Effects of apolipoprotein e on n methyl d aspartate receptor signalling the roles of ageing and chicken extract

... 2000) There are 14 Chapter 1: Introduction isoform-specific neurodegeneration and as age-dependent human effects apoE3 of prevents human kainic apoE on acid-induced neurodegeneration in comparison... regions more intensely than non-carriers and exhibit compensatory neural recruitment (Han and Bondi, 2008) This phenomenon occurs even in non-demented older adults suggesting ε4 carriers undergo... than age-matched apoE3 counterparts The NR2B expression levels show an unexpected decrease in the hippocampus at middle age which extend into the cortex at old age The age-dependent and region-specific

EFFECTS OF APOLIPOPROTEIN E ISOFORMS

DECLARATION

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

me in its entity I have duly acknowledged all the sources of information

which have been used in the thesis

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

YONG SHAN MAY

23rd January 2014

ACKNOWLEDGEMENTS

I would like to express my gratitude to my supervisor, Dr Wong Boon Seng for his guidance and supervision throughout my years of PhD study He has taught me to think critically and helped me to grow as an individual I am truly grateful to my TAC team members, Dr Lim Kah Leong and Dr Ramani, for the guidance provided in my project I also would like to thank Dr Low Chian Ming for his advice to overcome several obstacles that I experienced I extent

my gratitude to Dr Paramjeet Singh (Cerebros Pacific Limited) for providing the CE powder for my project

I sincerely thank my previous and current fellow lab mates, Ching Ching, Li Min, Hong Heng, Ray, Shiau Chen, Mei Li, Ira, Elizabeth, Cynthia, Pei Ling, Ket Yin, Bei En and past FYP students for making my lab experience a memorable and an enjoyable one Special thanks to Irwin, Alvin, Francis, Vanessa and Li Ren for their camaraderie and support All of them have been great mentors and friends, offering me advice and encouragement that helped

me to pull through the difficult times I will always remember the time that we have spent together and thank you for making a difference in my life

I express my heartfelt gratitude to my family members for their support and unconditional love Last but not least, I want to thank my boyfriend, Chee Wai, for being so understanding and supportive during my last two years of study

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

SUMMARY vi

LIST OF TABLES viii

LIST OF FIGURES ix

ABBREVATIONS xi

LIST OF PUBLICATION ……… xiv

Chapter 1: Introduction 1

1.1 Apolipoprotein E (ApoE) 1

1.1.1 Characteristics of ApoE 1

1.1.2 Expression and functions of apoE in CNS: astrocytic vs neuronal apoE 2

1.1.3 Differences between apoE isoforms 4

1.1.4 ApoE isoforms in synaptic plasticity 5

1.1.5 ApoE receptors 7

1.2 Alzheimer’s disease (AD) 9

1.2.1 AD pathogenesis and progression 9

1.2.2 Hippocampus in synaptic plasticity and memory formation 10

1.2.3 Ageing and synaptic plasticity 12

1.2.4 Genetic risk factor for sporadic AD: apolipoprotein E (apoE) variants 13

1.3 N-methyl-D-aspartate receptor (NMDAR): a key player in learning and memory 15

1.3.1 Characteristics and expression of NMDAR 16

1.3.2 Functions of NMDAR 19

1.3.3 Modulation of NMDARs by phosphorylation and dephosphorylation mechanisms 20

1.3.4 Opposing roles of NMDAR subunits in synaptic plasticity 21

1.3.5 NMDAR in ageing 22

1.3.6 NMDAR in AD 23

1.3.7 NMDAR in excitotoxity 24

1.4 α-amino-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPAR) 25

Chapter 2: Materials And Methods 28

2.1 Common reagents and materials 28

2.2 Animal model 28

2.2.1 Tissue preparation for protein analysis 29

2.2.2 Preparation of brain homogenates 29

2.2.3 Protein quantitation of brain lysates 30

2.2.4 Western blotting 30

2.2.5 Tissue preparation for immunofluorescence 32

2.2.6 Immunofluorescence 32

2.3 Cell culture 35

2.3.1 Immortalization and transfection of apoE-knockout neuronal cells 35

2.3.2 CE treatment 36

2.3.3 Cell lysis 37

2.3.4 Protein quantitation of cell lysates 37

2.3.5 Western blotting 37

2.3.6 Calcium assay 39

2.4 Statistical analysis 40

Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 41 3.1 Introduction 41

3.1.1 Human apoE gene-knockin (apoE-KI) mice model for investigating effects of apoE isoforms on NMDAR changes during ageing 41

3.1.2 Interactions between apoE and N-methyl-D-aspartate receptor (NMDAR) 47

3.1.3 Postsynaptic density (PSD) proteins/ NMDAR-associated proteins (NAPs) 48

3.1.3.2 PSD95 50

3.1.3.2 Calcium/calmodulin-dependent protein kinase II (CaMKII) 51

3.1.4 Signalling pathways coupled to NMDAR 53

3.1.4.1 Protein kinase C (PKC) pathway 53

3.1.4.2 Protein kinase A (PKA) pathway 55

3.1.4.3 Ras/mitogen-activated protein kinase (MAPK) pathway 56

3.1.5 CREB in learning and memory 59

3.2 Objectives of study 61

3.3 Results 63

3.3.1 Expression of human apoE in brains of huApoE-knockin (apoE-KI) mice across three time points 65

3.3.2 Expression of apoE receptor and PSD95 in brains of huApoE-KI mice across three time points 66

3.3.3 NMDAR subunits phosphorylation profile in brains of huApoE-KI mice across three time points 68

3.3.4 PKA and PKC signalling profile in brains of huApoE-KI mice across three time points 71

3.3.5 GluR1 and αCaMKII expression profile in brains of huApoE-KI mice across three time points 73

3.3.6 ERK-CREB signalling pathway in brains of huApoE-KI mice across three time points 76

3.3.7 Expression of human apoE in neurons and astrocytes of huApoE- KI mice brains across three time points 79

3.3.8 Protein expression level of NMDAR subunits in brains of huApoE-KI mice across three time points 85

3.4 Discussion 89

3.4.1 ApoE4 isoform downregulates expression level of total apoE but upregulates neuronal apoE production with increasing age 89

3.4.2 ApoE4-isoforms decreases expression of apoE receptor i.e LRP1 and postsynaptic protein PSD95 95

3.4.3 ApoE4 spatio-temporally regulate NMDAR expression and phosphorylation profiles during ageing 99

3.4.4 Modulation of ERK and CREB activity in apoE4-KI mice is mediated via PKC but not PKA signalling pathway 107

Chapter 4: Impacts of apoE isoforms in cellular responses to chicken extract (CE) treatment 119

4.1 Introduction 119

4.1.1 Cyclic nucleotide phosphodiesterase (PDE) and CREB 119

4.1.1 Chicken extract (CE): potential PDE inhibitor 120

4.1.2 Beneficial effects of CE to mental health 121

4.2 Objective of study 124

4.3 Results 125

4.3.1 Chronic expression of apoE in huApoE stable cells 125

4.3.2 Effects of CE treatment on expression of human apoE in

huApoE stable cells 126

4.3.3 Effects of CE treatment on basal intracellular calcium level in huApoE stable cells 127

4.3.4 Effects of CE treatment on phosphorylation of NR1 subunit in huApoE stable cells 129

4.3.5 Effects of CE treatment on PKA and PKC signalling profile in huApoE stable cells 131

4.3.6 Effects of CE treatment on GluR1 and αCaMKII expression profile in huApoE stable cells 134

4.3.7 Effects of CE treatment on ERK-CREB signalling in huApoE stable cells 136

4.4 Discussion 138

4.4.1 Different intracellular calcium responses in huApoE-transfected neurons and downregulation of apoE4 expression upon CE treatment 138

4.4.2 Upregulation of PKC pathway in huApoE3-transfected neurons and downregulation of PKA pathway in huApoE4-transfected neurons upon CE treatment 141

4.4.3 ERK-CREB signalling in CE-treated huApoE3- and huApoE4-transfected neurons 145

4.3 Conclusion 152

4.4 Future Directions 154

BIBLIOGRAPHY 162

Appendix 225

SUMMARY

Apolipoprotein E4 (apoE4) isoform has been shown to accelerate cognitive decline in human and mouse models during ageing compared to apoE3 isoform Mice expressing human apoE4 display impaired learning and memory and glutamatergic neurotransmission Despite the ongoing studies to look for preventive measures and therapeutic strategies, researchers have yet

to unravel the complex underlying mechanisms and rectify the pathological effect of apoE4 in learning and memory

My study shows that apoE4 regulates expression of NMDAR subunits and its activity in a temporal and region-specific manner during ageing when compared with apoE3-knock in (apoE3-KI) mice Western blotting analyses show an increased phosphorylation of NR1 subunit particularly at Ser896 in young apoE4-KI mice In contrast, this phosphorylation is downregulated in old apoE4-KI mice when compared with apoE3-KI mice The tyrosine phosphorylation of NR2A of apoE4-KI mice is reduced regardless of age whereas there is no difference in NR2B activity between apoE3- and apoE4-

KI mice across all time-points Immunofluorescence studies demonstrate an increase in NR1 signal intensity in the hippocampus and cortex at week 12 followed by downregulation of its total expression at week 72 in the hippocampus Similarly, NR2A subunit expression levels in most hippocampal subregions and cortex of apoE4-KI mice are always lower than age-matched apoE3 counterparts The NR2B expression levels show an unexpected decrease in the hippocampus at middle age which extend into the cortex at old age The age-dependent and region-specific modulation of the NMDAR subunits is correlated to the source of apoE4 production This suggests that the increasing neuronal apoE4 production at old age particularly in the CA3 and cortical area, exerts a detrimental effect on NMDAR expression due to the neurotoxicity of apoE4 fragments Hence, the downregulated phosphorylation and region-specific expression of NMDAR of apoE4-KI mice may partially explain their impaired behavioural performances at old age compared to age-matched apoE3 counterparts as observed by others

Analysis on NMDAR-associated proteins and the signalling pathways coupled

to NMDAR activity in apoE4-KI mice show that the postsynaptic density protein PSD95 and apoE receptor, LRP1, are downregulated in apoE4-KI mice

at all ages This suggests that a reduced NMDAR functionality mediated via these proteins Immunoblotting of Ca2+-sensitive kinases including αCaMKII, PKCα and PKA-Cα demonstrate an increased αCaMKII and PKCα activation with expected elevation in their common downstream target, GluR1 phospho-S831 without affecting PKA-Cα in young apoE4-KI mice In contrast, phosphorylation of αCaMKII, PKCα and GluR1 Ser831 are downregulated at old age The correspondence between signalling profiles of ERK-CREB versus αCaMKII and ERK-CREB versus PKCα strongly suggest their key roles played in facilitating ERK and CREB activation

Second part of my study investigated the effects of chicken extract (CE) on the NMDAR and its downstream signalling cascades in the context of apoE

isoforms in vitro as this dietary supplement has been shown to improve

cognition in human Howver, the underlying mechanisms are unclear Data from CE treatment of apoE-transfected neurons demonstrate the differential effects of apoE isoforms on intracellular Ca2+ responses and triggering of the

Ca2+-dependent signalling pathways In particular, PKA-Cα pathway is downregulated whilst PKCα pathway is upregulated in CE-treated apoE4 and apoE3 neurons respectively Moreover, the basal intracellular Ca2+ level, αCaMKII and GluR1 S831 are increased in apoE3 neurons whereas the opposite occurs for mock and apoE4-transfected neurons after treatment These might have led to enhanced ERK1/2 and CREB activity in apoE3 neurons but reduced ERK-CREB signalling in mock as well as apoE4 neurons

(564 words)

LIST OF TABLES

Table 2.1 List of primary antibodies used for immunofluorescence The source and the dilution factor used are as shown 34

Table 2.2 Composition of CE compound 37

Table 2.3 List of primary antibodies used for immunoblotting The source and the dilution factor used are as shown 38

Table 2.4 List of secondary antibodies used throughout the study The

purpose, source and dilution factor used are as shown 39

Table 3.1 Samples, brain regions of interest and findings of reviewed articles 46

Table 3.2 Recapitulative table on all significant comparisons between

huApoE3 and huApoE4 mouse lines 117

Table 4.1 Recapitulative table on the findings of CE treatment on mock, huApoE3 and huApoE4 stable cell lines 150

LIST OF FIGURES

Figure 1.1 Amino acid sequence of different apoE isoforms 2

Figure 1.2 Structure of NMDAR subunit 18

Figure 1.3 Model of bidirectional plasticity in AMPAR phosphorylation and dephosphorylation 27

Figure 3.1 Schematic diagram of the interactions between apoE and NAPs, and NMDAR-coupled signalling cascades 61

Figure 3.2 Expression level of huApoE in brains of apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 65

Figure 3.3 Protein expression level of LRP1 and PSD95 in brains of apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 66

Figure 3.5 Protein expression level of PKA-Cα and PKCα in brains of

apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 71

Figure 3.6 Protein expression level of GluR1 and αCaMKII in brains of

apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 73

Figure 3.7 Protein expression level of ERK and CREB in brains of apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 76

Figure 3.8 Cellular expression levels of huApoE in brains of apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 82

Figure 3.9 Expression level of total NMDAR subunit proteins in brains of apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 87

Figure 3.10 Model of age-dependent regulation of intracellular signalling pathways by apoE4 118

Figure 4.1 Protein expression level of apoE in mock and apoE-transfected neurons 125

Figure 4.2 Protein expression level of apoE in mock and apoE-transfected neurons 126

Figure 4.3 Basal intracellular calcium ion concentration in mock and transfected neurons 127

Figure 4.4 Phosphorylation level of NR1 subunit in mock and

apoE-Figure 4.8 Model of differential regulation of cellular responses to CE

treatment by apoE isoforms 151

Supplementary Figure 1 Protein expression levels of GFAP and NeuN in brains of apoE-KI mice models across three time points i.e 12, 32 and 72 weeks 225

Supplementary Figure 2 Protein expression level of LP1 in mock and transfected neurons 225

ApoER2 ApoE receptor 2

APP Amyloid precursor protein

ATP Adenosine triphosphate

BDNF Brain-derived neurotrophic factor

CaMK Calcium-calmodulin dependent kinase

cAMP Cyclic adenosine monophosphate

CNS Central nervous system

CREB cAMP-responsive element-binding protein

EDTA Ethylenediaminetetraacetic acid

EPSC Excitatory postsynaptic current

ERK Extracellular-signal regulated kinase

FDG-PET Fluorodeoxyglucose positron emission tomography

GFAP Glial fibrillary acidic protein

GLUT Glutamate transporter

GRF Guanine nucleotide Releasing Factor

GTP Guanosine triphosphate

HDL High density lipoprotein

HFS High frequency stimulation

ICD Intracellular domain

iGluR Ionotropic glutamate receptor

Ins(1,4,5)P 3 Inositol-1,4,5-triphosphate

JNK Jun-N-terminal kinase

KI Knockin

LDLR Low density lipoprotein receptor

LFS Low frequency stimulation

LRP1 LDL-related protein 1

LTP Long-term potentiation

mAchR Muscarinic acetylcholine receptor

MAGUK Membrane-associated guanylate kinase

MAP2 Microtubule-associated protein 2

MAPK Mitogen activated protein kinase

MCI Mild cognitive impairment

mGluR Metabotropic glutamate receptor

MK801 [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenxo

[a,d]cyclohepten-5,10-imine

mRNA Messenger ribonucleic acid

NMDAR N-methyl-D-aspartate receptor

NRHyper NMDAR hyperactivity

NRHypo NMDAR hypoactivity

NSE Neuron-specific-enolase

PDZ PSD95, disc large, zona occludens-1

PI3K Phosphatidylinositol 3-kinases

PKA-C Protein kinase A catalytic subunit

PMA Phorbol 12-myristate 13-acetate

PP1/2A Phosphatases 1,2A

PSD95 Postsynaptic density protein 95

RSK Ribosomal protein S6 kinase

SAP Synapse-associated-protein

SDS Sodium dodecyl sulfate

tPA Tissue plasminogen activator

U0126 dicyano-1,4-bis[2-aminophenylthio]butadiene

α2M Alpha-2 marcoglobulin

LIST OF PUBLICATION

S Yong, Q Ong, B Siew and B B Wong, Food Funct., 2014, DOI:

10.1039/C4FO00428K

ApoE4 makes up about 14% of the population and the rest are apoE2 (8%) In

a study of apoE polymorphisms and lipid profiles in three ethnic groups i.e Malays, Chinese and Indians in the Singapore population, ε3 allele is the most common (82%) followed by ε4 (10%) and ε2 (8%) (Tan et al., 2003) The three major isoforms predominantly expressed in the human population are apoE2, apoE3 and apoE4 These isoforms differ by two amino acids at position 112 and 158 in which apoE2 has two cysteines (Cys), apoE3 has Cys-

112 and Arg-158 (arginine) whereas apoE4 has Arg-112 and Arg-158 (Rall et al., 1982; Weisgraber, 1994) In general, apoE has two structural domains including a 22kDa N-terminal domain which binds to its receptor and a 10kDa C-terminal domain which is the lipid-binding site that can interact with other extracelullar proteins such as Aβ (Mahley and Rall, 2000) In the CNS, apoE exists in the form of discoidal-shaped high-density lipoprotein (HDL)-like particles containing phospholipids and cholesterol which is distinct from those

in the peripheral system (DeMattos et al., 2001a; Pitas et al., 1987) It is the major lipoprotein in the CNS among other apolipoproteins such as apoA, apoC, apoD and apoJ (also known as clusterin) whilst apoB is absent (Holtzman et al., 2012) Concentration of apoE in the cerebrospinal fluid (CSF) is 100-200

nM (Riemenschneider et al., 2002) and total concentration in brain extract is approximately 5 µg/mL (Haass and Selkoe, 2007)

Figure 1.1 Amino acid sequence of different apoE isoforms

The three major apoE isoforms differ in their amino acids at positions 112 and

158 which give rise to their distinct properties Each comprises two structural domains that bind to apoE receptors at the N-terminal and lipid particles such

Under pathological conditions, excitotoxic injury in CNS such as kainic acid treatment, oxidative stress and even ageing induces neurons to increase apoE production to initiate the repair process and mediate protection from these insults (Boschert et al., 1999; Huang, 2010; Mahley, 1988; Roses, 1997; Xu et al., 2006) However, proteolytical processing of neuronal apoE4 produces harmful fragments that induce neurodegeneration especially in ε4 carriers (Brecht et al., 2004; Huang, 2010; Huang et al., 2004; Mahley et al., 2006; Roses, 1997) which may underlie its correlation to the earlier onset of

Alzheimer’s disease (AD) (Boschert et al., 1999) Both in vitro and in vivo

studies have shown that neuronal apoE4 is more susceptible to cleavage by a neuron-specific, chymotrypsin-like serine protease generating neurotoxic fragments which are detrimental to the neurological repair or maintenance process compared to apoE3 (Huang et al., 2001; Tolar et al., 1999; Tolar et al., 1997)

The effect of the ε4 allele on the CNS apoE protein levels has been studied in both AD patient CNS and animal models, reporting controversial results showing either reduced levels (Beffert et al., 1999; Ong et al., 2014; Poirier, 2005; Riddell et al., 2008), no change (Fryer et al., 2005; Sullivan et al., 2004)

or increases (Fukumoto et al., 2003) in apoE expression compared to that of apoE4 individuals The reduced brain apoE level in ε4 carriers (Farrer et al., 1997; Poirier, 2005) and their predisposition to AD implies that apoE is required to sustain a certain level of cognitive function perhaps by maintaining synaptic integrity

It is suggested that adequate level of apoE may be crucial to regulate brain homeostasis during ageing as apoE expression increases in the liver in an age-dependent manner (Gee et al., 2005) However, it is unclear whether apoE expression changes in the brain with ageing in human whereas conflicting observation have been made from animal studies In rodents, apoE expression level decreases more than five-fold in the cortex and hypothalamus (Jiang et al., 2001) but increases in the hippocampus (Terao et al., 2002) of aged mice Aged rats also demonstrate elevated glial apoE expression in basal ganglia and corpus callosum (Morgan et al., 1999) In contrast, a recent study reported

there is no alteration in apoE mRNA and protein expression level in the cortex, hippomcapus and striatum of aging rat brains (Gee et al., 2006)

1.1.3 Differences between apoE isoforms

The replacement of Arg in apoE4 at position 112 has an impact on its structure and functions making it the least stable and a more pathological isoform compared to the other two Arg-112 mediates the interaction between Arg-61

at the N-terminus and Glu-255 (glutamine) at the C-terminus causing the whole molecule to exist as a partially folded intermediate or molten globule (Dong et al., 1994; Weisgraber, 1990) This unstable structure denatures at a lower temperature making it less concentrated in the CNS In fact there is a lower level of apoE in brain and serum of AD subjects whereby 40 to 80% of them have at least one ε4 allele (Farrer et al., 1997) ApoE4 has a lower cholesterol transport capacity and Aβ clearance ability compared to apoE2 and apoE3 leading to increased Aβ production (Dodart et al., 2005; Holtzman et al., 2000) Furthermore, apoE4 mediates some of the detrimental effects such as tau1 phosphorylation (Mandelkow and Mandelkow 1994), lysosomal leakage, mitochondrial dysfunction, neurodegeneration and cognitive deficits in AD (Buttini et al., 2002; Chang et al., 2005; Huang et al., 2001; Ji et al., 2002; Raber et al., 2002; Risner et al., 2006) ApoE4 has been shown to cause cytotoxicity in a dose- and time-dependent manner Significant neurotoxicity was detected 24 hours after apoE4 treatment and the percentage of cell death plateaus at 72 hours (Qiu et al., 2003)

Generation of neurotoxic fragments such as apoE4 (Δ1-272, Δ272-299, 242) are harmful to neurons and Neuro-2a cell lines derived from mouse neuroblastoma For instance, apoE4 (Δ1-272) is associated with

1 Tau is one of the microtubule-associated proteins that plays a role in the stabilization of neuronal microtubules and provides the tracks for intracellular transport However, when tau undergoes modifications such as phosphorylation, hyperphosphorylated tau will aggregate into paired helical fragments that coalesce into neurofibrillary tangles

neurofibrillary tangle-like structures in cortical and hippocampal neurons in transgenic mice (Harris et al., 2003; Huang et al., 2001) ApoE4 (Δ272-299) which is present in AD brains peaks at 6-7 month old in mice and impairs spatial learning and memory (Brecht et al., 2004; Harris et al., 2003) Furthermore, AD patients also have much higher levels of apoE fragments compared to non-demented controls (Harris et al., 2003; Huang et al., 2001) Thus under pathological conditions, it is worse for neurons to express apoE4 than to express no apoE at all, as opposed to apoE2 and apoE3 which are more stable and more effective in maintenance of neurons (Mahley, 1988; Morrow

et al., 2002) In other words, apoE4 has an adverse gain-of-function compared

to the other two isoforms (Buttini et al., 2000; Huang, 2006; Raber et al., 2000)

1.1.4 ApoE isoforms in synaptic plasticity

ApoE isoforms differentially modulate synaptic plasticity and learning and memory ApoE-targeted replacement mice (huApoE-TR) or human apoE-knockin (huApoE-KI) expressing human apoE isoforms have shown that huApoE3 mice have enhanced LTP compared to huApoE4 mice (Grootendorst

et al., 2005; Trommer et al., 2004) ApoE4 has been postulated to impair synaptic plasticity by reducing NMDAR-dependent Ca2+ influx mediated by apoE receptors in a dose-dependent manner Furthermore, apoE4 may impede apoE receptor recycling by sequestering the receptors intracellularly such that they are unable to be expressed on neuronal surfaces and trigger downstream signalling pathways upon ligand binding (Chen et al., 2010) Exogenously added apoE4 has been shown to reduce surface expression of apoE receptor 2 (apoER2) in primary neurons which functions as a dual receptor for apoE and

reelin, a ligand that modulates NMDAR functions both in vitro and in vivo

(Beffert et al., 2005) ApoE3 and apoE4 differ from each other in their influences on neurite extension (DeMattos et al., 1998; DeMattos et al., 2001b; Fagan et al., 1996; Handelmann et al., 1992; Nathan et al., 1995) In cultured dorsal root ganglion neurons and Neuro-2a cells, apoE3 together with β-VLDL stimulates neurite branching and extension as opposed to apoE4

(Holtzman et al., 1995; Nathan et al., 1994) This stimulatory effect on neurite outgrowth is also seen in rat hippocampal neurons mediated by astrocyte-derived apoE3 but not apoE4 (Sun et al., 1998) In organotypic hippocampal slice culture isolated from huApoE-TR mice model, apoE3 can stimulate neuronal sprouting which is inhibited by apoE4 (Teter et al., 2002) This contrasting effect of apoE4 is probably due to its facilitation of tubulin depolymerization in neuronal cells leading to microtubule instability (Nathan

et al., 1994) In addition, in vivo and in vitro studies show apoE4 impedes

synaptogenesis as apoE4 transgenic mice have reduced number of synapses per neuron (Cambon et al 2000), and lower density of dendritic spines than apoE3 mice (Dumanis et al., 2009; Ji et al., 2003) Similarly, exogenously added apoE4 or its proteolytic fragments decreases spine density in primary cortical neuronal cultures (Brodbeck et al., 2008) These structural deformities

in apoE4 mice perhaps contribute to learning and memory deterioration

In spite of all these, young apoE4-KI mice display enhanced plasticity compared to apoE3 mice but this effect disappears with age (Kitamura et al., 2004) Furthermore, this enhanced LTP may be mediated by postsynaptic properties as there is no difference in presynaptic release mechanisms between apoE3 and apoE4-KI animals Similarly, young human ε4 carriers exhibit higher intelligence and event-related potential (Yu et al., 2000) suggesting that apoE isoform modification of LTP may be age-dependent Studies that support the beneficial effect of apoE4 in early life show that school children with ε4 allele score better in verbal fluency scores and outperform ε2 peers in Rey Complex Figure Test (RCFT) copy trial (Alexander et al., 2007; Bloss et al., 2010) One of the interesting observations is that ε4 carriers tend to recruit task-related regions more intensely than non-carriers and exhibit compensatory neural recruitment (Han and Bondi, 2008) This phenomenon occurs even in non-demented older adults suggesting ε4 carriers undergo compensatory changes in order to achieve the same performance as non-carriers (Kukolja et al., 2010; Tuminello and Han, 2011) However, the beneficial role of apoE4 in early life remains controversial as there are contradictory findings indicating that 5 to 7-year-old ε4 carriers with sleep apnoea show impaired cognition and middle-aged adults do not differ in

cognitive performance (Dennis et al., 2010; Filbey et al., 2010; Gozal et al., 2007) Furthermore, additional environmental risk factors such as family history of AD and gender susceptibility may interact with ε4 allele to affect cognition as 7 to 10 year-old girls have lower spatial memory retention and lower visual recall scores on Family Pictures test (Acevedo et al., 2010; Bloss

et al., 2008) Perhaps the potential benefit of ε4 allele may be confined to a very narrow window in early life span and as the detrimental effects of apoE4 surfaces, the compensatory mechanism cannot sustain the premorbid cognitive performance levels and hence the condition worsens with age

1.1.5 ApoE receptors

ApoE binds to members of low density lipoprotein receptor (LDLR) family including very low density lipoprotein receptor (VLDLR), apoER2, LDL-related protein 1 (LRP1), LDL-related protein-1B (LRP-1B), multiple EGF repeat-containing protein-7 (MEGF7) and megalin (Brecht et al., 2004; Holtzman et al., 2012) These receptors have one or more ligand-binding domains, epidermal growth factor (EGF) and a cytoplasmic tail containing a consensus amino acid NPxY motif which acts as an interaction site for intracellular adaptor proteins that are further coupled to downstream transduction pathways (Beffert et al., 2005; Gotthardt et al., 2000; Hoe et al., 2006; Trommsdorff et al., 1998) Some of the functions of apoE receptors are utilization of mitogen-activated protein kinases (MAPK), tyrosine kinases, lipid kinases and ligand-gated ion channels such as glutamate receptors (Beffert et al., 2005; Bock and Herz, 2003)

LRP1 is one of the major apoE receptors that regulates CNS apoE levels It is

a 600 kDa receptor dimer consisting of an extracellular and intracellular domain which is 515 kDa and 85 kDa respectively It is expressed ubiquitously by hepatocytes, neurons, vascular smooth muscle, lung, macrophages and embryonic tissues (Bu et al., 1994; Ishiguro et al., 1995; Moestrup et al., 1992) Other than apoE, ligands that bind to LRP1 are activated α2-macroglobulin (α2M*), amyloid precursor protein (APP), tissue plasminogen activator (tPA), protease/protease inhibitor complexes,

lipoprotein lipase and platelet-derived growth factor (PDGF) It plays dual role

in endocytosis and regulation of signal transduction involving calcium currents, PDGF receptor signalling, phagocytosis of apoptotic cells and embryonic development (Boucher et al., 2003; Herz and Strickland, 2001; May et al., 2002; Wang et al., 2003; Yepes et al., 2003) Genetic deletion of

LRP1 causes embryonic lethality and hence tissue-specific LRP1 knockout

animals have been generated which exhibit pronounced tremor, ataxia, hyperactivity, dystonia and premature death without structural abnormalities

in these animals (Herz et al., 1992; May et al., 2004) They also show impaired brain lipid metabolism, age-dependent synaptic loss and neurodegeneration (Liu et al., 2010) This highlights the crucial role of LRP1

in modulation and turnover of synaptic proteins that contributes to the anomalies in morphology and phenotype in the absence of LRP1 As an endocytic receptor, LRP1 facilitates uptake and clearance of neural proteases such as neuroserpin from synaptic regions, as increased level of proteases will disrupt the balance between proteases and protease inhibitors leading to synaptic dysfunction (Makarova et al., 2003)

LRP1 has been linked to AD due to its binding to and clearance of Aβ as well

as APP metabolism (Beffert et al., 1999; Rebeck et al., 1993; Higuera et al., 2009), mediated via adaptor protein FE65 that binds to NPxY motif of LPR1 and APP (Kinoshita et al., 2001; Pietrzik et al., 2004; Trommsdorff et al., 1998) It is observed that decreased LRP1 expression in amyloid mouse model and brain capillaries of AD brain may contribute to impaired Aβ clearance (Deane et al., 2004; Van Uden et al., 2002) LRP1 undergoes proteolytic processing whereby the extracellular domain can be cleaved by β-secretase, BACE1, which also cleaves APP and generates the Aβ N-terminus, close to the transmembrane region resulting in the shedding of the extracellular domain (Quinn et al., 1999) The intracellular domain is then released from the plasma membrane by cleavage involving γ-secretase (May et al., 2002) This allows the free C-tail to translocate together with its adaptor proteins to other subcellular localizations Moreover, the circulating LRP1 may also serve as a peripheral 'sink' for Aβ clearance (Sagare et al., 2007)

Vázquez-1.2 Alzheimer’s disease (AD)

AD is the most common cause of dementia among the elderly (>65 years) and accounts for up to 75 percent of all dementia cases Global prevalence of dementia is estimated to be 3.9 % in people aged 60 years and above, and affects more than 25 million people in the world (Brookmeyer et al., 2007; Ferri et al., 2005; Wimo et al., 2003) Population ageing has become a global phenomenon In both developed and developing countries, the United Nation Ageing Program and the United States Centers for Disease Control and Prevention have projected that the aged population (65 years or more) in the world is expected to rise from 420 million in 2000 to nearly 1 billion by 2030, which is an increase from 7 to 12 percent of world population (Centers for Disease Control and Prevention, 2003) Similarly, a recent study reported the worldwide total number of 26.6 million AD patients will quadruple by the year 2050 (Brookmeyer et al., 2007) With the incidence rate of 5 million cases per year and the age-specific prevalence of AD doubling every five years after the age of 65 years, Alzheimer's disease has rapidly become a major public health crisis by imposing financial and societal burden as 43% of

AD patients require high level of care such as nursing homes and institutions The societal cost of dementia worldwide was estimated to be more than US$315 billion in 2005 (Wimo et al., 2007) Hence, much effort has been made to seek for effective therapeutic and preventive strategies as even a 1-year delay in the onset of and progression of AD will significantly reduce the global burden of this disease (Brookmeyer et al., 1998; Brookmeyer et al., 2007)

1.2.1 AD pathogenesis and progression

AD is a multifactorial disease which is subtyped into two groups i.e familial making up 5% of the cases, and sporadic, for the rest The former, also known

as early-onset AD, is caused by rare autosomal inherited mutations involving presenillin 1 and 2 (PS1 and PS2), and the amyloid precursor protein gene (APP), which typically occurs before age 65 The age of onset for the latter is

usually after 65 years old and the aetiology is a complex interplay between other genetic risk factors such as apolipoprotein E4 (apoE4), family history and environmental factors AD is characterized by several hallmarks including neuronal loss, synaptic damage, deposition of beta-amyloid (Aβ) and neurofibrillary tangles, loss of cholinergic activity and elevated oxidative stress which is detrimental to messenger ribonucleic acid (mRNA) as well as protein synthesis (Ding et al., 2005; Keller et al., 2005) AD progression has been characterized in different Braak stages I to VI (Braak and Braak, 1994) depending on the brain structures affected The related pathology is propagated in a typical topological pattern, originating in subcortical structures such as locus coeruleus (Braak and Del Tredici, 2011), and extends into entorhinal cortex at Braak stages I and II It subsequently progresses to the hippocampus and amygdala via the perforant pathway characterized as Braak stages III and IV, and eventually spreads into other cortical regions at Braak stages V and VI

Memory deficits are often displayed in mild cognitive impairment (MCI) before it progresses into dementia which is the main symptom of AD patients (Petersen, 2004) One of the methods to evaluate cognitive function is the investigation of spatial memory in hippocampus as it plays a crucial role in spatial memory performance (Clarke RE, 2007; Morris et al., 1982; Rolls, 2000) The hippocampus is important in synaptic plasticity, being the cellular basis for learning and memory and is particularly vulnerable to environmental insults (Sultana et al., 2010)

1.2.2 Hippocampus in synaptic plasticity and memory formation

Synaptic plasticity refers to activity-induced changes at appropriate synapses during memory formation and is necessary and sufficient for the information storage mediated by the brain area in which that plasticity is observed It occurs mainly in hippocampal sub-regions namely dentate gyrus (DG), cornus ammoni (CA1) and CA3 neurons There are three major circuits in the hippocampus to assist in formation of declarative memories which are the

perforant, mossy fibre (MF) and Schaeffer collateral (SC) pathway The first pathway extends from the entorhinal cortex to granule cells of DG; the second comprises of axonal projections from granular cells in DG to pyramidal cells

in hippocampal CA3; and the third pathway originates from CA3 and culminates at CA1 High frequency stimulation (HFS) of the perforant pathway induces long-lasting potentiation of synaptic transmission between perforant path fibres and dentate granule cells (Bliss and Lomo, 1973) DG is

a region capable of neurogenesis as it consists of newly formed neurons involved in learning and memory circuits (Zhao et al., 2006) The SC pathway involves formation of excitatory synapses between axons of CA3 pyramidal cells and dendrites of CA1 pyramidal cells and damage in the CA1 area is sufficient to prevent the formation of new memories (Auer et al., 1989; Zola-Morgan et al., 1986) This pathway displays a form of long-term potentiation (LTP) that requires Ca2+ influx via N-methyl-D-aspartate receptor (NMDAR),

a glutamate receptor that plays a key role in synaptic plasticity (Bliss and Collingridge, 1993; Collingridge et al., 1983; Lynch et al., 1983; Malenka and Nicoll, 1999) In contrast, LTP induced via the MF pathway can occur independently of NMDAR activation (Harris and Cotman, 1986; Johnston et al., 1992; Weisskopf and Nicoll, 1995; Zalutsky and Nicoll, 1990)

There are two forms of synaptic plasticity: LTP and long-term depression (LTD) (Norris et al., 1998) LTP is one of the functional indices of synaptic plasticity (Quan et al., 2010) and serves as a link between structural and behavioural outcome measures For instance, it is used to study the effects of apoE4 allele in increasing susceptibility to cognitive impairment at the level of dynamic functional changes in synaptic transmission (Trommer et al., 2004) LTP is divided into two temporal phases: early- (E-LTP) and longer lasting late-LTP (L-LTP) (Andersen et al., 1980; Frey et al., 1988; Huang and Kandel, 1994; Reymann et al., 1988) E-LTP is known as the induction phase which involves the release of neurotransmitters such as L-glutamate and excitation of glutamate receptors to allow calcium ion (Ca2+) influx Formation of Ca2+ and calmodulin (Ca2+/CaM) complex further activates glutamate receptors in a positive feedback loop to increase efficiency of ion fluxes Another source of

Ca2+ may come from intracellular stores released after the activation of

metabotropic receptors such as group 1 metabotropic glutamate receptors (mGluRs), adenosine 2A receptors (A2ARs) and muscarinic acetylcholine receptors (mAChRs) On the other hand, L-LTP is distinguished from the early phase by the events of protein synthesis Activation of the second messenger, cyclic adenosine monophosphate (cAMP), by Ca2+/CaM further phosphorylates protein kinase A (PKA) which then translocates into the nucleus to initiate gene transcriptions As mentioned before, synaptic plasticity

is bidirectional and is determined by the intracellular Ca2+ concentration Under basal conditions, Ca2+ concentration is 50 to 100 nanomolar (nM) which can increase to 10 to 100 micromolar (μM) upon stimulation When given a HFS, more Ca2+ influx is elicited and as intracellular Ca2+ level exceeds 5 μM, protein kinases are activated to phosphorylate synaptic proteins producing LTP On the other hand, a low-frequency stimulation (LFS) triggers less Ca2+ influx and a concentration of Ca2+ less than 1 μM will activate protein phosphatases leading to dephosphorylation of synaptic proteins and hence yields LTD

However, electrophysiological studies of cellular models of learning and memory do not always correlate with behavioural and cognitive deficits An example of inverse correlation is the mutation of postsynaptic density protein

95 (PSD95) which enhances LTP but reduces spatial learning and memory (Migaud et al., 1998) In such cases there seem to be no relationship between synaptic plasticity and behavioural changes in cognition

1.2.3 Ageing and synaptic plasticity

Younger or immature brains are more 'plastic' or receptive to external stimulation than the mature central nervous system (CNS) during induction of synaptic plasticity The underlying reason for this phenomenon is that there is

a critical period during postnatal development whereby the activity of structuring and re-organization of synaptic connectivity is at its peak (Keuroghlian and Knudsen, 2007) This has been demonstrated by the ability

re-to induce a change in receptive field of young animals by merely exposing

them to pure tones (de Villers-Sidani et al., 2007) On the other hand, adults need to be subjected to behavioural learning or exposed to plasticity-related neuromodulators such as acetylcholine for plasticity to occur (Kilgard and Merzenich, 1998) Besides that, rodents experience a reduction in plasticity with maturation in primary auditory cortex (A1) and primary visual cortex (V1)

as there is a decreased susceptibility to the induction of NMDA-dependent LTP with increasing age (Sato and Stryker, 2008) Furthermore, Jang and colleagues showed that cortical circuits of adults are more resistant to express changes in synaptic connectivity (Jang et al., 2009)

It is inevitable that synaptic plasticity and cognition declines with age (Brayne, 2007; Salthouse, 2009; Singh-Manoux et al., 2012) In rodents, cognitive dysfunction particularly in the spatial aspect of the learning and memory is an age-related process that affects both sexes (Benice et al., 2006; Zhao et al., 2009) and all apoE genotypes (Siegel et al., 2012) However, there is a gender-biased susceptibility as female mice performed worse in passive avoidance tasks (Benice et al., 2006) Furthermore, apoE genotype has an additional influence on the cognitive impairment which will be discussed in details in chapter three

1.2.4 Genetic risk factor for sporadic AD: apolipoprotein E (apoE) variants

There has been much evidence that ApoE4 poses as a genetic risk factor for

late-onset AD (Corder et al., 1993; Farrer et al., 1997; Strittmatter et al., 1993) Overall, apoE4 individuals have a 20% risk of getting AD compared to the other two isoforms by the age of 85 ε4 allele increases the risk of getting AD

in a gene-dose dependent manner as ε4 carriers have a three-fold higher chance of getting AD and the risk is twelve-fold higher for homozygotes when comparing within the group (Holtzman et al., 2012) In fact, this isoform accounts for 65 to 80% of sporadic AD cases (Farrer et al., 1997) The differential effect of apoE4 is also gender-specific as women with ε4 allele have a higher risk of developing AD compared to men (Acevedo et al., 2010; Breitner et al., 1988; Lautenschlager et al., 1996; Rao et al., 1994; van Duijn et

al., 1993) Fluorodeoxyglucose positron emission tomography (FDG-PET)studies indicate that there is less glucose utilization in normal and AD patients carrying ε4 allele compared to ε3 individuals (Reiman et al., 2004; Small et al., 1995) Not only are the old-aged ε4 carriers affected but middle-aged ones are also more susceptible to impaired glucose metabolism as well This may be due to the disruption of mitochondrial functions by neurotoxic apoE4 fragments through perturbing mitochondrial trafficking of organelles and promoting mitochondrial apoptotic pathways (Ji et al., 2002; Reynolds and Rintoul, 2004) The areas affected were mainly hippocampus and cortex which strikingly resembles AD pathogenesis (Reiman et al., 2001; Reiman et al., 2004) Taken together, apoE4 may exert its effect before the onset of AD and the modulation is region-specific

ApoE4 has been correlated to many hallmarks of AD including neurodegeneration ApoE4 produced by injured neurons is able to disrupt mitochondrial electropotential which in turn halts synaptogenesis and causes loss of synapto-dendritic connections in apoE mice models (Buttini et al., 1999; Li et al., 2004) In human brains, apoE4 dose is inversely proportional

to dendritic spine density in AD and aged normal controls (Ji et al., 2003) Another infamous hallmark of AD is the accumulation of Aβ and many studies have been done to dissect the role of apoE4 in Aβ metabolism ApoE4 can be found in amyloid plaques and neurofibrillary tangles (Riddell et al., 2008) and a combination of apoE4 and Aβ42 (the amyloidogenic fragment) treatment causes neurotoxicity in primary culture and impairs long-term potentiation (LTP) in hippocampal slices (Trommer et al., 2005) Furthermore, apoE4 mice have lower rates of Aβ clearance compared to apoE3 mice (Dodart et al., 2005; Holtzman et al., 2000) This supports the observations of increased Aβ deposition in apoE4 transgenic mice and humans (Fryer et al., 2005; Fryer et al., 2003; Rebeck et al., 1993)

Notably, apoE4 can also contribute to AD pathogenesis independent of Aβ Transgenic mice expressing apoE4 with or without expression of human β-amyloid precursor protein (hAPP), a precursor of Aβ, have decreased presynaptic terminals (Buttini et al., 2002; Holtzman et al., 2000) There are

isoform-specific and age-dependent effects of human apoE on neurodegeneration as human apoE3 prevents kainic acid-induced neurodegeneration in comparison to Apoe-null and apoE4 mice which were not protective (Buttini et al., 1999) These morphological changes are further translated into behavioural deficits as other models of transgenic mice which express human apoE4 specifically in neurons or astrocytes showed impaired working memory Not only are these effects age- and isoform dependent, there

is also an influence of gender as female neuron-specific-enolase (NSE)-apoE4 mice lacking mouse apoE and expressing human apoE4 in neurons demonstrated impairment in water maze and vertical exploratory test (Buttini

et al., 1999; Hartman et al., 2001; Raber et al., 1998) Notably, there is no accumulation of Aβ in these transgenic mouse lines suggesting that apoE4 causes abnormal morphological and behavioural changes independently of Aβ

In other words, apoE4 is sufficient to impair synaptic plasticity per se which

accounts for the learning and memory decline in AD

1.3 N-methyl-D-aspartate receptor (NMDAR): a key player in learning and memory

NMDAR-dependent activity is an eminent mechanism underlying LTP induction which is fundamental to formation of learning and memory (Bear and Malenka, 1994; Bliss and Collingridge, 1993) It has been widely implicated in excitatory synaptic transmission and plasticity, neuronal survival, maturation and migration (Balazs et al., 1989; Komuro and Rakic, 1993) NMDAR is a ligand- and voltage-gated glutamate receptor that is highly permeable to calcium ions, at least four to eight times higher compared to other ionotropic glutamate receptors (iGluRs) As activation of NMDAR requires not only binding of glutamate, an excitatory neurotransmitter, but also α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-induced postsynaptic membrane depolarization (Chen and Lipton, 2006; Kuryatov et al., 1994), these two glutamate receptors work in cooperation to facilitate the induction of synaptic plasticity

There are several signalling cascades implicated in synaptic plasticity that are regulated by Ca2+ influx through NMDAR channels (Madison et al., 1991; Micheau and Riedel, 1999; Soderling and Derkach, 2000) Some of the kinases such as PKC, PKA, cyclin-dependent-kinase 5 (cdk5), and Src-family tyrosine kinases (SFKs) are involved in phosphorylation of NMDARs and these interactions are coupled to the downstream extracellular signal-regulated kinase (ERK/MAPK) pathway which culminates in CREB-mediated gene transcription to influence neuronal survival and plasticity (Hardingham and Bading, 2010; Salter and Kalia, 2004; Sanz-Clemente et al., 2013) In the brain, 10 to 70 percent of NR1 and NR2 subunits are phosphorylated by PKC and PKA contributing to functional heterogeneity of NMDARs (Leonard and Hell, 1997)

1.3.1 Characteristics and expression of NMDAR

NMDAR is subjected to alternative splicing and mRNA editing giving rise to subunit diversity with at least eight isoforms in existence Functional NMDAR

is a heteromultimer (Wollmuth and Sobolevsky, 2004) comprising NR1, NR2A-2D and sometimes NR3A and 3B (Figure 1A) NMDAR is activated upon binding of glutamate to NR2 subunit and glycine, a co-activator, to NR1 subunit Under resting state, opening of NMDAR channel is inhibited by magnesium (Mg2+) block which is relieved when the membrane is depolarized

to threshold level and further triggers Ca2+ influx NMDA channel has slow kinetics due to delayed unbinding of glutamate and is subject to various modulations due to its sensitivity to extracellular microenvironment (Cull-Candy and Leszkiewicz, 2004; Paoletti, 2011; Traynelis et al., 2010)

In adult CNS, functional NMDARs are essentially made up of two NR1 and two NR2 subunits in which NR2A/NR2B are the two predominant ones co-existing with the mandatory NR1 subunit as the key players in synaptic plasticity (Watanabe et al., 1992; Wenzel et al., 1997a) This gives rise to formation of major diheteromeric receptor complexes with either NR1/NR2A

or NR1/NR2B combination, and also triheteromeric receptor complexes with

NR1/NR2A/NR2B subunits concentrated in the forebrain (Hatton and Paoletti, 2005), representing from 15% to less than half of the total receptor population (Al-Hallaq et al., 2007; Gray et al., 2011; Rauner and Köhr, 2011) The NR2 subunits dictate the characteristics of a receptor-ion channel complex as they influence the channel conductance, gating properties such as channel open probability and deactivation kinetics (Cull-Candy and Leszkiewicz, 2004; Paoletti, 2011; Traynelis et al., 2010) For instance, diheteromeric NR2A and NR2B-containing receptors are highly permeable to Ca2+ and generate high-conductance channel openings compared to NR2C and NR2D-contaning diheteromeric receptors NR1/NR2A receptors also have a higher open probability and faster deactivation compared to other NR2 subunits (Chen et al., 1999; Erreger et al., 2005) In contrast, incorporating NR3 subunit results

in low single channel conductance, Ca2+ permeability and Mg2+ block (Sasaki

et al., 2002) Hence, subunit composition of NMDARs affects the channel properties which in turn modulate neuronal functions

In situ hybridization studies demonstrated that mRNAs for NMDAR subunits

are differentially distributed throughout the brain NR1 and NR2A mRNAs and proteins are ubiquitously expressed throughout the brain (Paoletti et al., 2013), whilst NR2B mRNA is mostly found in forebrain regions such as the cortex and hippocampus On the other hand, NR2C mRNA is usually expressed in the cerebellum and mid- and hindbrains of mammals (Akazawa

et al., 1994; Monyer et al., 1994) NR1 subunit mRNA is further spliced into 8 isoforms: NR1-1a-4a and NR1-1b-4b which differ by the presence of three domains namely C1, C2 and C2’ (Dingledine et al., 1999) All NR1 molecules contain either C2 or C2’ domain while C1 domain is only present in NR1-1 and NR1-3 isoforms (Figure 1B) NR1 variants are differentially distributed as NR1-1 and NR1-4 have corresponding distribution, of which the former is found abundantly in cortex and hippocampus whereas NR1-2 is widely distributed (Paoletti, 2011) Protein expression pattern also differs developmentally and spatially for NR2 subunits as major changes occur during the first two postnatal weeks Initially NR2B and NR2D predominate in the neonatal brain but they are gradually replaced by mostly NR2A and NR2C subunits NR2A subunit expression in rodents begins at postnatal day 6 (P6)

and the ratio of NR2A to NR2B increases progressively with corresponding changes occurring at mRNA level (Hoffmann et al., 2000; Liu et al., 2004; Nase et al., 1999) NR2A is eventually distributed throughout the brain whereas NR2B is restricted to the forebrain (Monyer et al., 1994; Sheng et al., 1994) This can be inferred from speeding decay of NMDAR-induced excitatory postsynaptic current (EPSC) as NR2A-mediated EPSC is characterized by a faster deactivation compared with that of NR2B (Erreger et al., 2005) Furthermore sensitivity to NR2B antagonist is diminished at P7 NR2C appears at P10 and is found mainly in cerebellum as well as olfactory bulb while expression of NR2D is limited to brainstem (Paoletti et al., 2013)

Figure 1.2 Structure of NMDAR subunit

(A) The N-terminal domain (NTD) in the extracellular region consists of a tandem of large globular bi-lobed domains which is involved in allosteric modulation; and is linked to the agonist-binding domain (ABD) formed by two segments (S1 and S2) which binds glycine in NR1 and NR3 subunits and glutamate in NR2 subunits The transmembrane domain (TMD) is made of up three helices (M1, M2, M4) and a re-entrant loop (M2 or known as P element) that lines the ionic selectivity filter; whereas the intracellular C-terminal domain (CTD) is important for receptor trafficking, anchoring and coupling to downstream signalling molecules (B) Alternative splicing of exons 5, 21 and

22 in NR1 subunit gives rise to eight NR1 variants containing cassettes N1, C1,

C2 and C2’

(adapted from (Dingledine et al., 1999; Paoletti et al., 2013)

1.3.2 Functions of NMDAR

Since deletion of NR1, NR2A and NR2B causes neonatal lethality (Bach et al., 1995; Forrest et al., 1994; Kutsuwada et al., 1996), genetic manipulations of these subunits are made in regions of interest rather than the entire CNS in order to study their functions NR1 is the obligatory subunit in a functional NMDA channel and plays a crucial role in induction of LTP Pharmacological and animal studies using CA1-restricted NR1 knockout mice show that these animals lack NMDAR-mediated synaptic currents and LTP, which is further translated into impaired spatial memory (Nakazawa et al., 2003; Tsien et al., 1996) This effect is also seen with treatment of NMDAR antagonists Similarly, genetic perturbation of NR2A activation in CA1 cells attenuates LTP (Sakimura et al., 1995) and causes deficits in a series of behavioural studies such as operant discrimination, reversal learning and spatial working memory (Bannerman et al., 2008; Brigman et al., 2008) Likewise, forebrain- and corticohippocampal-deletion of NR2B as well as treatment with NR2B antagonist in rodents impair their performance in object recognition, lever pressing tasks, Morris water maze (MWM) and attentional tasks (Brigman et al., 2008; Duffy and Nguyen, 2003; Higgins et al., 2003)

Both NR1 and NR2A subunits contribute to neurite outgrowth (Le Greves et al., 2002) Postnatal modifications in NR2 subunit composition is implicated

in synaptic plasticity during development (Bear, 2003) NR2A is deemed more stable compared to NR2B subunit and thus may help to stabilize synapses while making structural and functional changes more difficult (Lavezzari et al., 2004; Petralia et al., 2005) In contrast NR2B is crucial to formation and retraction of dendritic spines and hence NR2B dominant synapses are more plastic (Ling et al., 2012) due to its increased mobility observed in cultured neurons (Groc et al., 2006) Site-directed mutagenesis and targeted truncation

of NR2A and NR2B C-terminal tails support their important roles in synaptic localization and NMDAR clustering as their C-termini contain PDZ (PSD95, disc large, zona occludens-1) binding motif that is associated with scaffolding protein (Lin et al., 2004; Steigerwald et al., 2000) Taken together NMDAR

plays a major role in the maintenance of structure and functions of the synapses

1.3.3 Modulation of NMDARs by phosphorylation and dephosphorylation mechanisms

Studies on LTP induction at the SC-CA1 synapse (Bashir et al., 1991; Bliss and Lomo, 1973; Madison et al., 1991) and LTD at the cerebellar parallel fibre-Purkinje cell synapse (Ito, 1989; Linden and Connor, 1993) have illustrated the functional consequences of protein phosphorylation on glutamate receptors NR1 subunit is subjected to phosphorylation at either serine (Ser) or threonine (Thr) residues while NR2A and NR2B subunits are phosphorylated at tyrosine residues Activation of serine residues on C-terminal C1 cassette of NR1 positively modulates trafficking of the subunit to the membrane surface and increases synaptic transmission (Tingley et al., 1997; Zou et al., 2000) Chiu and colleagues have shown that cocaine- and amphetamine-regulated transcript peptide (CARTp) increases phosphorylation

of serine residues at position 896 and 897 which subsequently activates extracellular-regulated kinase (ERK) via protein kinase C (PKC) and protein kinase A (PKA) pathway respectively (Chiu et al., 2009) Whilst phosphorylation of these two residues increases NR1 surface expression, another serine residue at position 890 which is also phosphorylated by PKC plays a role in receptor clustering (Tingley et al., 1997) Tyrosine phosphorylation of NR2 subunits increases ion gating and reduces endocytosis

of NMDARs (Salter and Kalia, 2004; Snyder et al., 2005) This results in overall enhancement of NMDAR function and synaptic strength Mice with truncated NR2A C-terminus without phosphorylation sites shared similar phenotype as the NR2A-knockout mice Their impaired CA1-LTP and contextual learning highlight the critical functions of C-terminus and its phosphorylation sites (Sprengel et al., 1998) Most of the studies are done on tyrosine phosphorylation of NR2B at residue 1472 as it is the major phosphorylation site involved in synaptic plasticity (Jiang et al., 2011a) This phosphorylation limits the clathrin-mediated endocytosis of NR2B subtypes

and localizes NR2B-containing NMDARs at postsynaptic density (Prybylowski et al., 2005) On the other hand, the activation of NMDARs can

be suppressed by dephosphorylation through serine and threonine phosphatases 1, 2A (PP1/PP2A), or 2B (calcineurin) (Lieberman and Mody, 1994; Wang et al., 1994) Tyrosine phosphatases may also downregulate NMDA channel opening probability as application of tyrosine phosphatase inhibitor increases channel opening rates in rat spinal neurons (Wang et al., 1996) Taken together, phosphorylation and dephosphorylation mechanisms modulate clustering and interactions of NMDAR with other intracellular proteins In addition to these mechanisms, NMDAR functionality is also influenced by mGluRs or by cAMP concentration which is activated by adenylate cyclase (Bleakman et al., 1992; Courtney and Nicholls, 1992; Koh

et al., 1991; Martin et al., 1997)

1.3.4 Opposing roles of NMDAR subunits in synaptic plasticity

Activation of NMDARs facilitates formation of both LTP and LTD (Bear and Malenka, 1994; Bliss and Collingridge, 1993) and there are several hypotheses regarding the roles of NMDAR in bidirectional plasticity Earlier studies have shown that the degree of NMDAR activation which is predetermined by the degree of stimulus, and subsequently the level of postsynaptic calcium elevation, governs the direction of NMDAR-induced synaptic plasticity (Cummings et al., 1996; Nishiyama et al., 2000) Recently, there has been increasing evidence that NR2 subtypes may also determine the polarity of synaptic plasticity When LFS is used to trigger LTD, there is a larger total charge transfer facilitated by NR1/NR2B receptors compared to NR1/NR2A receptors and vice versa when HFS is used to induce LTP (Erreger et al., 2005) Furthermore, in hippocampal slice preparations, inhibition of NR2A-containing NMDARs abolishes LTP whereas blockade or loss of NR2B subunits prevents formation of LTD but not LTP (Brigman et al., 2008; Liu et al., 2004) With the postnatal developmental switch from NR2B to NR2A subunit (Monyer et al., 1994; Mutel et al., 1998) and the increasing difficulty

to induce LTD at these synapses (Errington et al., 1995; Kemp et al., 2000), as

well as impaired LTP in NR2A-deficient mice (Köhr et al., 2003; Sakimura et al., 1995), all of these suggest the dominant role of NR2A-containing NMDARs in LTP However, conflict arises as some studies argue that NR2B

is also required for LTP (Barria and Malinow, 2005; Berberich et al., 2005; Clayton et al., 2002; Tang et al., 1999; Weitlauf et al., 2005) Application of NR2B-specific antagonist, ifenprodil, attenuates LTP in immature hippocampal slices and anterior cingulate cortex of juvenile mice ranging from few days to few weeks of age (Barria and Malinow, 2005; Lu et al., 2001; Zhao et al., 2005) On the other hand, overexpression of NR2B subunit in 4- to 6-month-old transgenic mice enhances hippocampal LTP (Tang et al., 1999) Discrepancies may be caused by differences in developmental and regional NMDAR subunit expression, LTP and LTD induction protocols, or poor specificity of selective antagonists

1.3.5 NMDAR in ageing

Ageing has been negatively associated with NMDAR binding densities and functionality (Barnes, 2003; Foster, 2012; Magnusson et al., 2010) In fact, NMDAR is more susceptible to the deleterious effects of ageing than other glutamate receptors and the degree of vulnerability is different for specific NMDAR subunits (Magnusson et al., 2010) In rodents, there is a general deterioration of NR1 and NR2B subunit while NR2A subunit is relatively being spared during ageing (Magnusson, 2000; Magnusson et al., 2002) Particularly, protein expression of NR1 and NR2B subunit in the hippocampus, cerebral cortex (Clayton and Browning, 2001; Eckles-Smith et al., 2000; Magnusson et al., 2002) and crude synaptosomes2 from frontal cortex (Magnusson et al., 2007) decline with age However, few contrasting studies reported that only NR2B in the frontal cortex of mice (Ontl et al., 2004); or NR2A and NR2B but not the NR1 subunit in the hippocampus and cortex (Sonntag et al., 2000; Zhao et al., 2009); show age-related decrement It is clear that NR2B is the most vulnerable subunit to the effects of ageing than the rest (Magnusson, 2000; Magnusson et al., 2007; Ontl et al., 2004; Zhao et al.,

2 sealed presynaptic structures

2009) with a greater reduction within the synaptic membrane as compared to the whole homogenate of the frontal lobe (Zhao et al., 2009) This suggests that ageing process may also modulate synaptic localization of the NR2B

subunit

1.3.6 NMDAR in AD

NMDAR dysfunction or hypofunction has been linked to AD pathology and loss of NMDAR is not only subunit- but also brain region-specific Constant administration of NMDA antagonist over a period of time in rats renders NMDAR to a chronic hypofunctional state that causes neuronal death in cerebrocortical and limbic regions This damage particularly affects pyramidal and multipolar neurons in hippocampus, cortex and amygdala Neurodegeneration involving excitotoxic retraction of dendritic spines leads to synaptic loss which is most strongly correlated with cognitive dysfunction in

AD (Corso et al., 1997) In NMDAR hypoactivity (NRHypo) animal model, there is no deposition of amyloid plaque suggesting that this mechanism is capable of generating neuropathological symptoms of AD independently of

Aβ (Olney et al., 1997) In postmortem human AD brain, there is reduced NR1 in frontal cortex and hippocampus but not amygdala (Amada et al., 2005); and reduced NR2A and NR2B (Bi and Sze, 2002) in hippocampus whereas NR2C and NR2D do not seem to be affected (Hynd et al., 2004) Furthermore, there is a differential and subunit-specific decrease of NR1 and NR2B with increasing AD severity (Mishizen-Eberz et al., 2004) In the hippocampi of schizophrenic patients there is also downregulation of NR1 mRNA level and these patients exhibit impairment in verbal memory as well as executive functioning and attention (Gao et al., 2000) This suggests that loss of NR1 may correlate with cognitive deficits in AD

Several factors may contribute to NRHypo in AD including age and NMDAR hyperactivity (NRHyper) during early stages of the disease (Olney et al., 1997) Normal ageing has been negatively associated with NMDAR functionality across species such as mice, rats, monkeys and human Advancing age may also interact with other genetic risk factors such as apoE

genotype that can exacerbate NRHypo state preceding neurodegeneration in

AD brain

1.3.7 NMDAR in excitotoxity

It is proposed that chronic NRHyper state can lead to excitotoxic insults on synapses and deletion of NMDARs upon neuronal death thus plummeting into NRHypo state (Olney et al., 1997) Under normal circumstances, NMDAR channels should open only for brief periods of time to allow mainly influxes of

Ca2+ and other cations However, hyperactivity of glutamate receptor or prolonged exposure to elevated glutamate concentrations relieves the Mg2+block and causes excessive amounts of Ca2+ influx into neurons Some of the factors that sensitize NMDAR to glutamate are oxidative stress and impaired energy metabolism of which the latter causes a partial membrane depolarization that eliminates Mg2+ inhibition In such cases, even normal levels of glutamate will be able to drive abnormal currents In cultured neurons, nitric oxide (NO) pathway generates free radicals that disrupt glycolytic metabolism and triggers NRHyper via the same mechanism as dysregulated energy metabolism Persistent intracellular Ca2+ overload affects mitochondrial metabolism and leads to production of free radicals, resulting in

a positive feedback loop in sensitization of NMDAR, activation of degrading enzymes such as caspases and release of apoptotic factors (Olney, 1994; Rothman and Olney, 1995) These events destroy cellular components and ultimately lead to synaptic damage and cell death (Dawson et al., 1991; Naskar et al., 1999) promoting NRHypo state This ultimately escalates to a variety of neurological diseases such as AD, Huntington’s disease, Parkinson’s disease, amylotrophic lateral sclerosis (ALS), etc on a long-term basis (Choi, 1992; Cull-Candy et al., 2001; Olney, 1971, 1994) Hence, tight regulation of NMDAR function is critical for optimal activity in response to external stimulation, but at the same time to avoid excessive activation as excitotoxicity may contribute to neurodegeneration