned
media from cortical astrocytes (CTA) and from cerebellar astrocytes (CBA). In line with
the results discussed earlier, the conditioned media from PA-treated CTA increased tau

hyperphosphorylation in CTN (Figure 5.8A). The conditioned media from PA-treated

CBA, however, did not increase the phosphorylation of tau in CTN (Figure 5.83).

Furthermore, we had previously shown that FFA metabolism by astroglia results in
increased reactive oxygen species (ROS) production in neurons 337’ 338. The elevated
ROS, in turn, were found to be involved in causing BACEl upregulation and tau

337' 338, suggesting a central role of oxidative stress in

hyperphosphorylation in neurons
the FFA-induced damage. Thus, elevated FFA metabolism associated with higher FACS
activity in the affected regions (basal forebrain, cortex and hippocampus) may lead to
increased oxidative stress in these regions compared to unaffected ones (cerebellum). In

this context, it is interesting to note that in AD brains, oxidative stress markers are higher

in the affected regions than in the unaffected ones 339’ 34°. The increased oxidative stress

85

may be particularly damaging to the basal forebrain cholinergic neurons as these neurons
have been shown to lack an important anti-oxidative enzyme, seleno-glutathione
peroxidase 3'”. This may account, in part, for the cell type-specific damage observed in
AD pathology; substantial loss of basal forebrain cholinergic neurons is a distinct feature
of AD 342_ Furthermore, these basal forebrain cholinergic neurons, through their long
ascending projections, innervate cortical and hippocampal regions 343 and the increased
oxidative stress in these regions of AD brain may lead to the degeneration of these
projections from the cholinergic neurons. In this context, it is also interesting to note that

A8 plaques, hallmarks of AD, have been shown to be specifically located where the

projection of the basal forebrain cholinergic neurons degenerate 344' 3“.
(A) CTN-CTA (B) CTN-CBA
. .mu)”- 1!. ‘ *
AT8 a“ AT8 a... _ .
Actin “d Ami“ m“
C PA
C PA
160 a
140 120
120 100 1
100 so ‘
80 60
6o 40
40 20
20 o
o

   

Figure 5.8. Differential effects of cortical and cerebellar astroglia on cortical neurons. Cortical
neurons (CTN) were treated with conditioned media from (A) cortical astroglia (CTA) and (B) cerebellar
astroglia (CBA). Western blot analysis of hyperphosphorylated tau was performed using AT8 antibody.
Cortical astroglia but not cerebellar astroglia were involved in PA-induced tau hyperphosphorylation in
neurons. Data represent mean i SD. of three independent experiments. Student‘s t-test was used for
analyzing differences between difierent treatment groups. *. p<0.05 compared with control.

86

5.4 CONCLUSIONS

In conclusion, astroglial ceramide was found to be involved in the observed PA-astroglia-
induced ROS production in neurons. Astrolglial ceramide was also found involved in the
PA-astroglia-induced hyperphosphorylation of tau in neurons. Although astroglial
ceramide activated both GSK-3a/B and cdk5 in neurons, only GSK-3a/f3 was found to be
involved in PA-astroglia-induced hyperphosphorylation of tau. Furthermore, Astroglial
ceramide increased BACE] levels and consequent amyloidogenic processing of APP
leading to the production of AB40/42. Thus, the present results establish a central role of
astroglial fatty acid metabolism and consequent increase in the de novo synthesis of
astroglial ceramide in causing two of the major pathophysiological changes associated

with AD, tau hyperphosphorylation and AB production.

87

CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS

6.1 Conclusions

The objective of this dissertation was to investigate the potential involvement of saturated
fatty acids in causing AD-associated pathophysiological and abnormal metabolic
changes. Our studies established a complex functional interaction between neuronal and
non-neuronal (astroglial) cells, leading to the AD-specific abnormalities under the
conditions of pathologically elevated levels of saturated fatty acids. It was shown that
saturated fatty acids had no direct deleterious effects on neurons; however, they caused
increased oxidative stress in neurons through astroglial mediation. Fatty acid-induced
oxidative stress played a central role in the hyperphosphorylation of tau and
amyloidogenic processing of APP, two of the important characteristics of AD pathology.
Both metabolic modeling (MFA) and experimental data suggested a key role of astroglial
ceramide in causing F FA-induced, AD-associated abnormalities in neurons. Note that this
was the first-ever attempt to apply MFA to comprehensively study the primary astroglial
metabolism. Our data place “astroglial fatty acid metabolism” at the center of the
pathogenic cascade in AD and also suggest “astroglial ceramide” as a potentially

important target for therapeutic intervention in AD.

Based on our findings, we hypothesize the following sequence of cellular events by
which saturated FFAS may play a central role in the pathogenesis of AD (Figure 6.1).
The brain experiences chronically elevated levels of saturated FFAS. Saturated FFAS are
taken up and metabolized by astroglia, downregulating astroglial GLUTl levels and

glucose metabolism. Astroglial FFA metabolism also results in increased levels of

88

ceramides. Ceramides then induce secretion of cytokines (e.g. IL-IB, TNF-a etc.) or
other signaling molecules (e.g. NO, due to increased expressed iNOS expression) by
astroglia, which may induce ROS production in neurons. Increased oxidative stress in
neurons causes BACEI upregulation and GSK-3 activation resulting in increased AB
production and hyperphosphorylation of tau, respectively. The sequence of events
suggested here is similar to that observed in AD pathology- decreased glucose
metabolism is an early event, which together with increased oxidative stress precedes the
pathophysiological changes observed in AD (NFTS and AB plaques). Furthermore, the
present “FFA-AD” hypothesis also provides a possible explanation for the region-specific

and cell type-specific damage observed in AD (Chapter 5).

     
     
   
      
   

Astrocyte
,‘ APP

Secreted

Factor(s) ceramide f

A Abetn

Production
A

RACF'l
A

ROS ‘
Tau‘

Phosphorylnlion ‘ i ' ,- No direct effect
(GSK—E) ‘ ‘_ , on neurons

Neuron

Figure 6.1. The “FFA-AD” hypothesis. Proposed cellular mechanism by which astroglial FFA
metabolism may play a central role in causing pathophysiological and metabolic changes associated with
AD.

89

The low level of ceramide production in cerebellar astroglia as compared to cortical and

hippocampal astroglia 336

, may play a key role in the little damage observed in
cerebellum of AD brain as opposed to other regions. Due to the low flux of saturated
FFAS through sphingolipid pathway in cerebellar astroglia, FFAS may be diverted to
other metabolic pathways, e. g. B-oxidation of fatty acids, which may lead to the increased
production of ketone bodies. Here it is interesting to note that ketone bodies have been
shown to act as anti-oxidants in that they protect neuronal cells form AIS-induced

oxidative damage 346. This may further explain why cerebellum is relatively spared in

AD.

It is also noteworthy that the single-most factor strongly correlated with AD is aging.
Therefore, any hypothesis to explain AD pathology must provide an explanation for the
correlation between aging and incidence of AD. Increased oxidative stress has been
strongly associated with aging, which might be a result of decreased anti-oxidant
enzymatic capacity with aging 54' 347. Under these conditions, enhanced production of
ROS induced by saturated FFAS may make the brain more vulnerable to increased
oxidative damage as compared to age-matched controls with low levels of saturated
FFAS. Thus, our present hypothesis may explain the link between age and the higher

incidence of AD.

Despite these supporting data, our “FFA-AD” hypothesis is unable to explain the AD
pathology comprehensively at this juncture. Specifically, although our cell-culture studies

showed potential involvement of saturated fatty acids in causing both the

90

amyloidogenesis and the hyperphoshorylation of tau, in animals high fat diet has been
shown to cause only increased AB production but not the tau hyperphosphorylation and

the NFT formation associated with it 122424

. The reason behind these disparities between
in vitro and in vivo findings is not well understood. In addition, some studies suggest the
tau protein abnormalities initiate the AD cascade, while others emphasize AB deposits as

the causative factors in AD 348. Our present data is unable to shed any light on this

ongoing debate.

Furthermore, in the present studies we established a central role of FA—induced oxidative
stress in causing AD-associated abnormalities. However, oxidative stress has been shown
to be central to many other brain diseases, e. g. Parkinson's disease 349, in addition to AD.
Therefore, it is not clear how FFA-induced oxidative stress is specific to AD pathology.
In other words, it is not clear how FFA-induced oxidative stress would lead to AD-
specific changes but would not induce abnormalities associated with other diseases, in
which oxidative stress plays a key role. All the high fat diet animal models show only
AD-specific changes in their brain. Our current data does not provide any explanation for

this important, fundamental question.

In addition, AD follows a specific spatio-temporal pattern of neurodegeneration, where
neurodegeneration starts at the basal forebrain and with time proceeds to entorhinal
cortex, hippocampus, parts of limbic system and associative cortex 350. The basal
forebrain cholinergic neurons lack an important anti-oxidative enzyme, seleno-

4

glutathione peroxidase 3 '. This may explain their higher vulnerability to the FFA-

91

induced increased oxidative stress. It is not clear, however, how saturated fatty acids

would contribute to the characteristic spatio-temporal neuronal damage observed in AD.

F urtherrnore, our present data explain the potential involvement of saturated fatty acids in
the increased AB production. However, it is not just the increased production of AB, but
also its aggregation that is important in AD pathology. To be specific, neither monomeric
nor mature aggregated polymeric forms, but the intermediate oligomeric forms of AB are
responsible for the AD-associated neurotoxicity 35'. In fact, a very recent study showed
that accelerating AB fibrillization which reduced AB oligomer levels, helped in reducing
functional deficits in AD mouse models 352. Our present data do not answer if (and how)

saturated fatty acids play a role in the AB oligomerization.

Here, it is also important to note that the risk of AD is higher in women as compared to
men 353. It is not clear if saturated fatty acids play any role in the gender-specificity
associated with AD. The decreased level of estrogen hormone in menopausal women has
been suggested to increase the risk for AD in women 354. It is not clear at present, if there
is any correlation between saturated fatty acids and estrogen levels, that may lead to
increased risk for AD development in women as compared to men, under the condition of

elevated levels of saturated FFAS.

Finally, our present studies established a central role of astroglia in causing FFA-induced,
AD-associated abnormalities. The critical role of astroglia in AD pathology has been

suggested previously by many studies. Increased expression of iNOS in astroglia and

92

consequent increase in NO levels has been shown to stimulate AB production and
hyperphosphorylation of tau in neurons 355' 356. Furthermore, reactive astrogliosis
associated with AD has been suggested to induce glutamate release, which may lead to

excitotoxicity and cell death in neurons 357' 358.

In addition, astroglial apoptosis has been
associated with AD and AB has been shown to induce astroglial cell death 359’ 360. This
astroglial cell death may result in the “loss of good function”, to support the neurons
under normal conditions. Together these data place astroglia at the centre stage of AD
pathology. However, the major limitation of all these studies, including ours, is that the
astroglia are used in these studies as a whole population. It would be worthwhile to
investigate the possible involvement of Type I and Type II astroglia separately, in

causing AD-associated damage. These data may prove invaluable in finding novel clues

that may further help in establishing the in-depth disease mechanism.

6.2 Future Directions

Our cell-culture based studies presented in this dissertation have provided important
information regarding the key role of saturated fatty acids in causing AD-associated
abnormalities. As discussed above, many questions remain unanswered and thus, need
further scientific investigation. The focus of the future investigation will be specifically

on the following studies as discussed below.

6.2.1 In vivo studies
Future work should focus on animal studies where cerebral FFA metabolism will be

studied in terms of the de novo synthesis of ceramide at the regional (e.g. cortex,

93

hippocampus vs. cerebellum) as well as sub-cellular (astroglia vs. neurons) levels in
response to various diets and stimuli in APP transgenic mice, e.g. Tg2576. This line of
mice expresses human APP695 with the 670/671 “Swedish” double mutation and show
clear age-dependent AB deposition and memory deficits 39. It would be a significant step
forward in AD research if saturated FFAS are shown to exert their risk for the
development of AD in vivo, through a similar mechanism as observed in the current in
vitro studies. Furthermore, the potential importance of ceramide as a therapeutic target
for AD should also be further studied in these animals by using pharmacological
inhibitors of de novo synthesis of ceramide, e.g. L-CS, D-serine, myriocin (ISP-l),
fumonisin Bl, viridiofungin A, sphingofungin B or lipoxarnycin. Decreased AB
production and its deposition in the brains of these mice would serve as measures of the

potential protective effects of these ceramide inhibitors.

6.2.2 Delivery of ceramide inhibitors through the blood-brain barrier (BBB)

With the use of ceramide inhibitors in vivo, one of the greatest challenges, which also
presents a great research opportunity, is to find ways for efficient transport of these
inhibitors across the BBB. In its neuroprotective role, the BBB prevents the delivery of
many important therapeutic agents to the brain; more than 98% of currently available
therapeutics cannot pass through the BBB 361. Future studies should investigate the use of
novel delivery systems, e.g. nanoparticles, as carriers of ceramide inhibitors to the brain.
In addition to their ability to cross the BBB, these vehicles should also be engineered so

as to deliver ceramide inhibitors specifically to astroglia, e.g. by attaching astroglia-

94

specific antibody (GFAP) to nanOparticles. In addition to their use in AD, these

successful delivery vehicles will also prove useful in treating other brain diseases.

6.2.3 Studying pathways downstream to ceramide

One of the major limitations with ceramide as a therapeutic target is its important role as
a signaling molecule in many physiological processes. In addition, the currently available
ceramide inhibitors mentioned earlier have very high toxicities, weak inhibition activity
and also exhibit low specificity 362' 363. Thus, it would also be worthwhile to focus future
studies on the pathways downstream of ceramide generation that may be involved in
FF A-induced, AD-associated abnormalities, e.g. ceramide-induced secretion of

inflammatory cytokines or other signaling molecules such as NO from astroglia.

95

APPENDIX

1. Brain cells from older animals

In our studies we used cortical neurons and astroglia from 1-2-day old rat pups. As AD is
strongly associated with aging, use of brain cells from older animals would be more
appropriate for these AD studies. However, it is difficult to isolate the cortical cells from
the brains of older rats and their viability reduced significantly; the cortical cells isolated

from 7-day old rat pups started dying after 2 days in culture.

   

Day 1 Day 2

2. Trans-well experiments

In our studies, the conditioned media from astroglia were transferred to neurons, so the
two cell types did not share the same growth enviromnent. Physiologically, however,
neurons and astroglia are in close proximity and share common growth environment
where secreted factors fiorn both the neurons and astroglia may affect both these cell
types. We investigated the possible effect of potential factors secreted from the neurons

that may affect astroglia, which in turn may modulate the observed astroglial effects on
96

the neurons using trns-well tissue culture. The astroglia were plated in the well inserts
and neurons in the wells. The cells were then treated with either 0.2mM PA or 4% BSA
(control) and levels of BACE] and phosphorylated tau in neurons were studied. We
found that PA-treatment significantly increased levels of BACE] and phosphorylated tau

in neurons, similar to our non-trans-well experiments.

W

tau (AT8) “M” '~

 

BACE1 w W

‘4‘“ arm"! PHI-t. we!"

actin n -
(3 PA

3. Exogenous addition of ceramide as a positive control

Our studies showed a central role of astroglial ceramide in the FFA-astroglia-induced,
AD-associated pathophysiological changes observed in neurons. The role of astroglial
ceramide was confirmed by HPLC measurement of elevated ceramide and also by using a
pharmacological inhibitor of ceramide synthesis in astrolgia. As a positive control, we
exogenously added 1051M C-6 ceramide (a synthetic analog of ceramide, Sigma) to
astroglia for 24hr, followed by the transfer of the astroglia-conditioned media to neurons
(24hr treatment). We found that C-6 ceramide significantly increased levels of BACEl

97

and phosphorylated tau in neurons, thus further emphasizing role of ceramide in causing

AD-associated abnormalities.

tau (AT8) H'- H
BACE1 m,

actin - .1

C ceramide

 

98

LIST OF PUBLICATIONS

This thesis is based on our following original publications-

1)

2)

3)

4)

5)

Patil, S. and Chan, C., “Palmitic and Stearic fatty acids induce Alzheimer-like
hyperphosphorylation of tau in primary rat cortical neurons”, Neuroscience

Letters, 384: 288-293 (2005).

Patil, S., Lufang, S., Masserang, A. and Chan, C., “Palmitic acid-treated
astrocytes induce BACE] upregulation and accumulation of C-terrninal fragment

of APP in primary cortical neurons”, ”, Neuroscience Letters, 406: 55-59 (2006).

Patil, S., Li, Z. and Chan, 0, “Cellular to Tissue Informatics: Approaches to
Optimizing Cellular Function of Engineered Tissue", Advances in Biochemical

Engineering / Biotechnology, eds. K. Lee and D. Kaplan, 102: 139-159 (2006).

Patil, S., Melrose, J. and Chan, C., “Involvement of astroglial ceramide in

palmitic acid-induced Alzheimer-like changes in primary neurons”, (Accepted,

European Journal of Neuroscience).

Patil, S., Balu, D., Melrose J. and Chan, C., “Brain region specificity of palmitic

acid-induced Alzheimer-like changes in primary neurons”, (In Preparation).

99

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