CHOLESTEROL INDUCED GENE EXPRESSION CHANGES IN THE BRAIN AND CEREBRAL VESSELS

IPA network showing the network with the second largest number of upregulated focus genes in the MCA of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control gr

CHOLESTEROL INDUCED GENE EXPRESSION CHANGES IN THE BRAIN

AND CEREBRAL VESSELS

LOKE SAU YEEN

(B.Sc (Hons), NUS)

SUPERVISOR: ASSOCIATE PROFESSOR ONG WEI YI

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Declaration Page

DECLARATION

I hereby declare that the thesis is my original work and it has been written by

me in entirety I have duly acknowledged all the sources of information which

have been in used in the thesis

This thesis has also not been submitted for any degree in any university

previously

Loke Sau Yeen

22 January 2015

Acknowledgements

ACKNOWLEDGEMENTS

Firstly, I would like to express my heartfelt gratitude to my supervisor,

Associate Professor Ong Wei Yi, Department of Anatomy, National

University of Singapore (NUS), who proposed the topic of my research and provided priceless guidance throughout my entire Ph.D program His indefatigable teachings, determination and support have inspired me to do well

in my research Secondly, I would like to extend my appreciation to Professor

Bay Boon Huat, Head of Department of Anatomy, for granting me an

invaluable opportunity to pursue my postgraduate study at NUS, and for his support in my entire candidature

I would also like to acknowledge my ex-colleagues, Ms Ng Pei Ern

Mary, for her exceptional assistance in the in vivo work and Dr Kazuhiro

Tanaka, for his constructive suggestions I am also thankful to Dr Wu Ya Jun,

Ms Chan Yee Gek and Ms Pan Feng for their remarkable expertise in

electron microscopy, confocal microscopy and histology, respectively; Mdm

Ang Lye Gek Carolyne, Mrs Diljit Kaur D/O Bachan Singh and Mdm Teo

Li Ching Violet for their administrative support

A heartfelt thanks to all my colleagues and friends in the Histology Lab,

Table of Contents

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i 

TABLE OF CONTENTS ii 

SUMMARY xi 

LIST OF TABLES xiv 

LIST OF FIGURES xvi 

LIST OF ABBREVIATIONS xxiv 

PUBLICATIONS xxxiii 

CHAPTER I INTRODUCTION 1 

1.  Cholesterol 2 

  Physicochemical Properties of Cholesterol 3 

  Cholesterol in the Brain 4 

1.2.1.  Biosynthesis of Cholesterol 6 

1.2.2.  Regulation of Cholesterol Biosynthesis 9 

1.2.3.  Transport and Storage of Cholesterol 11 

1.2.4.  Elimination of Cholesterol 11 

1.2.5.  Cholesterol and Blood Brain Barrier (BBB) 12 

2.  Oxysterols 14 

  Distribution of Oxysterols 17 

  Metabolism of Oxysterols 17 

  Elimination of Oxysterols 18 

  Oxysterols and Cholesterol Homeostasis 19 

  Other Roles of Oxysterols in the Brain 20

Table of Contents

3.  Cholesterol, Oxysterols and Neurological Disorders 23 

  Excitotoxicity and Neurodegeneration 23 

3.1.1.  Cholesterol and Oxysterols in KA-Induced Excitotoxicity and Neurodegeneration 24 

  Intracranial Large Artery Disease (ICLAD) 27 

3.2.1.  Middle Cerebral Artery (MCA) 28 

3.2.2.  Mechanisms of Intracranial Atherosclerosis 30 

3.2.3.  Pathological Characteristics of ICLAD 32 

3.2.4.  ICLAD and Stroke 33 

3.2.5.  Risk Factors of ICLAD 34 

3.2.5.1.  Hypercholesterolemia 34 

3.2.5.2.  Hypertension 35 

  Alzheimer’s Disease (AD) 38 

3.3.1.  Frontal Cortex (FC) 40 

3.3.2.  Amyloid-β (Aβ) 41 

3.3.3.  Cholesterol in the Pathogenesis of AD 44 

3.3.3.1.  Cholesterol Modulation of Aβ Biosynthesis 44 

3.3.3.2.  Cholesterol Modulation of Aβ Aggregation and

Table of Contents

4.  Animal Models of Atherosclerosis and AD 51 

  Cholesterol-Fed Rabbit Model of Hypercholesterolemia 51 

  2-Kidney, 1-Clip (2K1C) Model of Hypertension 53

CHAPTER II AIMS OF THE PRESENT STUDY 56

CHAPTER III EXPERIMENTAL STUDIES 59

SECTION 1 GENE EXPRESSION ANALYSES OF THE RAT PREFONTAL CORTEX AFTER CHOLESTEROL OR OXYSTEROL TREATMENT 60 

1.  Introduction 61 

2.  Material and Methods 63 

  Animal and Treatment 63 

  RNA extraction 64 

  DNA microarray analyses 64 

  Network analyses 65 

  Real-time RT-PCR 66 

  Western blot 67 

  Immunohistochemistry 68 

  Electron microscopy 69 

3.  Results 70 

  Microarray data collection and analyses 70 

  DEGs exclusive to 7β-HC treatment 71 

  DEGs exclusive to 7-KC treatment 71 

Table of Contents

  DEGs common to 7β-HC and 7-KC treatments 71 

  DEGs common to 7β-HC and cholesterol treatments 72 

  DEGs common to 7-KC and cholesterol treatments 72 

  DEGs common to 7β-HC, 7-KC, and cholesterol treatments 72 

  Networks for downregulated DEGs common to 7β-HC and 7-KC treatments 73 

  Networks for upregulated DEGs common to 7β-HC and 7-KC treatments 77 

 Real-time RT-PCR 79 

 Western Blot 81 

 Immunolocalization of OXTR in the PFC 83 

4.  Discussion 86

SECTION 2 GENE EXPRESSION ANALYSES OF THE RABBIT CEREBRAL VESSELS AFTER HYPERCHOLESTEROLEMIA AND/OR HYPERTENSION 91 

1.  Introduction 92 

2.  Material and Methods 94 

Table of Contents

  Electron microscopy 100 

  Real-time RT-PCR 100 

  Western blot 101 

  Histochemistry and immunohistochemistry 102 

3.  RESULTS 104 

  Body weight, mean arterial pressure (MAP) and serum total cholesterol levels 104 

  Microarray analyses in the MCA 107 

3.2.1.  Microarray analyses of the ‘hypertension only’ group 107 

3.2.2.  Microarray analyses of the ‘hypercholesterolemia plus sham’ group 115 

3.2.3.  Microarray analyses of the ‘hypercholesterolemia plus hypertension’ group 121 

3.2.4.  Microarray analyses of the ‘common area’ between ‘hypercholesterolemia plus sham’ and ‘hypertension only’ groups 125 

3.2.5.  Microarray analyses of the ‘exclusive area’ in the ‘hypercholesterolemia plus hypertension’ group 130 

  Electron microscopy of the MCA 136 

  Vascular changes in the aorta 138 

3.4.1.  Real-time RT-PCR 138 

3.4.2.  Western blot 139 

3.4.3.  Histochemistry and immunohistochemistry 140 

4.  Discussion 142 

Table of Contents

SECTION 3 GENE EXPRESSION ANALYSES OF THE RABBIT

FRONTAL CORTEX AFTER HYPERCHOLESTEROLEMIA AND/OR

HYPERTENSION 152 

1.  Introduction 153 

2.  Material and Methods 155 

  Animals 155 

  Measurement of body weight, mean arterial pressure (MAP) and serum total cholesterol 156 

  Tissue harvesting and RNA extraction 156 

  DNA microarray analyses 157 

  Network analyses 157 

3.  Results 158 

  Body weight, mean arterial pressure (MAP) and serum total cholesterol levels 158 

  Microarray analyses in the FC 158 

3.2.1.  Microarray analyses of the ‘hypercholesterolemia plus sham’ group 158 

3.2.2.  Microarray analyses of the ‘hypercholesterolemia plus

Table of Contents

SECTION 4 COMPARISON OF GENE EXPRESSION ANALYSES BETWEEN THE RABBIT CEREBRAL VESSELS AND FRONTAL CORTEX AFTER HYPERCHOLESTEROLEMIA AND/OR

HYPERTENSION 189 

1.  Introduction 190 

2.  Material and Methods 192 

  DNA microarray analyses 192 

  Network analyses 192 

  Immunohistochemistry 193 

3.  Results 194 

  Microarray analyses of the ‘hypercholesterolemia plus sham’ group in the FC and MCA 194 

3.1.1.  Microarray analyses of the ‘exclusive area’ in the FC 194 

3.1.2.  Microarray analyses of the ‘exclusive area’ in the MCA 196  3.1.3.  Microarray analyses of the ‘common area’ between the FC and MCA 197 

  Comparison of the DEGs from the hypercholesterolemia and/or hypertension groups in the FC and MCA 201 

  Immunohistochemistry 203 

4.  Discussion 206 

Table of Contents

SECTION 5 GENE EXPRESSION REGULATION BY STEROL

REGULATORY ELEMENT BIINDING PROTEIN 216 

1.  Introduction 217 

2.  Material and Methods 219 

  Treatment with SREBP inhibitors 219 

  Real-time RT-PCR 219 

  Immunofluorescence 220 

3.  Results 222 

  Human endothelial EA.hy926 cells 222 

3.1.1.  Real-time RT-PCR analysis of APP expression after treatment with SREBP inhibitors 222 

3.1.2.  Real-time RT-PCRT analysis of SERPINB2 expression after treatment with SREBP inhibitors 225 

3.1.3.  Immunofluorescence analysis of Aβ after treatment with 10 μM 1,10-phenanthroline 227 

3.1.4.  Immunofluorescence analysis of SERPINB2 after treatment with 10 μM 1,10-phenanthroline 229 

  Human neuroblastoma SH-SY5Y cells 231 

Table of Contents

3.2.4.  Immunofluorescence analysis of SERPINB2 after

treatment with 10 μM 1,10-phenanthroline 235 

4.  Discussion 237

CHAPTER IV CONCLUSION 243 

CHAPTER V REFERENCES 249 

CHAPTER VI APPENDICES 289 

After 1-day post-intracortical injection of cholesterol or oxysterols i.e 7β-hydroxycholesterol (7β-HC) and 7-ketocholesterol (7-KC) at low dose in the rat prefrontal cortex (PFC), microarray analyses has identified 1365 differentially expressed genes (DEGs), which were commonly affected by both 7β-HC and 7-KC treatments Among these DEGs, downregulation was the

as the hypertensive rabbits, despite relatively low percentage of ‘common genes’ between the two conditions Upregulated common genes were related to

‘node molecules’ like hepatocyte nuclear factor 4A (HNF4A), serpin peptidase inhibitor, clade B, member 2 (SERPINB2), and amyloid precursor protein (APP) Increased levels of HNF4A mRNA and protein were verified in the aorta

In addition, hypercholesterolemia and hypertension are also risk factors for AD Using the same animal models of hypercholesterolemia and hypertension, gene expression alterations were analyzed in the frontal cortex (FC) In FC, hypercholesterolemia induced more gene expression changes than hypertension The comparison between gene expression profiles of the FC and MCA, surprisingly, revealed a majority of the downregulated DEGs in FC were found in MCA of the same hypercholesterolemic rabbits Likewise, common

‘node molecules’ were also noted in these tissues after exposure to hypercholesterolemia Interestingly, ‘node molecules’ that were affected by hypercholesterolemia and/or hypertension in both FC and MCA have been

Summary

associated with various pathways involving APP and its proteolytic fragments

A significantly increased Aβ-immunolabeled neurons was observed in the FC

of ‘hypercholesterolemia plus sham/hypertension’ rabbits

To investigate the possible regulation of APP and its related genes by the sterol regulatory element binding protein (SREBP) signaling, which may be affected during hypercholesterolemia, expression of APP/amyloid-β (Aβ) and

SERPINB2 after treatment with SREBP inhibitors were studied in vitro

Increased expression of APP/Aβ and SERPINB2 induced by SREBP inhibition were observed in the human endothelial and neuroblastoma cell lines, thus, implying a possible regulation of SREBP in the expression of these genes

In conclusion, cholesterol exerted different acute and chronic effects on gene expression changes in the brain and cerebral vessels in present study Acute intracortical administration of oxysterols at low concentration were capable of inducing more severe gene expression changes compared with cholesterol In contrast, consumption of a high cholesterol diet for an extended period chronically induced massive gene expression changes in the brain and cerebral vessels, possibly through the mediation of SREBP signaling, and it might affect similar pathways in both of these tissues Therefore, current results

List of Tables

LIST OF TABLES

Table 3.1 Top five associated network functions mapped by IPA for

downregulated DEGs in the PFC, which were found in common to 1-day 7β-HC and post-7-KC treatments 74 

Table 3.2 Downregulated DEGs in the PFC found in common to 1-day 7β-HC and post-7-KC treatments 75 

post-Table 3.3 Upregulated genes in the MCA found in common between

‘hypercholesterolemia plus sham’ and ‘hypertension only’ rabbits (each group

vs sham operated control group) with greater than 4-fold change 126 

Table 3.4 Downregulated genes in the MCA found in common between

‘hypercholesterolemia plus sham’ and ‘hypertension only’ rabbits (each group

vs sham operated control group) with greater than 4-fold change 127 

Table 3.5 Upregulated genes in the FC of the ‘hypercholesterolemia plus sham’ rabbits vs sham operated control rabbits with greater than 10-fold change 160 

Table 3.6 Downregulated genes in the FC of the ‘hypercholesterolemia plus sham’ rabbits vs sham operated control rabbits with greater than 10-fold change 162 

Table 3.7 Upregulated genes in the FC of the ‘hypercholesterolemia plus hypertension’ rabbits vs sham operated control rabbits with greater than 10-fold change 172 

Table 3.8 Downregulated genes in the FC of the ‘hypercholesterolemia plus hypertension’ rabbits vs sham operated control rabbits with greater than 10-

Table 3.11 Comparison of the DEGs between the FC and MCA of

‘hypercholesterolemia plus sham’, ‘hypercholesterolemia plus hypertension’, and ‘hypertension only’ rabbits (each group vs sham operated control group) 202 

List of Figures

LIST OF FIGURES

Figure 1.1 Structure of cholesterol 4 

Figure 1.2 Cholesterol biosynthesis pathway 8 

Figure 1.3 SREBP activation pathway 10 

Figure 1.4 Structures of major oxysterols 15 

Figure 1.5 The arteries of the base of the brain 29 

Figure 1.6 APP processing in non-amyloidogenic (A) and amyloidogenic (B) pathways 43 

Figure 3.1 Venn diagram showing the classification of DEGs in the PFC after 1-day post-treatment with 7β-HC, 7-KC, or cholesterol 70 

Figure 3.2 IPA network of downregulated genes found in common to 1-day post-7β-HC and post-7-KC treatments with DEGs with more than 2-fold change 76 

Figure 3.3 IPA network of upregulated genes found in common to 1-day post-7β-HC- and post-7-KC treatments with DEGs with more than 2-fold change 78 

Figure 3.4 Real-time PCR analyses in the rat PFC 80 

Figure 3.5 Western blot analysis of OXTR 82 

Figure 3.6 Light micrographs of OXTR-immunostained brain slices in the PFC 84 

Figure 3.7 Electron micrograph of OXTR-immunostained sections from the PFC 85 

Figure 3.8 The 2K1C model of hypertension 96 

List of Figures

Figure 3.9 MAP (A) and serum cholesterol levels (B) in ‘hypertension only’ rabbits 105 

Figure 3.10 MAP (A) and serum cholesterol levels (B) in

‘hypercholesterolemia plus sham’ and ‘hypercholesterolemia plus

hypertension’ rabbits 106 

Figure 3.11 Venn diagram of DEGs in the MCA of ‘hypertension only’ rabbits, ‘hypercholesterolemia plus sham’ rabbits, and ‘hypercholesterolemia plus hypertension’ rabbits; each group vs sham operated control rabbits 108 

Figure 3.12 IPA network showing the network with the largest number of upregulated focus genes in the MCA of the ‘hypertension only’ group,

compared with sham operated control group 110 

Figure 3.13 IPA network showing the network with the second largest

number of upregulated focus genes in the MCA of the ‘hypertension only’ group, compared with sham operated control group 111 

Figure 3.14 IPA network showing the network with the largest number of downregulated focus genes in the MCA of the ‘hypertension only’ group, compared with sham operated control group 113 

Figure 3.15 IPA network showing the network with the second largest

List of Figures

Figure 3.17 IPA network showing the network with the second largest

number of upregulated focus genes in the MCA of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control group 117 

Figure 3.18 IPA network showing the network with the largest number of downregulated focus genes in the MCA of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control group 119 

Figure 3.19 IPA network showing the network with the second largest

number of downregulated focus genes in the MCA of the

‘hypercholesterolemia plus sham’ group, compared with sham operated

control group 120 

Figure 3.20 IPA network showing the network with the largest number of upregulated focus genes in the MCA of the ‘hypercholesterolemia plus

hypertension’ group, compared with sham operated control group 122 

Figure 3.21 IPA network showing the network with the largest number of downregulated focus genes in the MCA of the ‘hypercholesterolemia plus hypertension’ group, compared with sham operated control group 124 

Figure 3.22 IPA network showing the network with the largest number of upregulated focus genes in the MCA between the ‘hypercholesterolemia plus sham’ and ‘hypertension only’ group (‘common area’), compared with sham operated control group 128 

Figure 3.23 IPA network showing the network with the largest number of downregulated focus genes in the MCA between the ‘hypercholesterolemia plus sham’ and ‘hypertension only’ group (‘common area’), compared with sham operated control group 129 

Figure 3.25 IPA network showing the network with the second largest

number of upregulated focus genes in the MCA of the ‘hypercholesterolemia plus hypertension’ group (‘exclusive area’), compared with sham operated control group 132 

Figure 3.26 IPA network showing the network with the largest number of downregulated focus genes in the MCA of the ‘hypercholesterolemia plus hypertension’ group (‘exclusive area’), compared with sham operated control group 134 

Figure 3.27 IPA network showing the network with the second largest

number of downregulated focus genes in the MCA of the

‘hypercholesterolemia plus hypertension’ group (‘exclusive area’), compared with sham group 135 

Figure 3.28 Electron micrographs of the MCA 137 

Figure 3.29 Real-time RT-PCR analysis of HNF4A in the aorta of control,

‘hypertension only’ rabbits; each group vs sham operated control rabbits 159 

Figure 3.33 IPA network showing the network with the largest number of upregulated focus genes in the FC of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control group 166 

Figure 3.34 IPA network showing the network with the second largest

number of upregulated focus genes in the FC of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control group 167 

Figure 3.35 IPA network showing the network with the largest number of downregulated focus genes in the FC of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control group 169 

Figure 3.36 IPA network showing the network with the second largest

number of downregulated focus genes in the FC of the ‘hypercholesterolemia plus sham’ group, compared with sham operated control group 170 

Figure 3.37 IPA network showing the network with the largest number of upregulated focus genes in the FC of the ‘hypercholesterolemia plus

hypertension’ group, compared with sham operated control group 178 

Figure 3.38 IPA network showing the network with the largest number of downregulated focus genes in the FC of the ‘hypercholesterolemia plus

hypertension’ group, compared with sham operated control group 179 

List of Figures

Figure 3.39 IPA network showing the network with the only upregulated focus gene in the FC of the ‘hypertension only’ group, compared with sham operated control group 181 

Figure 3.40 IPA network showing the network with the largest number of downregulated focus genes in the FC of the ‘hypertension only’ group,

compared with sham operated control group 182 

Figure 3.41 Venn diagram of DEGs in the FC and MCA of the

‘hypercholesterolemia plus sham’ rabbits vs sham operated control rabbits 195 

Figure 3.42 IPA network showing the network with the largest number of upregulated focus genes in the ‘common area’ of the FC and MCA

(‘hypercholesterolemia plus sham’ group vs sham operated control group) 198 

Figure 3.43 IPA network showing the network with the largest number of downregulated focus genes in the ‘common area’ of the FC and MCA

(‘hypercholesterolemia plus sham’ group vs sham operated control group) 200 

Figure 3.44 Immunohistochemical staining of Aβ in the FC of rabbit exposed

List of Figures

Figure 3.47 Real-time RT-PCR analysis of APP mRNA expression levels in human endothelial EA.hy926 cells after treatment with 1,10-phenanthroline in

a dose-dependent manner for 24 h 224 

Figure 3.48 Real-time RT-PCR analysis of SERPINB2 mRNA expression levels in human endothelial EA.hy926 cells after treatment with various SREBP inhibitors at 10 µM for 24 h 225 

Figure 3.49 Real-time RT-PCR analysis of SERPINB2 mRNA expression levels in human endothelial EA.hy926 cells after treatment with 1,10-

phenanthroline in a dose-dependent manner for 24 h 226 

Figure 3.50 Immunofluorescence labeling of Aβ in human endothelial

EA.hy926 cells after treatment with vehicle or 10 µM 1,10-phenanthroline for

24 h 228 

Figure 3.51 Immunofluorescence labeling of SERPINB2 in human

endothelial EA.hy926 cells after treatment with vehicle or 10 µM

1,10-phenanthroline for 24 h 230 

Figure 3.52 Real-time RT-PCR analysis of APP mRNA expression levels in human neuroblastoma SH-SY5Y cells after treatment with various SREBP inhibitors at 10 µM for 24 h 231 

Figure 3.53 Real-time RT-PCR analysis of SERPINB2 mRNA expression levels in human neuroblastoma SH-SY5Y cells after treatment with various SREBP inhibitors at 10 µM for 24 h 232 

Figure 3.54 Immunofluorescence labeling of Aβ in human neuroblastoma SY5Y cells after treatment with vehicle or 10 µM 1,10-phenanthroline for 24 h 234 

SH-List of Figures

Figure 3.55 Immunofluorescence labeling of SERPINB2 in human

neuroblastoma SH-SY5Y cells after treatment with vehicle or 10 µM phenanthroline for 24 h 236 

1,10-Figure 3.56 The association between cholesterol, SREBP, APP, Aβ and related genes 242 

APP-Figure 4.1 The summary of present study 248 

2K1C 2-kidney, 1-clip hypertension

2K2C 2-kidney, 2-clip hypertension

ABCG1 ATP-binding cassette, subfamily G, member 1

ACAT Acyl-coenzyme-A cholesterol acyltransferase

ADAM A disintegrin and metalloproteinase

ADAMTS17 ADAM metallopeptidase with thrombospondin type 1

motif, 17 AICD APP intracellular domain

List of Abbreviations

AMPA Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate ANKAR Ankyrin and armadillo repeat containing

ANOVA Analysis of variance

ANP Atrial natriuretic peptide

ATF6 Activating transcription factor 6

BACE1 β-site APP cleaving enzyme 1

C1QTNF1 C1q and tumor necrosis factor related protein 1

CAA Cerebral amyloid angiopathy

CARE Centre for Animal Resources

List of Abbreviations

CIDE Cell death-inducing DNA fragmentation factor 45-like

effector CIDEC Cell death-inducing DFFA-like effector c

CTCF Corrected total cell fluorescence

CYP11β 11β-hydroxycholesteroid dehydrogenase type 1

CYP27A1 Sterol 27-hydroxylase

DAB 3,3,-diaminobenzidine tetrahydrochloride

DEG Differentially expressed gene

ERK1/2 Extracellular signal-regulated kinase 1/2

FAM167A Family with sequence similarity 167, member A FAM184B Family with sequence similarity 184, member B FAM19A4 Family with sequence similarity 16 (chemokine (C-C

motif)-like), member A4

FCRL3 Fc receptor-like 3

FGFBP2 Fibroblast growth factor binding protein 2

List of Abbreviations

FSHR Follicle stimulating hormone receptor

GAPD Glyceraldehyde-3-phosphate dehydrogenase GAPDHS Glyceraldehyde-3-phosphate dehydrogenase,

spermatogenic GC-MS Gas chromatograph-mass spectrometry GNMT Glycine N-methyltransferase

GSK-3β Glycogen synthase kinase-3β

GWAS Genome-wide association studies

HC Hypercholesterolemia plus sham

HCHT Hypercholesterolemia plus hypertension

HDL High-density lipoprotein

List of Abbreviations

ICLAD Intracranial Large Artery Disease

INSIG Insulin-induce gene

IPA Ingenuity Pathway Analysis

LCAT Lecithin-cholesterol acyltransferase

LDLR Low-density lipoprotein receptor

LOC10035102 Glutamate dehydrogenase 1-like

LOC100352398 Nibrin-like

LOC100354966 Ribosomal protein S3a-like

LOC100357097 Progesterone receptor membrane component 1 LRP-1 LDLR-related protein 1

MAP Mean arterial pressure

MAPK Mitogen-activated protein kinase

MAPK1 Mitogen-activated protein kinase 1

MCA Middle cerebral artery

MMP1 Matrix metallopeptidase 1

List of Abbreviations

MPP+ 1-methyl-4-phenylpyridinium

NAA25 N(alpha)-acetyltransferase 25, NatB auxiliary subunit NACLAR National Advisory Committee for Laboratory Animal

NPPA Natriuretic peptide A

NTBS Nickel Tris-buffered saline

NTSR1 Neurotensin receptor 1

NUS National University of Singapore

ODF2 Outer dense fiber of sperm tails 2

OTUD6A OTU deubiquitinase 6A

List of Abbreviations

PDGF Platelet-derived growth factor

RIPA Radioimmunoprecipitation assay

RNASEL Ribonuclease L (2',5'-oligoisoadenylate

synthetase-dependent) ROPN1 Rhophilin associated tail protein 1

ROPN1L Rhophilin associated tail protein 1-like

SCAP SREBP cleavage activation protein

SERPINB2 Serpin peptidase inhibitor, clade B, member 2

List of Abbreviations

SERPINE1 Serpin peptidase inhibitor, clade E (nexin, PAI-1), member

1 SNARE Soluble N-ethylmaleimide-sensitive factor attachment

protein receptor SP110 SP110 nuclear body protein

SPARCL Stroke Prevention Aggressive Reduction in Cholesterol

Levels SRBI Scavenger receptor BI

SRE Sterol regulatory element

SREBP Sterol regulatory element binding protein

SULT2B1b Cholesterol sulfotransferase

SYK Spleen tyrosine kinase

TAF15 TAF15 RNA polymerase II, TATA box binding protein

(TBP TBS Tris-buffered saline

TBST Tris-buffered saline with 0.1% Tween-20

TFCP2L1 Transcription factor CP2-like 1

TIA Transient ischemic attack

List of Abbreviations

UGT2B13 UDP-glucuronosyltransferase 2B13 UGT UDP-glucuronosyltransferase uPA Urokinase plasminogen activator UPS Ubiquitin-proteasome system VSMC Vascular smooth muscle cell

Publications

PUBLICATIONS

Several portions of the present study have been published in the following international refereed journals:

1 Loke SY, Tanaka K, Ong WY (2013) Comprehensive gene expression

analyses of the rat prefrontal cortex after oxysterol treatment J Neurochem 124:770-781

2 Ong WY, Ng MP, Loke SY, Jin S, Wu YJ, Tanaka K, Wong PT (2013)

Comprehensive gene expression profiling reveals synergistic functional networks in cerebral vessels after hypertension or hypercholesterolemia PLoS One 8:e68335

3 Loke SY, Ong WY Comprehensive gene expression profiling reveals

functional networks in frontal cortex induced by hypercholesterolemia and/or hypertension and comparison with cerebral vessels (in preparation for publication)

Chapter I - Introduction

CHAPTER I INTRODUCTION <TO DELETE>

Chapter I - Introduction

1 Cholesterol

Sterol lipids are the main non-polar cell membranes lipids with cholesterol predominates in mammals (van Meer et al., 2008) Cholesterol is an essential structural component of eukaryotic cellular membrane, vital for membrane organization, dynamics and function (Liscum and Underwood, 1995; Liu et al., 2010) It modulates fluid membrane rigidity by limiting passive permeability and enhancing lipid bilayer’s mechanical durability (Simons and Ikonen, 2000) Lipid rafts are membrane regions with dynamic nanoscale assemblies enriched in sphingolipid, cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins (Hancock, 2006) They are thought to serve as a platform to co-localize proteins that participate in intracellular signaling pathways (Calder and Yaqoob, 2007), ion channel activities (Koyrakh et al., 2005) and vesicular exocytosis aided by secretory

proteins assembly (e.g soluble N-ethylmaleimide-sensitive factor attachment

protein receptors (SNAREs)) into lipid rafts (Ong et al., 2010) Cholesterol is responsible for lipid rafts maintenance in a liquid-ordered phase (Marwali et al., 2003) It maintains the raft assembly together and as a molecular spacer between sphingolipids hydrocarbon chains (Simons and Toomre, 2000) Besides that,

Chapter I - Introduction

between cellular compartments (Liu et al., 2010) It plays important role in cellular metabolism and signal transduction through interaction with membrane proteins and enzymes (Pfrieger, 2003b) Furthermore, it modulates the function and organization of membrane proteins and enzymes (Burger et al., 2000) In addition, cholesterol is an essential precursor and a source of bioactive molecules for diverse biological processes regulated in both the periphery and central nervous system (CNS) For example, cholesterol is required for the biosynthesis of oxysterol, sterol hormones, vitamin D and bile acids (Simons and Ikonen, 2000; Liu et al., 2010)

Physicochemical Properties of Cholesterol

Cholesterol structure is composed of three regions: a hydrocarbon tail (lateral chain), a ring structure region with four hydrocarbon rings (A, B, C and D) and a hydroxyl group (Figure 1.1) (Yeagle, 1985; Vejux et al., 2008) Cholesterol is mainly a hydrophobic molecule due to the steroid ring backbone, sterane However, cholesterol’s amphiphilic nature (due to the presence of 3-β hydroxyl moiety), causes cholesterol to be arranged in the membrane bilayer with its long axis vertical to the membrane’s plane The hydroxyl group is orientated to face the aqueous surroundings while the hydrophobic ring is parallel to the hydrophobic fatty acyl chains of the phospholipids (Yeagle, 1985)

Chapter I - Introduction

Figure 1.1 Structure of cholesterol (Vejux et al., 2008)

Cholesterol is asymmetrically distributed across the plane of the membrane with higher levels present in the cytosolic leaflet (Bach and Wachtel, 2003) In addition, it is more abundant in plasma membrane and late secretory pathways than in membranes of a certain subcellular organelles such as mitochondria (Brown and London, 2000)

Cholesterol in the Brain

The brain is the most cholesterol-rich organ in the body (Björkhem and

Chapter I - Introduction

of glial cells (20%) and neurons (10%) (Dietschy and Turley, 2004) For the former, cholesterol enrichment in myelin sheaths is needed for electrical signal transmission along axons (Dietschy and Turley, 2004) On the other hand, cholesterol in the neural membranes is essential in membrane organization, dynamics, function and sorting (Simons and Ikonen, 2000) Besides, it is also associated with the assembly and maintenance of lipid rafts (Simons and Ehehalt, 2002) Lipid rafts are involved in various activities in the CNS such as neuronal excitability and synaptic transmission (Tsui-Pierchala et al., 2002) Moreover, cholesterol is required for the development and function of neuron and synapse (Sun et al., 2014), as well as optimal neuroplasticity and behavior (Farooqui, 2011) Chelation of cholesterol by cyclodextrin has been shown to impair neuronal synaptic transmission and plasticity associated with activation

in CNS, neuronal function (e.g memory and neural oxidative stress reactions)

as well as development of neurodegenerative diseases (Pfrieger, 2003a; Nelson and Alkon, 2005)