Novel Biological Functions of Apolipoprotein-E

Novel biological functions of apolipoprotein-E

David Anthony Elliott

Submitted in total fulfilment of the requirements of the degree of

Doctor of Philosophy

Faculty of Medicine,

University of New South Wales

May, 2009

To my parents Judy & Ray Elliott

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ……………………………………………......

Date ……………………………………………......

SUPERVISOR STATEMENT

I hereby certify that all co-authors of the published or submitted papers agree to David Elliott submitting those papers as part of his Doctoral Thesis.

Signed ……………………………………………...... Dr Brett Garner

Date ……………………………………………......

III ACKNOWLEDGEMENTS

Firstly, I would like to thank my supervisor Brett Garner for his much appreciated support and encouragement throughout my candidature. I am also very grateful to my co-supervisor Woojin-Scott Kim for all of his support and assistance.

I would also like to extend my appreciation to the rest of my colleagues within the

Garner Group, the Prince of Wales Medical Research Institute and the Centre for

Vascular Research for their technical advice and, more importantly, for being a great bunch of people to work with.

Special thanks to Kayan Tsoi and Sandra Holinkova for assistance in running western blots, Glenda Halliday for provision of human tissue samples and David Jans for expert advice.

I am deeply grateful to my father, Ray, and girlfriend, Meagan, for the tremendous amount of love, support and encouragement they haven given to me throughout my candidature.

ABSTRACT

ApoE is a polymorphic protein that has been found to play many different roles in biological processes including lipid transport, neurobiology and immunoregulation.

ApoE occurs in the human population in three major isoforms; apoE2, apoE3 and apoE4. The apoE4 isoform has been identified as a major risk factor for several diseases including atherosclerosis and Alzheimer’s Disease, therefore a greater understanding of apoE biology is highly sought after. In my thesis, I have investigated several novel aspects of apoE biology. I have identified an association between increased apoE expression and apoptosis in a neuronal cell type and demonstrated that apoE becomes enriched within the neuronal apoptotic debris, consistent with a possible role for apoE in facilitating apoptotic debris clearance. A possible anti-apoptotic role of apoE in macrophages was assessed by reducing or eliminating apoE expression using siRNA and cells isolated from apoE knockout animals, respectively. The removal of apoE did not alter overall sensitivity to apoptosis, however, it did significantly increase staurosporine-induced caspase-3 activation. In other studies, the poorly understood accumulation of apoE within the nucleus was found to be enhanced during serum starvation and to localise in intra-nuclear structures that are distinct from interchromatin granule clusters. Analysis of apoE within the human brain revealed a correlation between fragmentation and the apoE3 isoform which was independent from

AD status and brain region examined. Additionally, a portion of brain apoE3 was found to be present in the form of disulphide-linked dimers. Collectively, these studies have further expanded the current knowledge of apoE biology in terms of its association with apoptosis, nuclear localization and structural differences between the apoE3 and apoE4 isoforms in the human brain.

TABLE OF CONTENTS

Originality statement ...……………………………………………………. III

Acknowledgements .………………………………………………………. IV

Abstract …………….…………….…………….…………………………... V

Publications arising from this thesis ……...………….………………….… IX

Journal articles ……….…………………...………….………………….… IX

Conference abstracts ……...………………………….………………….… IX

Oral presentations ………………...………….………………….… IX

Poster presentations ………………..………….………………….… X

Abbreviations ……...………….…………………………..…………….… XII

1. INTRODUCTION …………………………………………….…………. 1

1.1 GENERAL INTRODUCTION TO APOLIPOPROTEIN-E ……………. 1

1.1.1 Structure ……………………………………………………………..… 1

1.1.2 Regulation of apoE …………………………………………………..… 5

1.1.2.1 Cellular expression …………………………………………….…..… 5

1.1.2.2 Intracellular transport and processing ………………….…………….. 5

1.1.2.3 Transcriptional regulation …………………………………...……..… 7

1.1.3 ApoE is a multifunctional protein ………………………………..…..… 8

1.1.3.1 Lipid transport ………………………………………….…………..… 8

1.1.3.2 Immunomodulation ………………………………………………..… 11

1.1.3.3 Receptor interactions ………………………………………………… 14

1.2 ApoE AND MACROPHAGE BIOLOGY ……………………………..… 18

1.2.1 The macrophage ……………………………………………………...… 18

1.2.2 Atherosclerosis ………………………………………..……………...… 19

1.3 ASSOCIATION OF apoE WITH APOPTOSIS ………………………..... 20

1.3.1 Introduction to apoptosis …………………………………….………..... 20

1.3.2 A role of apoE in apoptosis? ………………………………………….... 25

1.4 ROLE OF apoE IN NEUROBIOLOGY ………………………...... 26

1.4.1 CNS lipid transport ……………………………………….…...... 26

1.4.2 Cognitive function ………………………...... 27

1.4.3 Role in nerve regeneration and repair ………………………...... 28

1.4.4 Neuronal expression of apoE ………………….……………...... 28

1.5 ApoE AND ALZHEIMER’S DISEASE ……...………………...... 30

1.5.1 Alzheimer’s disease background ……………………………...... 30

1.5.2 Generation of amyloid plaques …………………...…………...... 32

1.5.3 Alzheimer’s disease risk factors ………………………...... 35

1.5.3.1 Early-Onset Alzheimer’s Disease ………………………...... 35

1.5.3.2 Late-Onset Alzheimer’s Disease ………………….………...... 35

1.5.4 ApoE in the Alzheimer’s Disease brain ………….…………...... 36

1.5.4.1 ApoE levels in the AD brain ………………………...... 36

1.5.4.2 Interactions with amyloid beta ……………………………...... 38

1.5.4.3 Role of apoE in Neurofibrillary Tangle formation …………………... 41

1.5.4.4 Proteolysis of apoE ………………………………………………….. 41

VII

2. AIMS OF THIS THESIS ……………………………………………….. 44

3. RESULTS ………………………………….…………………………….. 45

3.1 RESULTS SUMMARY ………………………………………………… 45

3.2 PUBLICATIONS ……………………………………………………..… 48

3.2.1 Publication I …………………………………………………...……… 48

3.2.2 Publication II ………………………………………………………..… 54

3.2.3 Publication III ………………………………………………….……… 64

3.2.4 Publication IV ……………………………………………….………… 74

4. GENERAL DISCUSSION …………………………………………….. 128

4.1 POSSIBLE ROLES OF apoE IN APOPTOSIS ……………………….. 128

4.1.1 Cell survival …………………………………………………………. 128

4.1.2 Clearance of apoptotic debris ……………………………………….. 130

4.2 NUCLEAR TRAFFICKING OF apoE ……………….……………….. 131

4.3 STRUCTURAL DIFFERENCES BETWEEN apoE ISOFORMS

IN THE HUMAN BRAIN …………………………………..…..…….. 132

4.3.1 Fragmentation of apoE3 in the human brain ……………….……….. 133

4.3.2 Mechanism of apoE proteolysis in the brain ………………………... 136

4.3.3 Biological function of apoE fragments? …………………………….. 138

4.3.4 ApoE disulfide linked dimers ……………………………………….. 139

5. MAJOR CONCLUSIONS ……………………………………………. 141

6. REFERENCES ………………………………………………..………. 142

VIII

PUBLICATIONS ARISING FROM THIS THESIS

Journal articles

1. David A. Elliott, Woojin S. Kim, David A. Jans and Brett Garner. (2007) Apoptosis induces neuronal apolipoprotein-E synthesis and localization in apoptotic bodies.

Neuroscience Letters 416: 206-210.

2. David A. Elliott, Woojin S. Kim, David A. Jans and Brett Garner. (2008)

Macrophage apolipoprotein-E knockdown modulates caspase-3 activation without altering sensitivity to apoptosis. Biochimica et Biophysica acta 1780:145-53.

3. Woojin S. Kim , David A. Elliott , Maaike Kockx, Leonard Kritharides, Kerry-Anne

Rye, David A. Jans and Brett Garner. (2008) Analysis of apolipoprotein-E nuclear localization using green fluorescent protein and biotinylation approaches. The

Biochemical Journal 409: 701-9.

4. David A. Elliott, Kayan Tsoi, Sandra Holinkova, Sharon L. Chan, Woojin S. Kim,

Glenda M. Halliday, Kerry-Anne Rye, David A. Jans and Brett Garner. Isoform- specific processing of apolipoprotein-E in the human brain. Revised manuscript accepted for publication in Neurobiology of Aging, February 2009. Currently in print.

Conference abstracts

Oral presentations:

1. Elliott, D. A. ‘Isoform-specific processing of apoE in the human brain’ Kioloa

Neuroscience Colloquium, April 2008.

2. Elliott, D. A., Kim, W. S. and Garner, B. ‘Exploring the potential role of apolipoprotein-E in apoptosis’ Dementia, Ageing and Neurodegeneration DISeases

Group (DANDIS) conference held at the Prince of Wales Medical Research Institute,

January 2006.

Poster presentations:

3. Elliott, D. A., Kim, W. S. and Garner, B. ‘Post-translational modifications of apolipoprotein-E in the human brain’ International Conference on Alzheimer’s Disease,

Chicago, USA, July 2008.

4. Elliott, D. A., Kim, W. S. and Garner, B. ‘Post-translational modifications of apolipoprotein-E in the human brain’ Australian Neuroscience Society 28th Annual

Meeting, Hobart, January 2008.

5. Elliott, D. A., Kim, W. S. and Garner, B. ‘Potential role of apolipoprotein-E in clearance of apoptotic debris’ 37th Annual meeting of the Society for Neuroscience, San

Diego, USA, November 2007.

6. Elliott, D. A., Kim, W. S. and Garner, B. ‘Exploring the potential role of apolipoprotein-E in apoptosis’ Australian Society for Medical Research NSW Scientific

Conference, Sydney, June 2006.

7. Elliott, D. A., Kim, W. S. and Garner, B. (2006) ‘What is the role of apolipoprotein-

E in apoptosis?’ Australian Neuroscience Society 26th Annual Meeting, Sydney, Jan-

Feb 2006.

8. Elliott, D. A., Kim, W. S., Quinn, C. M., Kritharides, L., Kockx, M. and Garner, B.

‘Exploring the potential role of apolipoprotein-E in apoptosis’ Australian Vascular

Biology Society Meeting, Sydney, August 2005.

LIST OF ABBREVIATIONS aa amino acid

A amyloid

Ab Antibody

AD Alzheimer's Disease AIDS Acquired Immune Deficiency Syndrome

AIF apoptosis inducing factor

Apaf1 activating protease-activating factor 1

APC Antigen Presenting Cells apoA1 apolipoprotein-A1 apoA-II apolipoprotein-A2 apoB100 apolipoprotein-B100 apoE apolipoprotein-E apoER2 apoE receptor 2 apoD apolipoprotein-D apoJ apolipoprotein-J

APP amyloid precursor protein

BACE1 -site amyloid precursor protein cleavage enzyme 1

BBB blood brain barrier

BCA bicinchoninic acid

BSA bovine serum albumin

CatD Cathepsin D

CCM cell-conditioned medium

CHO Chinese hamster ovary

CNS Central Nervous System

CON Control

XII

CSF Cerebrospinal Fluid

Cys cysteine cyt c cytochrome c

DD death domains

DED death effector domain

EGF epidermal growth factor

Endo G endonuclease G

EOAD Early-onset Alzheimer’s Disease

Front Frontal cortex gHCl guanidine hydrochloride

GFP green fluorescent protein

Glu glutamate

GSL glycosphingolipids

HDL high density lipoprotein

Hippo Hippocampus

HI-Thr heat inactivated thrombin

HIV Human Immunodeficiency Virus

HSPG heparan sulfate proteoglycan

HSV Herpes Simplex Virus

IAP inhibitor of apoptosis protein

IDE insulin degrading enzyme

Il-6 interleukin 6

LBD lipid binding domain

LDLR low-density lipoprotein receptor

LOAD Late-onset Alzheimer’s Disease

XIII

LRP LDLr-related protein

LXR Liver X Receptor mAb monoclonal antibody

M-CSF Macrophage Colony Stimulating Factor

MED1 multiple start site element downstream 1

MEGF7 multiple EGF repeat-containing protein min minutes

MMP-14 matrix metalloproteinase-14 mRNA messenger RNA

NEP neprilysin

NFB nuclear factor kappa B

NFT neurofibrillary tangles

NLS nuclear localization sequence

NR non-reduced

Occip Occipital lobe

PAGE polyacrylamide gel electrophoresis

PARP poly (ADP-ribose) polymerase

PBS Phosphate Buffered Saline

PD Parkinson’s Disease

PHF paired helical filaments

POPC 1-palmitoyl-2-oleylphosphatidyl choline

PS phosphatidyl serine

PS presenilin

R reduced

RBD receptor binding domain

XIV

RXR Retinoid X Receptor sAPP soluble APP sAPP soluble APP

SDS Sodium Dodecyl Sulfate

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis

Ser serine siRNA short interfering RNA sLDLR soluble LDLR

SM sphingomyelins

SP signal peptide

STAT1 signal transducer and activator of transcription 1

STAT2 signal transducer and activator of transcription 2

TBI traumatic brain injury

TBS Tris Buffered Saline

TBS-X Tris Buffered Saline and Triton X-100

TF transcription factor

Thr Thrombin

Thr threonine

TNFR tumour necrosis factor receptor

U units

VLDL very low-density lipoprotein

VLDLR very low-density lipoprotein receptor

1. INTRODUCTION

1. GENERAL INTRODUCTION TO APOLIPOPROTEIN-E

1.1.1 Structure

Apolipoprotein-E (apoE) is a 299 amino acid (aa) (~34 kDa) protein that was first identified in the 1970’s as a constituent of plasma lipoproteins (Shore & Shore 1973,

Mahley 1988). It is encoded by the APOE gene located on chromosome 19 and is a member of the apolipoprotein multigene family (Li et al. 1988).

ApoE consists of 2 major domains, N-terminus 1-191aa and C-terminus 216-299aa, separated by a flexible ‘hinge’ region (Figure 1) (Aggerbeck et al. 1988, Wetterau et al. 1988). The N-terminal domain consists of 4 alpha-helix bundles and contains a region, residues 136-150, that is enriched in the basic amino acids arginine (Arg) and lysine (Lys). This region is termed the Receptor Binding Domain (RBD) and is involved in binding with heparan sulfate proteoglycan (HSPG) and a range of receptors

(discussed below). The C-terminal domain consists of amphipathic -helices which are responsible for the lipid-binding capability of apoE and are a common feature of all apolipoprotein family members. (Li et al. 1988). Residues 244-272 are most crucial to lipid binding and are thus referred to as the lipoprotein-binding domain (LBD). These two specialised domains enable apoE to be highly effective at binding lipids and controlling their transport and metabolism in a regulated manner.

ApoE occurs in the human population as three common isoforms apoE2, apoE3 and apoE4 which differ in their cysteine (Cys) and Arg composition at residues 112 and

158. ApoE2 contains Cys112, Cys158; apoE3 contains Cys112, Arg158; and apoE4 contains

Arg112, Arg158 (Figure 1) (Rall et al. 1982). The Cys residues present in apoE2 and

Figure 1.

Model of the apoE protein highlighting; N-terminal (1-191aa) and C-terminal (216-

299) domains; receptor binding domain (136-150); lipid binding domain (244-272); polymorphic sites at amino acids 112 and 158; the major thrombin cleavage site; O- linked glycosylation site, O-glycan; phosphorylation site, PO4. (Adapted from

Weisgraber et al., 1996).

2 apoE3 represent the only Cys residues present in the whole apoE protein, thus apoE4 is completely devoid of Cys. The frequency of the different APOE alleles in western populations is; 3 ~77%, 4 ~15% and 2 ~8%. This can vary across different human populations, however, 3 is consistently the most common (reviewed in (Mahley & Rall

2000)).

Several structural differences exist between the apoE isoforms. The presence of Arg112 in E4 disrupts the ionic association between Glu109 and Arg61, leaving Arg61 free to associate with Glu255. This leads to inter-domain interaction and a reduction in the lipid- binding capacity of apoE4. In apoE3 and apoE4 a salt-bridge bond exists between

Arg158 and Asp154. This is eliminated by substitution of Cys158 in apoE2, resulting in the formation of a salt-bridge between asp154 and arg150 which pulls the positively charged side-chain of arginine150 away from the RBD. This impairs the ability of apoE2 to interact with certain receptors (discussed below) (Figure 2). In addition, apoE4 is unable to form disulphide-linked bonds with other proteins due to its lack of a Cys residue. It has also been shown using chemical and thermal denaturation techniques that apoE4 is structurally less stable when compared to apoE3 (Morrow et al. 2000,

Clement-Collin et al. 2006).

The structure of apoE can also be altered by post-translational modifications.

Carbohydrate molecules can be attached to Thr194 via O-linked glycosylation (Zannis et al. 1986, Wernette-Hammond et al. 1989) and apoE contains a phosphorylation site at

Ser296 (Raftery et al. 2005). Also, the hinge region of apoE is susceptible to cleavage by proteolytic enzymes (refer to Figure 1) {Wetterau, 1988

Figure 2.

A) Schematic diagram highlighting the domain interaction that occurs with apoE4, but not apoE3. Arg112 in apoE4 disrupts the ionic association between Glu109 and Arg61, leaving Arg61 free to associate with Glu255. (Adapted from Mahley et al., 2006). B)

Three-dimensional model highlighting structural differences between the apoE3 and apoE2 isoforms. Boxed area represents the RBD. The salt bridge that exists between

Arg158 and Asp154 in apoE3 is disrupted by the Cys158 substitution in apoE2, leading to an ionic interaction between Asp154 and Arg150 which pulls Arg150 away from the

RBD. (Adapted from Mahley et al., 2000).

4 #71}. Stable fragments of apoE have also been detected in the human brain and this is discussed in more detail below.

1.1.2 Regulation of apoE

1.1.2.1 Cellular expression

ApoE expression has been reported in most tissues of the body, primarily the liver, brain and to a lesser extent in spleen, skin, ovaries, adrenal glands, lungs, kidney and muscle (Mahley 1988, Elshourbagy et al. 1985, Blue et al. 1983, Lin et al. 1986,

Driscoll & Getz 1984). The liver produces approximately 70% of the total apoE in the body, with the majority of this secreted into the plasma where it plays an important role in lipid transport. Hepatocytes and macrophages are a major source of apoE in the circulation and peripheral tissues (Mahley 1988). Astrocytes are the primary source of apoE in the brain, however, apoE is also expressed by oligodendrocytes, ependymal layer cells, microglia and, under certain physiological circumstances, in neurons (Bao et al. 1996, Diedrich et al. 1991, Han et al. 1994b, Ignatius et al. 1986, Metzger et al.

1996, Nakai et al. 1996, Nathan et al. 1994, Poirier et al. 1991, Xu et al. 2006).

1.1.2.2 Intracellular transport and processing

ApoE is translated as a 317aa protein containing an N-terminal 18aa signal peptide (SP) which is a characteristic feature of proteins destined for secretion. The SP directs apoE to the endoplasmic reticulum where the SP is cleaved, producing the 299aa apoE protein, which is then transported to the Golgi where it can undergo O-linked glycosylation before being either excreted, stored within the cell or degraded in the lysosome (Zannis et al. 1984, Zannis et al. 1986). Approximately 92% of apoE secreted by macrophages is glycosylated, compared to ~42% of intracellular and ~24% of

5 plasma apoE (Zannis et al. 1986). Interestingly, ablation of the Thr194 glycosylation site does not impair secretion, thus the significance of apoE glycosylation is currently unclear (Wernette-Hammond et al. 1989). Prior to excretion, apoE may reside at the cell surface in a storage pool with a relatively long turnover time (Zhao & Mazzone 2000).

The stability of apoE in this pool is mediated by binding to cell surface HSPG which can modulate both the rate of secretion and intracellular reuptake of apoE (Lucas &

Mazzone 1996, Ji et al. 1998). ApoE in this pool may also undergo recycling back to the Golgi for glycosylation prior to secretion. Exogenous apoE, complexed with lipid in lipoprotein particles, can also be endocytosed via receptor mediated interactions

(discussed in detail below), resulting in the delivery of lipids to the cell and either lysosomal degradation or recycling of apoE to the cell surface (Farkas et al. 2004, Fazio et al. 1999, Heeren et al. 2001). Alternatively, both exogenous and endogenous apoE can reside in intracellular vesicles that share features in common with lysosomes but do not result in protein degradation (also known as late endosomes and late lysosomes)

(Dekroon & Armati 2001, Dekroon & Armati 2002a, Ljungberg et al. 2003, Tabas et al.

1990, Tabas et al. 1991). These vesicles serve as intracellular storage pools whereby apoE can be bidirectionally transported to and from the cell surface.

The proportion of apoE that is stored in each cellular compartment or destined for degradation or secretion can vary depending on cell type (Mazzone et al. 1992, Duan et al. 1997, Fazio et al. 1999), lipoprotein profile of exogenous apoE (DeKroon & Armati

2001, Tabas et al. 1990, Tabas et al. 1991, Ljungberg et al. 2003) and a wide range of extrinsic cellular factors such as cytokines, fatty acids and growth factors (Dekroon &

Armati 2002b, Tedla et al. 2004, Quinn et al. 2004, Werb & Chin 1983, Werb et al.

1989, Zhao et al. 2002, Rensen et al. 2000). Steady state apoE concentration is

6 influenced by isoform differences with apoE4 being more susceptible to lysosomal degradation and less efficient at HSPG mediated endocytosis (DeKroon & Armati 2001,

Ji et al. 1998). Targeted replacement studies in mice have shown apoE4 is more rapidly turned over than apoE3 (Riddell et al. 2008).

Interestingly, there is evidence that a small pool of apoE can localize to the nucleus in hepatocytes, macrophages and ovarian cancer cells, however, the functional significance of this is not understood (Quinn et al. 2004, Chen et al. 2005, Panin et al.

2000).

1.1.2.3 Transcriptional regulation

The upstream promoter region of the APOE gene is known to contain several regulatory regions that can bind a variety of different transcription factors (TFs) (Chang et al.

1990, Adroer et al. 1997, Laffitte et al. 2001, Salero et al. 2003, Lahiri 2004, Du et al.

2005, Naukkarinen et al. 2005, Ramos et al. 2005). One of the most well characterised mechanisms of APOE upregulation is mediated by the Liver X Receptors (LXRs) and

Retinoid X Receptors (RXRs), usually in response to increased levels of cellular cholesterol (Laffitte et al. 2001). LXR and RXR are members of a large family of transcription factor proteins known as nuclear receptors which are regulated by binding various ligands. LXRs are regulated by oxidized forms of cholesterol, known as oxysterols, and intermediate products produced during cholesterol synthesis (Janowski et al. 1996, Janowski et al. 1999). RXRs are regulated by 9-cis-retinoic acid and polyunsaturated fatty acids (Heyman et al. 1992, Bourguet et al. 2000). LXR and RXR proteins form heterodimers which, upon ligand binding, control expression of target genes via interaction with specific LXR response elements present in the gene promoter

7 region of APOE (Laffitte et al. 2001) and several other genes, such as ABCA1 and

ABCG1, which play a role in cholesterol efflux and lipidation of apoE (Repa et al.

2000) (Venkateswaran et al. 2000a, Venkateswaran et al. 2000b).

Interestingly, the APOE regulatory region also contains binding sites for the anti- apoptotic TF nuclear factor kappa B (NFB) (Du et al. 2005) and several inflammatory response associated TFs interleukin 6 (Il-6), Il-6 responsive element-binding protein, multiple start site element downstream 1, signal transducer and activator of transcription 1 and signal transducer and activator of transcription 2 (reviewed in

(Lahiri 2004)).

1.1.3 ApoE is a multifunctional protein

1.1.3.1 Lipid transport

ApoE was first identified as a constituent of plasma lipoprotein particles where it plays a role in facilitating the transport and cellular uptake of lipids throughout the body

(Shore & Shore 1973, Mahley 1988). ApoE associates with chylomicron particles that are derived from the intestines and contain dietary lipids. The delivery of these lipids to the liver is mediated by apoE binding to receptor molecules present on the surface of hepatocytes, primarily the low density lipoprtoein receptor related protein (LRP) which is facilitated by binding with HSPG (Innerarity & Mahley 1978, Mahley & Ji 1999).

ApoE is also secreted from the liver in association with triglyceride rich very low density lipoprotein (VLDL) which, along with apoE containing chylomicron remnants, delivers lipids to the extra-hepatic cells of the body by either receptor-mediated uptake or hydrolysis by lipoprotein lipase which results in the liberation of free fatty acids

(Figure 3). The apolipoprotein apoB100 also plays a major role in this process. ApoE is

Figure 3.

Basic overview highlighting the role of apoE in lipoprotein metabolism. ApoE associates with several lipoproteins to facilitate lipid transport and delivery to cells mediated by receptor interactions; apoE-chylomicrons transport dietary lipids from the intestine to the liver via chylocmicron particles; apoE-VLDL/IDL/LDL transports lipids from the liver to peripheral cells (eg. macrophages); apoE-HDL participates in the reverse-transport of cholesterol to the liver where it can then be secreted from the body in the form of bile salts.

9 also involved in the transport of cholesterol from peripheral macrophages back to the liver where it can be eliminated from the body via bile salts, which contributes to a process commonly known as reverse cholesterol transport. In this process cholesterol is packaged in high density lipoprotein (HDL) particles. ApoE is not essential in this process, however, it does greatly enhance the cholesterol binding capacity of HDL

(Gordon et al. 1983, Koo et al. 1985) (Huang et al. 1994, Mahley & Innerarity 1983).

The primary apolipoprotein in HDL is apoA1 which delivers cholesterol to the liver via the SR-B1 receptor. The transfer of lipids from hepatocytes and macrophages to lipoprotein particles is facilitated by membrane transport proteins, particularly ABCA1 and ABCG1 (Jessup et al. 2006). ApoE also mediates the redistribution of lipids between cells within tissue, a well characterized example being in peripheral nerve regeneration (discussed below in section 1.4.3).

The differences between apoE isoforms can have variable effects on lipoprotein metabolism. The affinity of apoE2 for the LDL receptor (LDLR) is relatively weak, being ~2% that of apoE3 and apoE4 (Schneider et al. 1981, Weisgraber et al. 1982).

This results in apoE2 homozygous individuals being susceptible to the lipoprotein disorder type III hyperlipoproteinemia (Mahley et al. 1999). Population studies have revealed that apoE4 is associated with increased levels of total cholesterol and LDL

(Utermann et al. 1984, Utermann 1987). The underlying mechanism responsible for this is not entirely understood, however, it may be related to the fact that apoE4 displays preferential binding to triglyceride rich lipoproteins such as VLDL, whereas apoE2 and apoE3 display preferential binding with HDL (Steinmetz et al. 1989, Utermann 1987,

Weisgraber 1990). Also, it has been suggested that apoE4 may alter the rate at which dietary fats are catabolised, leading to down-regulation of LDLR and thus increased

10 plasma LDL and cholesterol levels (Gregg et al. 1986). This property of E4 may be a factor in the increased incidence of heart disease associated with this allele (Davignon et al. 1988, Menzel et al. 1983, Stengard et al. 1995).

ApoE is involved in a diverse range of biological processes beyond lipoprotein metabolism and the different isoforms have been associated with modifying the risk of developing several diseases (Table 1).

1.1.3.2 Immunomodulation

It is now well established that apoE possesses potent immunomodulatory properties, with several studies attributing anti-inflammatory properties to apoE and isoform differences being associated with altering the virulence of certain pathogens. Evidence for this arose from studies showing that LDL particles could either promote or inhibit the proliferation and activation of T lymphocytes, an event which is important for mounting an immune response (Cuthbert & Lipsky 1984b, Cuthbert & Lipsky 1986).

The stimulation of proliferation is dependent on the delivery of fatty acids to the cell via the LDLR. In contrast, the inhibitory effect is not associated with LDLR interaction or the delivery of lipids to the cell. Interaction with another receptor, or group of receptors, is responsible for this effect, with the transferrin receptor and cell surface HSPG being possible candidates (Hui & Harmony 1980b, Hui & Harmony 1980a, Cuthbert &

Lipsky 1984b, Cuthbert & Lipsky 1984a). The component of LDL responsible for conferring the inhibitory effect is apoE (Hui et al. 1980, Curtiss & Edgington 1976,

Curtiss & Edgington 1981), more specifically, the RBD region within apoE.

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12 In vitro studies have shown that mimetic peptides derived from the RBD (136-150) are sufficient to inhibit T cell activation (Clay et al. 1995, Cardin et al. 1988, Dyer et al.

1991). Furthermore, several studies have demonstrated that the RBD of apoE has potent anti-inflammatory effects on microglial cells (the primary immuno-regulators in the brain) and on systemic immune responses in mice (Lynch et al. 2003) (Lynch et al.

2005) (Laskowitz et al. 2001) (Li et al. 2006) (Singh et al. 2008). These effects are mediated, at least in part, by binding with members of the LDLR family. More specifically, in microglia this is dependent on VLDLR and LRP1, not LDLR

(Pocivavsek et al. 2008).

Other studies have revealed that apoE knockout (apoE-/-) in mice results in significantly enhanced inflammatory responses both basally and in response to inflammatory stimuli, with increased levels of several pro-inflammatory cytokines; tumor necrosis factor alpha, interferon gamma, IL-6, interleukin 12 and fibrinogen (Ali et al. 2005, Grainger et al. 2004, Laskowitz et al. 2000, Tenger & Zhou 2003, Mace et al. 2007). Also,

Antigen Presenting Cells (APC), such as macrophages isolated from apoE-/- mice, display increased levels of the cell surface co-stimulatory molecules CD40, CD80 and

MHC-II compared to wild type (WT) mice and this results in an enhanced capacity to stimulate T lymphocytes (Tenger & Zhou 2003).

There is evidence that apoE can modulate host susceptibility to various bacterial, viral and protozoan pathogens (Urosevic & Martins 2008). ApoE-/- mice are more susceptible to infection by Klebsiella pneumoniae and Listeria monocytogenes (de Bont et al. 1999, de Bont et al. 2000, Roselaar & Daugherty 1998). These differences appear to be unrelated to the hypercholesterolemia present in apoE-/- mice as they are not observed in

LDLR-/- mice, which are also hypercholesterolemic when fed a diet high in fat and cholesterol (Netea et al. 1996, Ishibashi et al. 1993). Differences in disease susceptibility exist between humans carrying different apoE isoforms. ApoE4 carriers are more susceptible to developing Herpes Simplex Virus (HSV) induced herpes labialis (cold-sores) (Itzhaki et al. 1997, Burgos et al. 2002) and human immunodeficiency virus (HIV) positive 4/4 patients are afflicted with a more rapid disease progression (Burt et al. 2008). In vitro studies have also shown that cells are more easily infected by HIV when in the presence of apoE4 (Burt et al. 2008). HSV and

HIV both require interaction with cell surface HSPG to facilitate cell entry, thus it has been speculated that apoE interaction with HSPG disrupts this process (Herold et al.

1991). Further evidence for this comes from cell culture studies of HIV infection where apoE peptides, derived from the heparin binding/ RBD, inhibit HIV cellular attachment

(Dobson et al. 2006). The malaria protozoan also utilizes cell surface HSPG to gain entry into cells and there is evidence that apoE2 carrying individuals are more susceptible to infection (Wozniak et al. 2003).

1.1.3.3 Receptor interactions

The RBD of apoE enables binding with a range of receptors to mediate functions such as the delivery of lipids to cells and cellular signaling. ApoE binding partners are predominantly the LDL receptor family, but also include HSPG, transferrin receptor and the alpha-7 nicotinic acetylcholine receptor (Gay et al. 2007).

ApoE is a ligand for the seven members of the LDLR family in mammals: LDLR, Very

Low Density Lipoprotein Receptor (VLDLR), apoE receptor 2 (apoER2), multiple epidermal growth factor (EGF) repeat-containing protein (MEGF7), LRP, LRP1B and

14 megalin (also referred to as LRP2) (Beffert et al. 2004, Rebeck et al. 2006). This family of receptors share several common structural domains, including ligand-binding domains consisting of negatively charged cysteine-rich ligand-binding repeats (which bind the positively charged RBD of apoE), EGF homology domains with YMTD motifs

(propeller domains) and cytoplasmic domains containing at least one NPxY motif

(Figure 4). Some members of this family play a well established role in the endocytosis of lipids, as discussed earlier. However, ligand binding to these receptors can also mediate cell signaling pathways that regulate a variety of cellular/physiological responses ranging from the inflammatory response in microglia, migration of endothelial cells, neurite outgrowth, microtubule stabilisation, synaptic plasticity, calcium homeostasis and kinase activation (Beffert et al. 2002, Beffert et al. 2004, Hui

& Basford 2005). The cytoplasmic NPxY motif can link receptor binding with intracellular signaling by binding with adaptor proteins containing phosphtyrosine binding (PTB) domains, such as Disabled-1 (Dab1) (Howell et al. 1999, Trommsdorff et al. 1998). This leads to a cascade of intracellular signalling events which results in either the activation or deactivation of several key regulatory enzymes including cyclic adenosine monophosphate (cAMP), Protein Kinase A (PKA), extracellular regulated kinase 1/2 (ERK 1/2), c-Jun N-terminal kinase (cJNK), Src family kinases, phosphatidylinositol-3-kinase (PI3K), Akt and glycogen synthase kinase 3- (GSK-3 )

(Hoe et al. 2005, Hui & Basford 2005). The signaling pathways initiated by Reelin binding with apoER2 and VLDLR have been well studied and play an important role in microtubule organization and brain development (Hiesberger et al. 1999, Benhayon et al. 2003).

Figure 4.

Schematic representation of the seven LDLR family members in mammals. Common structural features are shown. (Reproduced from Beffert et al., 2004).

16 Soluble forms of the LDLR family (sLDLR), which consist of the extracellular domain detached from the cell surface, have been identified in vivo in the plasma and CSF. sLDLR are produced by either extracellular proteinases or alternate mRNA splicing

(Garcia-Touchard et al. 2005, Xing et al. 2003). The generation of sLDLR can be increased by ligand binding and phorbol esters (Hoe & Rebeck 2005). The ability of sLDLR to interact with ligands including apoE has been confirmed (Quinn et al. 1997,

Koch et al. 2002, Ronacher et al. 2000, Bajari et al. 2005, Yamamoto et al. 2008), however, the physiological role of these receptors has not yet been determined.

Physiological roles have been found for other transmembrane proteins that can release soluble extracellular domains, thus it seems highly likely that sLDLR may have analogous functions. These include, antagonizing membrane bound receptors by competing for ligand binding, regulating receptor population, inhibiting extracellular proteinases and modulating the activity of cytokines and growth factors (reviewed in

(Rebeck et al. 2006)).

ApoE binding to cell surface HSPG plays a role in the endocytosis of lipids and the transduction of cellular signals, which may be involved in the immunomodulatory responses discussed above. HSPG can also enhance LRP endocytosis of apoE by either forming a complex with LRP or entrapping apoE at the cell surface before transferring it to LRP for endocytosis (Mahley & Ji 1999).

The ability of apoE to interact with these receptors can vary due to isoform differences.

As mentioned above, apoE2 is ~98% less effective than apoE3 and apoE4 at binding the

LDLR. In contrast, the binding of apoE2 with LRP is equivalent to that of apoE3, while apoE4 is slightly less efficient (Ruiz et al. 2005). When apoE3 forms a disulphide

17 linked homodimer or heterodimer with apoA-II, which may represent up to 55% of total plasma apoE, LDLR binding activity is reduced by ~80% and ~70%, respectively

(Weisgraber & Shinto 1991). ApoE4 is less efficient at binding HSPG (Ji et al. 1994,

Kowal et al. 1990). Also, the binding of apoE to apoER2 and VLDLR promotes receptor cleavage and the generation of soluble receptors in an isoform dependent manner, with apoE2 being the most effective followed by apoE3 then apoE4 (Hoe &

Rebeck 2005).

1.2 ApoE AND MACROPHAGE BIOLOGY

1.2.1 The macrophage

The macrophage is a key component of the immune system. It is the primary cell type in the body responsible for the phagocytic removal and destruction of pathogens and dead tissue. Macrophages originate from myeloid progenitor cells, produced in the bone marrow, which give rise to monocytes that are dispersed throughout the body in the blood. Monocytes then enter tissues in response to chemokine signals and differentiate into macrophages (Janeway 1999). There are various factors that promote differentiation and maintenance of macrophage populations in the tissue environment, the most important being Macrophage Colony Stimulating Factor (M-CSF) (Stanley et al. 1994).

Bacterial, viral and parasitic/protozoan pathogens can be tagged by complement and immunoglobulin proteins of the immune system. This directs binding with cell surface receptors present on macrophages and mediates phagocytosis. Macrophages may also bind directly with bacterial cell surface polysaccharides (Taylor et al. 2005). Pathogens are endocytosed into a phagosome which then fuses with lysosomes to form the

18 phagolysosome where the pathogens are destroyed by free radicals and enzymes. This results in the macrophage becoming activated and secreting several cytokines involved in promoting an inflammatory response and attracting additional monocytes/macrophages. Macrophages also play a role as Antigen Presenting Cells

(APC) whereby pathogen derived peptides are displayed on the cell surface, enabling recognition by T and B lymphocytes as part of an adaptive immune response (Janeway

1999).

Macrophages are attracted to sites of tissue injury and play an important role in the removal of apoptotic cells, mediated by signals such as phosphatidyl serine (PS) exposure on the surface of apoptotic cells (discussed below) and residual debris from necrotic cell death (Fadok et al. 1992). In this situation macrophages play an important role in storing cholesterol and lipids derived from dead cells and redistributing them to regenerating cells. This process is facilitated by the production and secretion of apoE from the macrophage (Ignatius et al. 1986); (Snipes et al. 1986), a good example of this process is observed in the injury and regeneration of peripheral nerves (discussed below). The importance of this apoE-mediated role in macrophage biology is underlined by the fact that apoE can represent up to 25% of the total protein secreted by the macrophage (Kayden et al. 1985, Basu et al. 1981, Basu et al. 1982, Werb et al.

1986, Garner et al. 1997).

1.2.2 Atherosclerosis

Atherosclerosis is a disease of the vascular system that is the leading cause of mortality worldwide (Ross 1993). The key feature of atherosclerosis is inflammation and the presence of atheromatous plaques in the endothelial wall of arteries. Atheromatous

19 plaques consist of macrophages that have become enlarged and enriched with cholesterol and oxidized lipids, and are referred to as foam cells. Debris from apoptotic foam cells and cholesterol crystals can build up in these plaques, forming a necrotic core which makes the plaque unstable and likely to rupture. Plaque rupture can obstruct arterial blood flow causing tissue ischemia, the underlying mechanism behind the two most common causes of death, stroke and myocardial infarction (Hansson & Libby

2006, Ross 1993).

ApoE plays a protective role against atherosclerosis by promoting the efflux of cholesterol from endothelial foam cells (via reverse cholesterol transport, discussed above) and most probably attenuating the chronic state of inflammation (via anti- inflammatory properties discussed above) (Ross 1993). Possession of the apoE4 isoform, which is less efficient at promoting reverse cholesterol transport, is associated with an increased risk of developing atherosclerosis (Davignon et al. 1988, Menzel et al. 1983, Stengard et al. 1995). Also, apoE-/- mice are hypercholesterolemic and will develop severe atherosclerosis when fed a diet high in fat and cholesterol, whereas WT mice are resistant. The protective role of macrophage apoE is highlighted by the reversal of atherosclerosis in apoE-/- mice following transplantation of bone marrow from WT mice (Zhang et al. 1992, Plump et al. 1992, Boisvert et al. 1995).

1.3 ASSOCIATION OF apoE WITH APOPTOSIS

1.3.1 Introduction to apoptosis

Apoptosis is a tightly regulated form of cell death that plays an essential role in controlling the population of cells present in the tissues of multicellular organisms (Kerr

20 et al. 1972). In contrast to unregulated or accidental cell death, termed necrosis, apoptosis is genetically programmed and occurs following a well coordinated sequence of morphological and biochemical events (Ellis et al. 1991). The apoptotic cell undergoes cytoplasmic shrinkage, chromatin condensation, inter-nucleosomal cleavage of DNA, externalization of PS on the membrane surface and the ‘blebbing’ off of apoptotic bodies which consist of membrane-enclosed particles containing intact organelles. PS acts as an ‘eat me’ signal that directs phagocytes and neighbouring cells to efficiently phagocytose the apoptotic bodies and thus avoid inducing an inflammatory response (Savill & Fadok 2000). There are several other molecules that also act as ‘eat me’ signals, these include apolipoprotein J (apoJ)/clusterin, surface sugars and complement binding proteins (Bartl et al. 2001, Gardai et al. 2005, Botto et al. 1998). Necrosis on the other hand is often caused by acute trauma and is characterized by cell swelling and rupture of the plasma membrane, resulting in the release of noxious cellular material and induction of inflammation in the surrounding tissue.

Apoptosis plays a vital role in removing damaged, infected and unnecessary cells in the adult organism. It is also essential for the pruning of superfluous cells during development, for example, removing the webbing between fingers and the removal of excess neurons in the CNS (Yuan & Yankner 2000). Dysfunction in the regulation of apoptosis can result in major pathological consequences. An excess of apoptosis has been associated with diseases such as AD, PD, Huntington’s Disease, ischaemic damage and AIDS, while insufficient apoptosis can lead to cancer and autoimmune disorders (Cotman et al. 1994, Cribbs et al. 2004, Matsui et al. 2006, Yuan & Yankner

2000, Vogelstein & Kinzler 2004).

Apoptosis can be induced via extrinsic or intrinsic pathways, which activate a cascade of events culminating in cell death, predominantly mediated through caspase proteins

(Figure 5). The extrinsic pathway is stimulated by cell surface receptors, known as death receptors, belonging to the tumour necrosis factor receptor (TNFR) superfamily, of which Fas and TNFR1 are the best-characterised (Ashkenazi & Dixit 1998, Chen &

Goeddel 2002, Wajant 2002). Ligand binding with these receptors results in activation of intracellular domains termed death domains (DD) and death effector domains (DED), triggering a cascade of intracellular events. Intracellular adaptor proteins are also recruited to enhance this effect. The extrinsic pathway is used predominantly by the immune system to control populations of activated T lymphocytes after an immune response and destroy virus-infected and cancerous cells (Janeway 1999). The intrinsic pathway is stimulated by the disruption of intracellular homeostasis from sources such as; radiation, DNA damage, lysosomal rupture, oxidative stress, growth factor withdrawal and toxic chemicals. These stressors induce mitochondrial damage which then triggers the apoptotic cascade (Brunk et al. 1997, Antunes et al. 2001, Green &

Reed 1998) (Zhao et al. 2003) (Erdal et al. 2005).

Apoptosis is mediated primarily by the caspase family of proteins which are cysteine proteases that cleave protein substrates following aspartate residues (Thornberry &

Lazebnik 1998, Cohen 1997). They are all synthesized as inactive proenzymes

(procaspases) that become activated after cleavage at a conserved aspartate residue, resulting in the release of an inhibitory N-terminal prodomain. In humans there are 11 members of the caspase family and they fall into three major categories, cytokine

EXTRINSIC Apoptotic PATHWAY stimuli Death Ligand Receptor INTRINSIC PATHWAY Death Domain Mitochondria

Bcl-2 Adaptor protein Cyt c Caspase-8 NF-B Apaf1 Smac/DIABLO

Caspase-9 IAP

Caspases-3,6,7

AIF Endo G

APOPTOSIS

Nucleus

Figure 5.

Schematic overview depicting the key features of apoptosis. In the extrinsic pathway, the binding of ligand to cell-surface death receptors activates intracellular adaptor proteins which then trigger the caspase cascade. The intrinsic pathway is activated by a range of apoptotic stimuli (eg. radiation, oxidative stress, growth factor withdrawal) that can disrupt intracellular homeostasis and induce mitochondrial damage. Pro- apoptotic factors released into the cytosol from damaged mitochondia can either activate the caspase cascade (cyt c), directly induce DNA fragmentation (AIF, Endo

G) or block other anti-apoptotic proteins such as IAP (Smac/DIABLO). There are also many other proteins that can promote or repress the apoptotic cascade, with Bcl-2 and

NFB shown as examples.

23 activators, apoptosis executioners and apoptosis initiators. Caspases 1, 4, 5 and 13 activate cytokines and play a role in inflammation, with only a minimal role in apoptosis. The apoptosis executioner caspases (caspases 3, 6 and 7) mediate the morphological changes characteristic of apoptosis by cleaving a wide range of cellular substrates, of which over 250 are known (Fischer et al. 2003). Two well studied examples of this include; cleavage of the microtubule-associated protein gelsolin, facilitating cytoplasmic contraction and blebbing of apoptotic bodies (Kothakota et al.

1997); and cleavage of the DNA repair and genome integrity protein poly (ADP-ribose) polymerase (PARP), allowing DNA fragmentation (Nicholson 1996). The executioner caspases also cleave the aspartate residue present within procaspases, thus caspases can activate each other in a cascade-like fashion.

Initiator caspases (caspases-2, 8, 9 and 10) are responsible for activating the executioner caspases and thus triggering the caspase cascade. Initiator caspases are themselves activated by signals from the extrinsic and intrinsic apoptotic pathways. In the extrinsic pathway, procaspase-8 is recruited to the activated DD and DED becoming activated and proceeding to activate executioner caspases (Boldin et al. 1996, Chinnaiyan et al.

1995, Muzio et al. 1998). In the intrinsic pathway cytochrome c (cyt c) is released from the mitochondria, activating protease-activating factor 1 (Apaf1) which then binds several procaspase-9 proteins to the form the apoptosome complex whereby caspase-9 becomes activated, thus activating the executioner caspases (Muzio et al. 1998).

The regulation of apoptosis is also modulated by several other proteins. Some well studied examples include; Bcl-2 which blocks the release of cyt c from the mitochondria, inhibitor of apoptosis proteins (IAP’s) that directly inhibit activated

24 caspases and Smac/DIABLO which is released from the mitochondria to bind and inhibit IAP’s (Fadeel et al. 1999, Deveraux et al. 1997, Du et al. 2000, Verhagen et al.

2000). Additionally, apoptotic stimuli can activate the transcription factor NF-B, targeting it to the nucleus where it induces the upregulation of multiple anti-apoptotic genes (Karin & Lin 2002). Also, apoptosis can occur independently from caspase activation, whereby apoptosis inducing factor (AIF) and endonuclease G (Endo G) are released from the mitochondria and migrate directly to the nucleus, resulting in chromatin condensation and DNA fragmentation (Susin et al. 1999, Li et al. 2001).

This however is less common.

1.3.2 A role of apoE in apoptosis?

Recent studies have identified an association between apoE and apoptosis. ApoE is upregulated during the induction of apoptosis in human fibroblasts (Quinn et al. 2004) and THP-1 macrophages (Tedla et al. 2004). In addition, cellular apoE expression is increased following ischemic injury, peripheral nerve crush injury and kainic acid treatment and it is possible that at least some of the cells expressing apoE during these injurious conditions may be apoptotic (Ignatius et al. 1986, Boschert et al. 1999)

(Snipes et al. 1986, Hall et al. 1995, Kida et al. 1995, Horsburgh & Nicoll 1996)

(Ishimaru et al. 1996, Ali et al. 1996, Kitagawa et al. 2001).

The exact function that apoE performs in apoptosis is currently unclear, but recent studies suggest that apoE may inhibit apoptosis (DeKroon et al. 2003, Chen et al.

2005). Interestingly, Chen et al., have shown that apoE is highly expressed in malignant ovarian carcinoma cells, whilst being undetectable in benign counterparts (Chen et al.

2005). When siRNA was used to inhibit apoE expression in OVCAR3 cells, cell cycle

25 arrest and apoptosis was induced (Chen et al. 2005). Additional comparisons of gene expression in cancerous and healthy tissues (using the Serial Analysis of Gene

Expression databases and proteomics approaches) revealed that apoE expression is increased in several types of cancer, including breast, pancreatic, stomach, colon, prostate and liver (Hough et al. 2000, Yokoyama et al. 2006) . Also, the promoter region for the APOE gene contains specific response elements for the anti-apoptotic transcription factor NF-B (Du et al. 2005). These studies implicate a potential role for apoE in the inhibition of apoptosis in general; however, direct evidence for this is lacking and this issue therefore remains to be resolved. Other studies have provided evidence that apoE may facilitate the phagocytosis of apoptotic bodies by macrophages

(Grainger et al. 2004). Macrophages from apoE-/- mice are impaired in their ability to ingest apoptotic cells and there is a systemic increase in uncleared apoptotic bodies in these mice (Grainger et al. 2004).

1.4 ROLE OF apoE IN NEUROBIOLOGY

1.4.1 CNS lipid transport

The transport of lipids throughout the CNS is carried out predominantly by specialised

HDL-like lipoproteins, unlike the rest of the body where a broad range of lipoprotein densities are utilised (Pitas et al. 1987b, Borghini et al. 1995). ApoE is the most abundant apolipoprotein in the CNS, followed by apoAI and to a lesser extent apoA-II, apoJ and apoA-I (Ladu et al. 2000, Montine et al. 1998, Thomas et al. 2003). There is virtually no exchange of apoE or cholesterol between the CNS and the periphery, due partly to the impermeable nature of the blood brain barrier (BBB) (Bjorkhem &

Meaney 2004). Studies on liver transplant recipients revealed that apoE present in the

26 plasma adopts the same isoform as that of the donor liver, whereas the isoform of brain apoE remains unchanged (Linton et al. 1991). Thus, apoE containing lipoprotein particles and cholesterol are almost entirely synthesized in the CNS and are responsible for both the delivery and removal of cholesterol from cells within the CNS (Rebeck et al. 1998). The majority of CNS apoE is expressed by astrocytes and secreted as a nascent lipoprotein (Elshourbagy et al. 1985, Pitas et al. 1987a, Fagan et al. 1999).

Lipids and cholesterol are transferred to apoE via the membrane bound lipid transport proteins ABCA1 and ABCG1 which are expressed on astrocytes, microglia and neurons

(Wahrle et al. 2004, Karten et al. 2006, Kim et al. 2007). The transport of lipids and cholesterol to cells within the CNS is then mediated by the interaction of apoE with cell surface receptors, analogous to lipid transport in the periphery. Several apoE binding receptors are present in the CNS; neurons express LDLR, LRP, ApoER2 and VLDLR; microglia express VLDLR and LRP; astrocytes express LDLR and LRP (reviewed in

(Rebeck et al. 2006)). Lipoproteins are secreted into the CSF via the choriod plexus

(Strazielle & Ghersi-Egea 2000) and approximately 20% of apoE in the CSF, from

E3/E3 individuals, is present as a disulphide linked apoE homodimer or apoE-apoA-II heterodimer (Rebeck et al. 1998).

1.4.2 Cognitive function

The importance of apoE in brain function is highlighted by mouse studies where APOE knockout results in memory deficits and decreased synaptic density (Masliah et al.

1995, Gordon et al. 1995, Gordon et al. 1996). Targeted replacement with the human 3 allele, but not 4, ameliorates these deficits indicating that functional differences exist between apoE isoforms in the brain (Raber et al. 1998).

1.4.3 Role in nerve regeneration and repair

As mentioned previously (in macrophage biology section), monocytes and macrophages accumulate in regions of cellular injury and are responsible for clearance of cellular debris and redistribution of lipids to recovering cells via an apoE mediated process. A well documented observation of this phenomenon is seen in the peripheral rat sciatic nerve following injury by crush or transection (Mahley 1988, Ignatius et al. 1986,

Snipes et al. 1986). Macrophages converge to the site of injury and secrete large amounts of apoE, attaining levels of up to 5% of total soluble protein in the nerve sheath. Cholesterol and lipids scavenged from cellular and myelin debris accumulate within macrophages to be later released in apoE-lipid complexes for redistribution to injured and regenerating neurons. The production of apoE peaks after ~8 days then gradually returns to baseline levels after 8 weeks when regeneration is complete. ApoE delivers lipids to neurons via surface LDL receptors resulting in a ~100 to ~200 fold increase in neuronal intracellular apoE levels. The injured neuron regenerates its axon by sprouting neurites which migrate down the neurolemmal tube. The tips of these neurites are particularly enriched in LDL receptors. Interestingly, the presence of apoE4 is associated with inhibited neurite outgrowth in the peripheral nervous system (Nathan et al. 1994, Nathan et al. 1995) and a similar phenomenon most likely occurs with injured neurons in the CNS. APOE knockout in mice is associated with impaired clearance of cholesterol rich axonal debris following entorhinal cortex lesion (Fagan et al. 1998) and impaired recovery following closed head injury (Chen et al. 1997).

1.4.4 Neuronal expression of apoE

Although it has been known for quite some time that apoE can be present in neurons and accumulate following injury, it was believed that this was derived from non-

28 neuronal cells, chiefly macrophages in the periphery and astrocytes in the CNS (Boyles et al. 1985, Poirier et al. 1991, Diedrich et al. 1991, Zarow & Victoroff 1998).

However, more recently it has become clear that CNS neurons are capable of synthesizing apoE and that this is typically confined to the frontal cortex and hippocampal regions of the brain (Diedrich et al. 1991, Xu et al. 1999a, Xu et al. 1998,

Xu et al. 1999b, Han et al. 1994b, Bao et al. 1996, Harris et al. 2004, Metzger et al.

1996). Interestingly, neuronal specific regulatory elements have been identified in the

APOE gene proximal promoter (Ramos et al. 2005). Neuronal expression of apoE is increased in response to stressful stimuli such as cerebral infarction (Aoki et al. 2003), hypoxia and excitotoxicity induced by kainic acid or glutamate (Boschert et al. 1999,

Xu et al. 2006). CNS neurons of APOE knockout mice are more susceptible to damage induced by close head injury and focal cerebral ischemia (Chen et al. 1997, Laskowitz et al. 1997), and apoE possesses neuroprotective properties which include enhanced regeneration of synapses (analogous to apoE in peripheral nerves), immunomodulation and anti-oxidant activity (Kitagawa et al. 2002b, Kitagawa et al. 2002a, Miyata &

Smith 1996). Thus it is possible that neuronal expression of apoE promotes the survival of neurons in response to specific stressful stimuli.

1.5 ApoE AND ALZHEIMER’S DISEASE

1.5.1 Alzheimer’s disease background

Alzheimer’s disease is a progressive neurodegenerative illness that leads to dementia and death. The risk of developing AD increases with age and is the most common form of dementia, accounting for approximately 60% of all dementia cases (Blennow et al.

2006). There are currently ~27 million people suffering from AD worldwide and, due to increases in human longevity and the ageing population, this figure is estimated to leap to ~107 million by the year 2050 (Brookmeyer R 2007). Thus AD poses a major problem to society in terms of personal suffering and financial burden. Unfortunately there are currently no treatments available to prevent or slow the progression of this debilitating illness.

In 1906 Dr Alois Alzheimer was the first person to recognise the unique behavioral and pathological characteristics that distinguish AD from other forms of dementia.

Behavioural symptoms typically begin with deterioration of short term memory whilst other cognitive faculties remain relatively intact. This progressively worsens and is accompanied by a loss of executive function (decision making and planning), language skills, social skills, motor function and culminates in the patient requiring constant support until death (Waldemar et al. 2007). These symptoms are caused by degeneration and atrophy of brain regions involved with performing these mental activities, commencing in the hippocampus, entorhinal cortex and temporal cortex, followed by the frontal cortex and eventually most of the cerebral hemisphere (Figure

6) (Braak et al. 1998). Atrophy is accompanied with severe neuronal cell loss, synaptic dysfunction, inflammation, microglial activation, oxidative stress and large deposits of extracellular amyloid plaques and intracellular neurofibrillary tangles. The

Figure 6.

Diagrams illustrate the progressive spread of amyloid plaque and NFT associated degeneration (represented by dark blue shading) in the cerebral hemisphere during

AD. (Adapted from the following source: www.alz.org).

31 accumulation of plaques and tangles are the defining pathological features of AD when compared to other forms of dementia and is the benchmark by which a diagnosis of AD is confirmed post-mortem (Mirra et al. 1991).

1.5.2 Generation of amyloid plaques

Amyloid plaques are composed of the amyloid beta (A) peptide which is derived from the amyloid precursor protein (APP) (Kang et al. 1987, Masters et al. 1985). APP is a transmembrane protein expressed as one of 3 splice variants; 695, 751 or 770aa. All variants are expressed in both neuronal and non-neuronal cells throughout the body, however, the 695aa variant is predominantly expressed in neurons (Haass et al. 1991,

Selkoe et al. 1988). APP is proteolytically cleaved by membrane associated enzyme complexes at three locations termed the , and secretase cleavage sites (Figure 7).

Cleavage of APP follows one of two proteolytic pathways, the major one being the non- amyloidogenic pathway and the minor one, the amyloidogenic pathway (Busciglio et al.

1993, Esch et al. 1990, Shoji et al. 1992, Sisodia et al. 1990). In the non-amyloidogenic pathway APP is first cleaved at the -secretase site by the enzyme complex ADAM10 and then at the intra-membrane -secretase site by a protease complex containing presenilin (PS) 1 and 2 (Wolfe et al. 1999, Steiner et al. 2002). This results in the production of a non-toxic peptide fragment, sAPP. While in the amyloidogenic pathway APP is first cleaved at the -secretase site by -site amyloid precursor protein cleavage enzyme 1 (BACE1) and then at the -secretase site to produce a non-toxic extracellular protein, sAPP, and the neurotoxic A peptides (Vassar et al. 1999).

Amyloidogenic cleavage of APP is enhanced within regions of the membrane known as lipid raft domains that are enriched in cholesterol, glycosphingolipids (GSL) and sphingomyelins (SM) (Hooper 2005). Two major species of A, either 40 (A-40) or 42

Figure 7.

Overview of APP processing. In the non-amyloidogenic pathway APP is cleaved at the secretase site by ADAM to produce sAPP whereas in the amyloidogenic pathway APP is cleaved at the and secretase sites by BACE and presenilins (PS), respectively, to produce sAPP and neurotoxic A peptides. Amyloidogenic processing is favoured within lipid raft microdomains. Abbreviations are explained in the text.

33 (A-42) residues in length, are produced as a consequence of alternate cleavage at the

-secretase site (Verdile et al. 2007, Beel & Sanders 2008). However, cleavage to produce A-40 is typically favoured ~95% of the time.

The mechanism by which A monomers form A plaques is not entirely understood, however, there is evidence to suggest it is a sequential process with several intermediate steps; firstly, A monomers form oligomers, followed by protofibrils, fibrils and finally plaques (Podlisny et al. 1995) (Thal et al. 2006) (Finder & Glockshuber 2007). A-42 is considered the pathological species of A as it is more hydrophobic and thus more prone to aggregate into soluble oligomers (Jarrett et al. 1993). A-40 also becomes associated with late-stage plaques, though to a lesser extent (Iwatsubo et al. 1994).

Possible mechanisms involved in the removal of monomeric A from the brain include degradation by insulin degrading enzyme (IDE), neprliysin (NEP) and endothelial converting enzyme (reviewed in (Bates et al. 2008, Carson & Turner 2002)). Also, monomeric A may be effluxed across the BBB into the peripheral circulation with evidence suggesting that LRP1 and LRP2 facilitate the uptake of cerebral-interstitial A by cells at the BBB (Shibata et al. 2000) (Bell et al. 2007).

Amyloid plaques in the AD brain are associated with regional inflammation, neuronal cell death and neuronal dysfunction. Inflammation is characterized by microglial activation, astrocytosis, increased levels of proinflammatory cytokines and complement proteins of the innate immune system (McGeer & McGeer 1995, Rogers et al. 1992).

Neurons suffer from neuritic injury, disrupted calcium homeostasis, oxidative damage, disrupted synaptic function and apoptotic cell death (Mattson et al. 1992, Behl et al.

1994, Harris et al. 1995, Cotman et al. 1994, Matsui et al. 2006) (Selkoe 2002).

Insoluble amyloid plaques were initially believed to be responsible for mediating these toxic effects, however, a growing body of evidence now suggests that intermediate soluble A oligomers may be the culprits (Hsia et al. 1999, Mucke et al. 2000, Buttini et al. 2002, Walsh et al. 2002, Hartley et al. 1999, Lambert et al. 1998). Thus, insoluble amyloid plaques, the major pathological hallmark of AD, most likely represent an end- product of the disease process as opposed to a causative agent.

1.5.3 Alzheimer’s disease risk factors

1.5.3.1 Early-Onset Alzheimer’s Disease

Early-onset Alzheimer’s Disease (EOAD) is defined as AD that manifests in patients who are younger than 65 years of age (Selkoe 2001, Blennow et al. 2006). It represents

~5-10% of all AD cases and is highly heritable in an autosomal dominant fashion.

EOAD is typically caused by mutations within genes that encode for components of the

APP processing pathway. This results in an increased amount of A-42 being produced, either by a shift towards the amyloidogenic pathway, or, an increase in the ratio of A-

42 to A-40. The majority of identified EOAD mutations occur in the presenelin 1 and

2 genes (PS1 and PS2) and the , and secretase cleavage sites within the APP gene

(APP) (Goate et al. 1991, Schellenberg et al. 1992, Sherrington et al. 1995, St George-

Hyslop & Petit 2005).

1.5.3.2 Late-Onset Alzheimer’s Disease

Late-onset Alzheimer’s Disease (LOAD), also known as sporadic AD, is the most common form of AD, representing ~90-95% of all cases. Onset occurs in people aged

65 years and older with the incidence increasing exponentially with age (Selkoe 2001,

Blennow et al. 2006). Besides the most obvious risk factor, increased age, there is

35 evidence suggesting that several other environmental and lifestyle factors may influence risk, these include; diet, exercise, alcohol consumption, hypertension, diabetes, history of head injury, chronic dental infection, educational attainment and infection with HSV

(Lahiri 2006, Itzhaki et al. 1997, Mayeux 2003, Mortimer et al. 2003, Jellinger 2004,

Luchsinger & Mayeux 2004b, Luchsinger & Mayeux 2004a, Kamer et al. 2008). The only major genetic risk factor associated with LOAD is possession of the APOE 4 allele. 4 increases LOAD risk in a dose-dependent manner whereby carriers of one or two copies of the 4 allele have a ~5 or ~10 fold increase, respectively, in risk of developing AD (Corder et al. 1993, Strittmatter et al. 1993a). Therefore, while the frequency of 4 in the general population is ~15%, in AD populations it is ~55%

(Figure 8). In contrast the 2 allele is associated with decreased AD risk (Roses et al.

1995). Despite intense research the exact mechanism by which an alteration in one residue, Cysteine Arginine at 112, of 4 confers this increased risk is yet to be elucidated. However, apoE has been found to function in an isoform dependent manner in several biological processes involved in AD pathogenesis.

1.5.4 ApoE in the Alzheimer’s Disease brain

1.5.4.1 ApoE levels in the AD brain

Several studies have suggested that the levels of apoE protein and mRNA are increased in the AD brain, however, the level of apoE in individual samples overlaps considerably between AD and control (Yamada et al. 1995) (Yamagata et al. 2001) (Thomas et al.

2003, Laws et al. 2002). The question of whether brain apoE levels are further influenced by isoform remains unclear with different studies associating apoE4 with either increased (Thomas et al. 2003, Fukumoto et al. 2003) (Yamagata et al. 2001),

Figure 8.

Graphical representation of APOE allele frequencies in the general population and the

AD patient population. The APOE 4 allele is present at a much greater frequency in the AD patient population.

37 decreased (Bertrand et al. 1995) (Beffert et al. 1999b) (Glockner et al. 2002)

(Ramaswamy et al. 2005) (Riddell et al. 2008) or unchanged (Sullivan et al. 2004,

Fryer et al. 2005) apoE levels.

1.5.4.2 Interactions with amyloid beta

The binding of apoE to A was first discovered by studies demonstrating co- localisation of apoE with amyolid plaques in the AD brain (Strittmatter et al. 1993a,

Beyreuther & Masters 1991, Namba et al. 1991, Wisniewski & Frangione 1992). This occurs primarily through interaction with the LBD of apoE (residues 244-272)

(Strittmatter et al. 1993b). Initial in vitro studies using lipid-free apoE suggested that apoE4 binds A with a higher avidity than apoE3 (Strittmatter et al. 1993b). However, further studies using lipid-associated apoE, which most likely represents a more physiologically relevant model, found that apoE3 binds A with ~20 fold greater avidity than apoE4 (LaDu et al. 1994). Also, the disulphide linked apoE/apoE and apoE/apoA-II dimers, formed by apoE2 or apoE3 but not apoE4, bind A with a greater avidity than apoE monomer (Aleshkov et al. 1997, Yamauchi et al. 1999, Yamauchi et al. 2000). It is hypothesised that isoform differences in A binding may be related to isoform differences in the ability of apoE to modulate A clearance and toxicity in the brain (Jordan et al. 1998, Yamauchi et al. 2000). Total A levels are greater in AD patients who carry the 4 allele (Schmechel et al. 1993). Also, A levels are acutely increased following traumatic brain injury (TBI) with this effect being much greater in

4 allele carriers (Nicoll et al. 1995).

ApoE plays a role in enhancing the clearance and degradation of A in the brain. A recent study demonstrated that apoE enhances the extracellular degradation of A by

IDE and the intracellular degradation by NEP in microglial cells. This effect was dependent on lipidation of apoE and apoE4 was less effective than apoE2 and apoE3

(Jiang et al. 2008) (Figure 9). Also, the binding of apoE to A forms a complex that can interact with apoE receptors, resulting in endocytosis and removal across the BBB or intracellular degradation in microglia and astrocytes. ApoE isoform can influence this process with apoE4 generally being the least effective (Gylys et al. 2003, Rebeck et al. 1993, Urmoneit et al. 1997, Beffert et al. 1999a, Cole & Ard 2000, Yang et al. 1999,

Koistinaho et al. 2004, Deane et al. 2008).

Independent from the clearance of A, apoE can also reduce the detrimental effects of

A, with apoE4 being the least effective. This has been demonstrated in several in vitro studies focused on the impact of A on neuronal survival, synaptic dysfunction, free radical damage and pro-inflammatory activation of microglia (Manelli et al. 2004, Ji et al. 2002, Ji et al. 2006, Trommer et al. 2005, Lauderback et al. 2002, Miyata & Smith

1996).

In tg mouse models of AD, expression of mutant hAPP results in intense A deposition which, interestingly, is reduced on an apoE-/- background (Bales et al. 1999). This indicates that murine apoE facilitates A plaque formation. In stark contrast, the addition of human apoE isoforms to this mutant hAPP/apoE-/- model results in a further dealy in A deposition (apoE2 > apoE3 > apoE4) (Holtzman et al. 1999) (Holtzman et al. 2000) (Fagan et al. 2002). The cellular mechanism(s) underlying these effects, and the observed inter-species difference, are not entirely understood, however, these findings highlight the influential role apoE plays in modulating A deposition.

Figure 9.

Illustration summarises the role of lipidated apoE in the degradation of soluble A.

Nascent-free apoE secreted from astrocytes and microglia is lipidated by ABCA1.

The activation of LXR upregulates expression of both apoE and ABCA1, leading to increased levels of lipidated apoE. Lipidated apoE promotes the degradation of extracellular A by IDE. Also, apoE and A can be engulfed by microglia, via pinocytosis, where lipidated apoE enhances the degradation of A by NEP in late endosomes/lysosomes. (Reproduced from Jiang et al., 2008).

40 1.5.4.3 Role of apoE in Neurofibrillary Tangle formation

In addition to amyloid plaques, neurofibrillary tangles (NFT) are a major pathological hallmark of AD found intracellularly within neurons in brain regions affected by AD.

NFT consist of bundles known as paired helical filaments (PHF) which in turn are composed of the microtubule associated protein tau (Fig. 16) (Kosik et al. 1986, Nukina

& Ihara 1986, Wood et al. 1986, Wischik et al. 1988, Lee et al. 1991). Studies have demonstrated that tau binds and stabilizes microtubules, however, when tau is phosphorylated it becomes disassociated from microtubules and consequently more likely to aggregate and form PHF (Grundke-Iqbal et al. 1986, Patrick et al. 1999). This is believed to be the reason why NFT are associated with microtubule instability and neurotoxicity (Lewis et al. 2000, Rapoport et al. 2002). In vitro studies have shown that apoE3, but not apoE4, can bind to tau and promote microtubule stability (Strittmatter et al. 1994, Nathan et al. 1995, Roses et al. 1996). In vivo, apoE must enter the cytoplasm in order to interact with tau. This has been reported to occur in human neurons (Han et al. 1994a) (Roses et al. 1996), however, the underlying mechanism is not known. It is hypothesised that apoE3 binding to tau helps prevent phoshporylation thereby promoting tau microtubule association and therefore microtubule stability. The inability of apoE4 to perform this function is believed to be the underlying reason why E4 is unable to promote neurite outgrowth following nerve injury (Nathan et al. 1995).

Furthermore, the absence of Apoe in the mouse brain results in microtubule deficiency and hyperphosphorylation of tau (Masliah et al. 1995, Gordon et al. 1996).

1.5.4.4 Proteolysis of apoE

It has been recognised that apoE undergoes proteolytic cleavage in the brain to form truncated fragments, some of which preferentially associate with NFT and amyloid

41 plaques (Aizawa et al. 1997, Cho et al. 2001, Huang et al. 2001, Harris et al. 2003).

Studies by Huang and colleagues suggested apoE3 and apoE4 are both cleaved in the brain to form a carboxy terminal truncated ~29 to 30 kDa fragment and that the relative amount of this fragment is increased in AD (Huang et al. 2001). A quantitatively minor group of fragments in the range of ~14 to 20 kDa was also detected but only in AD subjects expressing the apoE4 isoform (Huang et al. 2001). This same group performed detailed studies in transgenic mice which indicated that neuronal expression of human apoE3 was accompanied predominantly by formation of the ~29 kDa fragment in the brain, whereas expression of apoE4 was accompanied by the lower molecular weight fragments (Brecht et al. 2004). Importantly, transgenic expression of these forms of apoE in astrocytes (the principal source of apoE in the human brain) did not result in detectable apoE fragmentation in mouse brain (Brecht et al. 2004). Similarly, transgenic expression of human apoE3 or apoE4 under the control of the endogenous mouse apoE promoter did not result in detectable apoE fragmentation in the brains of young (12 to

20 weeks old) mice (Riddell et al. 2008). Thus, in summary, there is currently no general consensus on whether the cleavage of apoE is influenced by factors such as isoform and AD.

The key proteolytic enzyme(s) generating apoE fragments in vivo is still not known, although different potential candidates have been identified with some studies indicating a chymotrypsin-like serine protease (Harris et al. 2003) and others an aspartic protease such as cathepsin-D (CatD) (Marques et al. 2004, Zhou et al. 2006). Other plausible enzymes include thrombin and the matrix metalloproteinase MMP-14 as they both proteolyse apoE, generating fragments ranging from 28 to 10 kDa in size, and are present in the brain (Castano et al. 1995, Aizawa et al. 1997); (Tolar et al. 1997,

Smirnova et al. 1997, Mhatre et al. 2004, Arai et al. 2006, Park et al. 2008, Candelario-

Jalil et al. 2008, Toft-Hansen et al. 2007).

The amino acid sequences of these fragments have not been characterized and their biological roles are not yet established. However, it is possible they are playing a physiological role in the brain as several studies have demonstrated that truncated apoE

(and apoE-mimetic peptides) exert potent bioactive properties that regulate neuronal and glial signaling (Gay et al. 2007, Hoe et al. 2005) and (depending on the fragments analysed) may promote neurodegeneration (Crutcher et al. 1994, Clay et al. 1995, Tolar et al. 1999, Tolar et al. 1997, Huang et al. 2001, Chang et al. 2005, Wellnitz et al.

2005) or stimulate neuroprotective and anti-inflammatory pathways (Aono et al. 2003,

Laskowitz et al. 2001, Li et al. 2006, Singh et al. 2008, Lynch et al. 2003, Lynch et al.

2005). These properties are mediated by interaction with cell surface receptors including members of the LDL receptor family and the alpha-7 nicotinic acetylcholine receptor.

2. AIMS OF THIS THESIS

The overall purpose of my thesis was to investigate novel biological properties and functions of apoE, with the following aims addressed:

- To investigate a potential expression of apoE in neuronal apoptosis.

- To investigate a possible anti-apoptotic role of apoE.

- To investigate the mechanisms and characteristics of apoE nuclear localization.

- To investigate isoform specific generation of apoE fragments in the human brain.

3. RESULTS

3.1 RESULTS SUMMARY

Many novel aspects of apoE biology were uncovered during my candidature.

Publications-I and II were focused on the recently observed association between apoE and apoptosis. In Publication-I, the human SKNSH neuronal cell line was used to investigate potential changes in apoE expression during apoptotsis which occurs as a consequence of extended culture (up to 5 days) without replenishing trophic factors.

After 3 days, apoE mRNA and protein levels were significantly increased by 6- and 8- fold, respectively, and this correlated with increased caspase-3 activation, TUNEL positivity and the formation of apoptotic bodies. Furthermore, analysis of cellular debris that accumulated in the culture supernatants indicated that apoE levels became progressively concentrated in apoptotic bodies, consistent with the possibility that apoE may play a role in the clearance of apoptotic bodies through apoE-receptor interactions.

A previous study by Chen et al., (Chen et al. 2005) had suggested that apoE expression may be associated with apoptosis resistance and this possibility was further examined in

Publication-II. Macrophages were used to address this issue as they constitutively express apoE and are relatively resilient to apoptosis. Using siRNA, human monocyte derived macrophage (hMDM) apoE mRNA and protein was reduced by 97% and 61%, respectively. ApoE knockdown significantly increased staurosporine-induced caspase-3 activation by 78% without altering cell survival or apoptosis as assessed by TUNEL analysis and morphological changes. Thus, although there was an increase in activation of one of the major executioners of the apoptotic cascade, caspase-3, this did not lead to an increase in apoptosis, as assessed by final-stage markers of this process (eg. TUNEL and morphological changes).This result was confirmed using murine bone marrow

45 derived macrophages (mBMDM) from apoE-/- and WT mice. In these experiments, staurosporine-induced caspase-3 activation was significantly increased by 49% in apoE-

/- mBMDM and this was not associated with differences in TUNEL signal, annexin-V binding or DNA fragmentation.

The localization of apoE to the nucleus is a largely unexplored aspect of apoE biology, with previous data indicating that it may be increased during times of cellular stress. In publication-III the factors associated with apoE nuclear localization were examined using live-cell imaging of CHO (Chinese-hamster ovary) cells that constitutively expressed apoE-GFP (green fluorescent protein). A small proportion of apoE-GFP was found to be associated with the nucleus in living cells and this was increased with serum starvation. The potential nuclear targeting of exogenous apoE was investigated using biotinylated apoE in either a lipid-free state or as part of a lipidated discoidal complex. Exogenous apoE localised to the nucleus but only when it was applied to cells as part of a lipidated complex and this was observed equally for all three apoE isoforms.

ApoE displayed a distinctly punctuate pattern of localization within the nucleus. I clearly demonstrate that nuclear apoE is not associated with nucleoli or the nuclear sub- structures interchromatin granule clusters/nuclear speckles.

Structural differences between the apoE3 and apoE4 isoforms in the human brain were investigated in publication-IV. Human hippocampus, frontal cortex (gray and white matter), occipital lobe and cerebellum samples were homogenized into fractions that were soluble in Tris-buffered saline (TBS), Triton X-100 or guanidine hydrochloride and analysed by apoE western blot. Approximately 20% of apoE in the apoE3 brains was detected as fragments which were predominantly in the TBS-soluble fractions. In

46 contrast the extent of apoE4 fragmentation was significantly lower and this pattern was consistently observed in all brain regions examined and was not influenced by AD status. The striking difference between apoE3 and apoE4 fragmentation in the TBS- soluble homogenate was also observed when hippocampal samples were homogenized directly in a detergent-rich buffer. The possibility that CatD may play a role in the cleavage of brain apoE was examined. In vitro cleavage of recombinant apoE with

CatD generated fragments that were similar to those detected in the human apoE3 brain, however, kinetic studies did not reveal apoE isoform-specific differences in proteolytic susceptibility.

Analysis of apoE using non-reducing conditions demonstrated that apoE3 disulphide- linked dimers are present in the brain. The possibility that this may be a post-mortem artefact is unlikely as dimers were also observed in rapidly processed SKNSH neuronal cells and rabbit brain.

3.2 PUBLICATIONS

3.2.1 Publication I

David A. Elliott, Woojin S. Kim, David A. Jans and Brett Garner. (2007) Apoptosis induces neuronal apolipoprotein-E synthesis and localization in apoptotic bodies.

Neuroscience Letters 416: 206-210.

Declaration

I certify that I have performed the majority of the research work and writing contained in the following publication and that reproduction in this thesis does not breach copyright regulations.

David Anthony Elliott

Neuroscience Letters 416 (2007) 206–210

Apoptosis induces neuronal apolipoprotein-E synthesis and localization in apoptotic bodies David A. Elliott a, Woojin S. Kim a, David A. Jans b, Brett Garner a,c,∗ a Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia b Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia c School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia Received 21 December 2006; received in revised form 1 February 2007; accepted 5 February 2007

Abstract Neuronal apoptosis is crucial for central nervous system development and also contributes to neurodegenerative disease. Apolipoprotein-E (apoE) regulates brain lipid transport and specific neuronal functions and previous research, investigating non-neuronal cell types, identified an association between apoptosis and increased apoE expression. In the present study we used the human SK-N-SH neuronal cell line to investigate potential changes in apoE expression during apoptosis which occurs as a consequence of extended culture (up to 5 days) without replenishing trophic factors. Standard and real-time PCR analysis indicated a significant 6-fold increase in apoE mRNA after 3 days which was correlated with caspase-3 activation, TUNEL positivity and the formation of apoptotic bodies. ApoE protein levels were low in the absence of apoptosis but increased by 8-fold when apoptosis was induced. Analysis of cellular debris that accumulated in the culture supernatants indicated that apoE levels became progressively concentrated in apoptotic bodies. These data indicate that apoE is up-regulated during neuronal apoptosis and raise the possibility that apoE may play a role in the clearance of apoptotic bodies through apoE–receptor interactions. © 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Apoptosis; Neurodegeneration; Caspase-3; Apolipoprotein-E; Apoptotic bodies

Apolipoprotein-E (apoE) plays a crucial role in brain lipid while the exact mechanisms remain unclear, there is evi- metabolism and neurobiology and exists in humans as one of dence that apoptosis contributes to neurodegenerative disease three common isoforms, E2, E3 or E4 [23,25]. The APOE4 geno- including AD [7,8,21,35]. Although previous studies have type is associated with increased risk of late-onset Alzheimer’s independently focused on either neuronal apoptosis or apoE disease (AD) [6]. ApoE is also an immunoregulator, a stabilis- expression, there is very little information regarding potential ing component of neuronal microtubules, and a component of alterations in apoE expression during neuronal apoptosis. amyloid plaques associated with AD [19,27]. In the brain, apoE To address this we utilised the widely studied human SK-N- is predominantly expressed by astrocytes; however, expression SH neuroblastoma cell line. Previous studies have shown that in neurons can be induced during nerve regeneration after injury a variety of toxic insults, including 1-trichloromethyl-1,2,3,4- and in growth and development of the CNS [11,13]. terahydro-␤-carboline [1], myristicin [17], staurosporine [22], We reported that apoE is transcriptionally induced in apop- thimerosal [12] and etorphine [34], all induce apoptosis in SK- totic ﬁbroblasts and is over-expressed in apoptotic macrophages N-SH neurons. While each of these pro-apoptotic stimuli utilise [24,29]. In both cases, apoE expression was correlated with distinct initiating signals, the activation of caspases and DNA caspase-3 activation and additional morphological and biochem- fragmentation (mostly assessed using the TUNEL assay) are ical markers of apoptosis. Interestingly, caspase-3 dependent common features of neuronal apoptosis reported in these reports. apoptosis plays a central role during CNS development and, Neuronal apoptosis can also be induced by the removal of trophic factors from culture medium (see [35]); thus prolonged (up to

∗ 5 days) culture of SK-N-SH neurons without supplementing or Corresponding author at: Prince of Wales Medical Research Institute, Barker replacing medium results in classic signs of apoptosis charac- Street, Randwick NSW 2031, Australia. Tel.: +61 2 939 91024; fax: +61 2 939 91005. terised by caspase-3 activation and DNA fragmentation [33]. E-mail address: [email protected] (B. Garner). We used this method to assess the possible induction of apoE

expression during neuronal apoptosis. Importantly, our results heat-inactivated at 95 ◦C for 10 min immediately prior to addi- indicate that apoE is up-regulated during neuronal apoptosis and tion to the cell lysate. Cell protein was measured by the BCA raise the possibility that apoE may play a role in the clearance method [24]. of apoptotic bodies through apoE-receptor interactions. Apoptotic debris (isolated as described above) was washed Cell culture media and additives were from Invitrogen (Mel- three times with PBS and incubated at 4 ◦C for 1 h in PBS con- bourne, Australia) or Sigma (Castle Hill, Australia). Human taining 1.0% (v/v) horse serum and rabbit anti-human apoE neuronal SK-N-SH cells were grown in DMEM, 10% FCS, polyclonal antibody diluted 1/1000, followed by further wash- 2 mM glutamine, and 100 IU/ml penicillin and 100 ␮g/ml strep- ing and a 30 min incubation at 4 ◦C with Alexa 568-conjugated tomycin. Cultures were routinely grown in 75 cm2 flasks at 37 ◦C goat anti-rabbit IgG antibody 1/1000 (Invitrogen). Apoptotic in 5% CO2 and plated into either 6-well or 12-well plates for debris was also treated with non-immune rabbit IgG (DAKO) use in experiments. as a negative control and this did not result in significant stain- A morphological and biochemical analysis of apoptosis in ing. The apoE immunostained apoptotic debris was examined SK-N-SH neurons cultured for up to 5 days without replenishing using a Nikon TE2000 microscope and a 60× oil objective. DIC media was previously published [33]. In the present study, apop- and epifluorescence (Ex 570 nm, Em 573–648 nm) images were tosis was assessed by measuring caspase-3-activity in cell lysates collected and the diameter of apoptotic bodies was determined after addition of 20 ␮M Ac-DEVD-7-amino-4-methylcoumarin using Image Pro Plus software (Media Cybernetics). (Pharmingen, San Diego, CA) and measurement of the liber- Experiments were performed in triplicate or quadruplicate ated fluorophore 7-amino-4-methylcoumarin (AMC) at Ex 380; and repeated at least twice. Significance was determined using Em 435 nm [24]. Apoptosis was also assessed using an apopto- the 2-tailed Student’s t-test for unpaired data and P < 0.05 con- sis ELISA kit based on the TUNEL (terminal deoxynucleotidyl sidered significant. transferase nick end labelling) technique following the manu- Five-day culture of SK-N-SH neurons without media replen- facturer’s instructions (Cell Death Detection ELISA-Plus kit; ishment induces apoptosis as assessed by caspase-3 activity Roche Applied Biosciences). and TUNEL [33]. In agreement with this, our time-course RNA extraction and standard and real-time PCR were as experiments showed that at t = 0 and at day 1, caspase-3 activ- described [15]. ApoE primers were 5-GTCGCTTTTGGGAT- ity and TUNEL signal remained low (Fig. 1). However, at TACCTGC-3 (forward), 5-CCGGGGTCAGTTGTTCCTC-3 day 3 and day 5, caspase-3 activity and TUNEL signal were (reverse). Amplification was carried out with 30 cycles of denat- increased (Fig. 1). This was accompanied by apoptotic mor- uration (94 ◦C, 30 s), annealing (60 ◦C, 30 s), and extension phological alterations including cellular contraction, nuclear (72 ◦C, 30 s). Product size was 162 bp. The level of expression condensation and membrane blebbing (data not shown). After was normalised using the housekeeping gene ␤-actin: 5- GAATTCTGGCCACGGCTGCTTCCAGCT-3 (forward), 5- AAGCTTTTTCGTGGATGCCACAGGACT-3(reverse). ApoE was detected by Western blotting as described [24]. Briefly, adherent cells from 6-well plates were lysed in 200 ␮l lysis buffer (10 mM Tris–HCl, 10 mM Na2PO4/NaHPO4,pH 7.5, 130 mM NaCl, 1% Triton-X-100, 10 mM NaPPi). Simi- larly,1.5 ml cell culture media (containing apoptotic debris) was collected and centrifuged at 16,100 × g at 4 ◦C for 5 min. The supernatants were removed and the pellet (where present) lysed in 200 ␮l lysis buffer. Samples were then mixed with Laemmli sample buffer, run on 10% PAGE gels and transferred to PVDF membranes. Protein loading and transfer efficiency were monitored by Ponceau S staining. The membranes were incubated for 16 h at 4 ◦C with a rabbit anti-human apoE polyclonal antibody (DAKO) diluted 1/1000, followed by a 1 h incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (DAKO). Blots were developed using enhanced chemiluminescence (Amersham Pharmacia), and the membranes were exposed to X-ray film (Fuji), developed, scanned and signal intensity quantified using NIH Image software [24]. ␮ Thrombin digestion was performed using 15 g of protein Fig. 1. Induction of apoptosis in SK-N-SH neurons. Apoptosis was induced by from apoptotic (day 3) cell lysates and incubating with 2 U of prolonged culture of neurons without media replenishment. Cells were seeded ◦ thrombin (Sigma) in PBS at 37 C for 16 h. Three control condi- in 12-well plates and protein (grey bars), caspase-3 activity (white bars) and tions were used to ensure that the protein fragmentation observed TUNEL signal (black bars) assessed at the times indicated (A). Caspase-3 and was due to thrombin-specific cleavage; cell lysates were either TUNEL data derived from panel “A” expressed after correction for cell protein ◦ (B). Data or means ± S.E. of at least 3 values and are representative of three stored at −20 C for the 16 h incubation period, Incubated at * ** *** ◦ experiments. P < 0.05, P < 0.01, P < 0.001; significantly different to “day 37 C with PBS only, or incubated with thrombin that had been 0”. 50 208 D.A. Elliott et al. / Neuroscience Letters 416 (2007) 206–210

the predicted 22 kDa N-terminal domain confirmed the identity of the major 35 kDa band as apoE (Fig. 3). Attempts to generate quantitative data for day 5 mRNA were not successful due to the variable quality of mRNA (both apoE and housekeeping genes) at this time point most likely as a result of apoptosis-related RNAase activation and concomitant RNA degradation [4].In contrast, apoE protein was clearly and consistently detectable at high levels at day 5 (see below). The data in Fig. 1A indicated that cell protein levels (recov- ered from cells remaining attached to the culture dish) decreased when apoptosis was induced. This decrease in cell protein was at least partially explained by the appearance of cellular debris particles/apoptotic bodies that were present in the culture medium (particularly at day 5, data not shown) that did not remain attached to the culture dish. In order to investigate whether apoE associates (preferentially or otherwise) with apoptotic bodies, medium from the neuronal cultures was collected at days 1, 3 and 5 and apoptotic bodies were purified by low speed centrifugation and apoE levels determined by Western blotting. Consistent with the data shown in Fig. 1, analysis of cell protein Fig. 2. Expression of apoE mRNA and protein in SK-N-SH neurons undergoing by Ponceau-S staining indicated reduced protein levels remain- apoptosis. Cells were cultured as described in Fig. 1 and apoE mRNA expression ing attached to the culture dishes at days 3 and 5 compared to determined by standard (A) and quantitative real-time PCR (B) at the times indicated. Data are representative of 3 values (A) or are means ± S.E. of 3 values day1(Fig. 4A). In contrast, protein derived from apoptotic bod- (B), representative of 4 experiments. ies was scarcely detectable at day 1 while robustly detected at days 3 and 5 (Fig. 4A). Western blot analysis confirmed that day 3, cellular protein levels declined whereas caspase-3 and neuronal apoE levels were significantly increased with apopto- TUNEL activities remained high (when corrected for cell pro- sis (by approximately 5-fold) and revealed that apoE was highly tein, Fig. 1). concentrated in day 5 neuronal apoptotic bodies (Fig. 4B and In order to assess the possible up-regulation of apoE during C). neuronal apoptosis parallel studies were conducted where cells Microscopic examination of immunostained apoptotic debris were harvested for measurement of apoE mRNA and protein. At indicated a heterogeneous population of membrane bound bod- t = 0 and at day 1, apoE mRNA was detected by both standard ies that varied in diameter from 2 to 18 ␮m(Fig. 5). While this and real-time PCR (Fig. 2A and B, respectively). ApoE protein size range is in general agreement with previous work [26],it at these early time points was detectable by Western blotting at is possible that the repeated washing steps during immunostain- levels that were close to the limits of sensitivity for this method ing reduced the recovery of apoptotic bodies < 2 ␮m diameter. It (see below); thus apoE mRNA and protein were detected albeit is clear though that apoE is an integral component of neuronal at low levels in non-apoptotic neurons. In contrast, at day 3, apoE apoptotic bodies and larger apoptotic cellular debris. was strongly expressed at the mRNA level and real-time PCR Previous work indicated that apoptosis was associated with analysis indicated that apoE mRNA levels increased more than increased apoE expression in fibroblasts and macrophages and 6-fold by day 3. Similarly, neuronal apoE protein was strongly that apoE overexpression may inhibit apoptosis in ovarian can- expressed by day 3 (Fig. 3). Several bands in addition to the cer cells [5,24,29]. Additional studies showed that macrophages major apoE band at ∼35 kDa were also detected by Western blot- from apoE null mice were impaired in their capacity to ingest ting and thrombin digestion of the major 35 kDa band to yield apoptotic cells [10]. Thus, there is an association of increased apoE expression with apoptosis in several cell types, although the precise role that apoE plays in the apoptotic pathway remains uncertain. The present data indicates that neurons also over-express apoE during apoptosis. The observed enrichment of apoE in apoptotic bodies is consistent with a potential role in apoptotic body clearance by phagocytic cells. The low density lipoprotein receptor related protein (LRP) utilises apoE as a ligand [3,31], is expressed in microglial cells [16,20] and plays a role in the clearance of apoptotic bodies [2,9,28]. It is therefore plausi- Fig. 3. ApoE protein identification confirmed by thrombin cleavage. Cells were ble that apoE may act in a manner analogous to calreticulin or cultured for 3 days as described in Fig. 1 and lysates assessed for apoE expression in the absence of thrombin or in the presence thrombin (Thr) or heat-inactivated apoJ bound to the surface of apoptotic bodies to activate LRP thrombin (HI-Thr) to confirm the identity of the major band at 35 kDa as apoE. on microglia to stimulate engulfment [2,9]. The uptake of apoE Data are representative of 2 experiments. enriched apoptotic bodies by LRP expressed on microglial cells 51 D.A. Elliott et al. / Neuroscience Letters 416 (2007) 206–210 209

Fig. 4. Expression of apoE protein in adherent SK-N-SH neurons undergoing apoptosis and in non-adherent apoptotic bodies. Cells were cultured as described in Fig. 1 and total protein levels in the adherent cells “C” and apoptotic bodies derived from media “M” were visualised by Ponceau-S staining (A) at the times indicated. The same blot shown in “A” was probed for apoE by Western blotting (B). The relative concentrations of apoE present in both the adherent cells (grey portion of bar) and the apoptotic bodies (black portion of bar) derived from the media samples was estimated by measuring the optical density of the blot Fig. 5. Characterization of apoptotic bodies after apoE immunostaining. Cells shown in “B” where the “day 1” sample is assigned a value of 1 (C). Data are were cultured as described in Fig. 1 and at day 5 apoptotic bodies derived from representative of 3 values, representative of 2 experiments. media were immunostained. Micrographs illustrate apoptotic bodies viewed using a 60× objective with differential interference contrast (DIC) (A) and epifluorescence (B). Distribution of apoptotic body diameter (n = 62) is illustrated (C). Mean and median diameter 6.4 and 6.0 ␮m, respectively. Scale bar = 10 ␮m. could therefore represent a novel pathway to clear neuronal apoptotic bodies and limit inflammation associated with AD Acknowledgements [18,32]. In the context of AD, apoE accumulation in amyloid plaque Supported by the Australian Research Council (Grant No. and neurofibrillary tangles may be a consequence of caspase- DP0557295 awarded to B.G. and D.A.J.). dependent neurodegeneration which shares features in common with the apoptotic pathway. The over-expression of apoE in AD brain [30] is consistent with this idea; although apoE4- References amyloid-␤ peptide complexes may induce neuronal apoptosis [1] R.S. Akundi, A. Macho, E. Munoz, K. Lieb, G. Bringmann, H.W. via an LRP-dependent process involving lysosomal rupture and Clement, M. Hull, B.L. Fiebich, 1-trichloromethyl-1,2,3,4-tetrahydro- caspase activation [14]. beta-carboline-induced apoptosis in the human neuroblastoma cell line In conclusion, this study reveals that apoE expression is SK-N-SH, J. Neurochem. 91 (2004) 263–273. induced in SK-N-SH neurons undergoing apoptosis and that [2] M.M. Bartl, T. Luckenbach, O. Bergner, O. Ullrich, C. Koch-Brandt, Mul- the apoE generated is strongly expressed on neuronal apoptotic tiple receptors mediate apoJ-dependent clearance of cellular debris into nonprofessional phagocytes, Exp. Cell. Res. 271 (2001) 130–141. bodies. An increased understanding of the function of apoE [3] U. Beisiegel, W. Weber, G. Ihrke, J. Herz, K.K. Stanley, The LDL-receptor- in neuronal apoptosis should provide further insight into the related protein, LRP, is an apolipoprotein E-binding protein, Nature 341 potential roles of apoE in the brain. (1989) 162–164. 52 210 D.A. Elliott et al. / Neuroscience Letters 416 (2007) 206–210

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3.2.2 Publication II

David A. Elliott, Woojin S. Kim, David A. Jans and Brett Garner. (2008) Macrophage apolipoprotein-E knockdown modulates caspase-3 activation without altering sensitivity to apoptosis. Biochimica et Biophysica acta 1780:145-53.

Declaration

I certify that I have performed the majority of the research work and writing contained in the following publication and that reproduction in this thesis does not breach copyright regulations.

David Anthony Elliott

Author's personal copy

Available online at www.sciencedirect.com

Biochimica et Biophysica Acta 1780 (2008) 145–153 www.elsevier.com/locate/bbagen

Macrophage apolipoprotein-E knockdown modulates caspase-3 activation without altering sensitivity to apoptosis ⁎ David A. Elliott a, Woojin S. Kim a, David A. Jans b, Brett Garner a,c,

a Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia b Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia c School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia Received 14 June 2007; received in revised form 9 October 2007; accepted 25 October 2007 Available online 7 November 2007

Abstract

Apolipoprotein-E (apoE) expression may be associated with apoptosis resistance. Since macrophages constitutively synthesize apoE we speculated that this may contribute to apoptosis resistance. Using siRNA, human monocyte derived macrophage (hMDM) apoE mRNA and protein was reduced by 97% and 61%, respectively. ApoE knockdown increased staurosporine-induced caspase-3 activation by 78% without altering cell survival or apoptosis as assessed by TUNEL analysis and morphological changes. This result was confirmed using murine bone marrow derived macrophages (mBMDM) from apoE null and wild type mice. In these experiments, staurosporine-induced caspase-3 activation was increased by 49% in apoE null compared to wild type mBMDM and this was not associated with differences in TUNEL signal, annexin-V binding or DNA fragmentation. ApoE is also important for cholesterol transport and macrophage cholesterol can regulate apoptosis. Knockdown of hMDM apoE inhibited basal cholesterol efflux by 20% without altering apolipoprotein-AI mediated cholesterol efflux over 24 h. Similarly, in apoE null mBMDM a non significant trend for a 16% reduction in basal cholesterol efflux was observed as compared to wild type mBMDM. In conclusion, apoE expression modulates capase-3 activity, but this has no significant impact on sensitivity to apoptosis and only a moderate impact on basal cholesterol efflux. © 2007 Elsevier B.V. All rights reserved.

Keywords: Apolipoprotein-E; siRNA; Macrophage; Caspase-3; Apoptosis; Cholesterol-efflux

1. Introduction condition, highlighting the crucial role of macrophage apoE in regulating atherosclerosis [10,11]. Apolipoprotein-E (apoE) is a ~34 kDa glycoprotein that ApoE also has several proposed functions beyond lipopro- participates in lipoprotein metabolism and cellular lipid tein metabolism such as in immunoregulation, cell cycle/ transport [1]. It is highly expressed in hepatocytes, macrophages proliferation, oxidative stress, stabilization of neuronal micro- and astrocytes, and can be inducibly expressed in other cell tubules, nerve regeneration, and is associated with amyloid types including fibroblasts, neurons and cancer cells [2–7].In plaques [12–15]. More recently, an association between apoE humans, apoE exists as three major isoforms E2, E3 and E4 and apoptosis has been identified. ApoE is upregulated during which differ in their cysteine/arginine composition at positions the induction of apoptosis in human fibroblasts [3], THP-1 112 and 158 [8]. ApoE4 is a risk factor for Alzheimer’s disease macrophages [16] and the neuronal cell line SK-N-SH [7]. [9] and atherosclerosis [8]. ApoE gene knockout (apoE-/-) mice ApoE is associated with apoptotic bodies and there is evidence develop severe atherosclerosis and transplantation of bone mar- that apoE may facilitate the phagocytic clearance of apoptotic row from wild type (WT) mice into these animals reverses this debris [7,17]. In addition, cellular apoE expression is increased following ischemic injury, peripheral nerve crush injury and kainic acid treatment and it is possible that at least some of the ⁎ Corresponding author. Prince of Wales Medical Research Institute, Barker Street, Randwick NSW 2031, Australia. Tel.: +61 2 939 91024; fax: +61 2 939 cells expressing apoE during these injurious conditions may be 91005. apoptotic [6,18–25]. An understanding of how apoE may E-mail address: [email protected] (B. Garner). modulate apoptosis could also provide important insights into

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factors that regulate atherosclerotic plaque stability. One of the humidified atmosphere containing 5% CO2. Macrophage colony stimulating most intensive fields investigating a role for macrophage apo- factor (M-CSF) (Peprotech, USA) was added to the media immediately after monocyte isolation at 125 ng/ml, and was removed and replaced with regular ptosis in human disease focuses on how macrophage apoptosis media after 3 days. impacts on atherosclerosis and specifically on atherosclerotic Murine bone marrow derived Macrophages (mBMDM) were obtained from plaque stability. It has been suggested that in late stages of both WT and apoE-/- adult male C57Bl/6J mice (Animal Resources Centre, atherosclerosis, macrophages may become apoptotic and that Canning Vale, WA, Australia). The mBMDM from 5 mice were pooled in each the debris contributes to the formation of the so-called “necrotic experiment. Briefly, bone marrow cells were collected from femoral and tibial ” bone shafts by flushing with Hanks buffered salt solution (Sigma), then core of the atheromatous plaque [26]. Macrophage apoptosis is centrifuged at 206 g for 5 min and resuspended in DMEM containing 10% fetal therefore thought to make plaques unstable and thus more likely calf serum (Gibco), supplemented with L-glutamine, penicillin and streptomycin to rupture causing acute thrombus formation, coronary occlu- as above. Cells were then cultured in 10 ml plastic Petri dishes at 37 °C, as sion and myocardial damage. It has been suggested that sta- above, for 18 h. Non-adhered monocytes were then collected, counted and 6 bilising advanced plaques through inhibition of macrophage seeded at 2×10 cells/ml in 12 well plates in the presence of M-CSF (125 ng/ ml). Media and M-CSF was replaced every 3 days. apoptosis could represent a novel therapeutic approach for human atherosclerosis [26]. Examination of the potential impact 2.2. Inhibition of apoE expression by siRNA of macrophage apoE on apoptosis is therefore relevant to the understanding of human atherosclerosis. Transfection of siRNA into hMDM was performed using commercially The exact function that apoE performs in apoptosis is cur- prepared siRNA duplexes (Invitrogen Stealth™, Carlsbad, CA, USA). The rently unclear, but recent studies suggests that apoE may inhibit sequence targeted by the siRNA was selected using the BLOCK-iT RNAi designer program (www.invitrogen.com/rnai). The apoE siRNA was targeted to apoptosis [27,28]. Interestingly, Chen et al. have shown that the following apoE specific sequence 5′-GGAGTTGAAGGCCTACAAATCG- apoE is highly expressed in malignant ovarian carcinoma cells, GAA-3′ and a random reorganisation of this sequence was used as the control whilst being undetectable in benign counterparts [28]. When siRNA 5′-GGAAAGTCCGGACATCTAAGTGGAA-3′. siRNA duplexes were siRNA was used to inhibit apoE expression in OVCAR3 cells, transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer’s μ cell cycle arrest and apoptosis was induced [28]. Additional protocol. Briefly, 500 pmol of siRNA was incubated with 3 l Lipofectamine 2000 in 200 μl of OPTIMEM (Gibco) for 20 min at 22 °C to allow the formation comparisons of gene expression in cancerous and healthy tis- of a transfection complex. The hMDM were then rinsed with PBS and exposed sues (using the Serial Analysis of Gene Expression databases to the transfection complex in 800 μl of antibiotic-free media (RPMI-1640 with and proteomics approaches) revealed that apoE expression is 10% human serum and glutamine) and applied to the cells for 6 h. This media increased in several types of cancer, including breast, pancre- was then removed, the cells rinsed with PBS and replenished with media atic, stomach, colon, prostate and liver [4,28,29]. These studies relevant to the assay being performed (see below). Transfection efficiency was confirmed by substituting the siRNA with fluorescently labeled BLOCK-iT further implicate a potential role for apoE in the inhibition of reagent, supplied with the kit, using the same transfection procedure and this apoptosis in general; however, direct evidence for this is lacking confirmed N95% transfection efficiency was achieved as assessed by fluores- and this issue therefore remains to be resolved. cence microscopy (data not shown). It is well known that macrophages are resistant to apoptosis and this is suggested to be at least partly due to increased 2.3. Assessment of apoptosis Inhibitor of Apoptosis Protein (IAP) expression [30–33]. Since For hMDM, apoptosis was induced immediately after the removal of the apoE is constitutively produced by macrophages at levels in the siRNA transfection complex by applying either 100 nM or 250 nM 6 order of 1 to 10 μg apoE/10 cells (and this can represent as staurosporine (Sigma) dissolved in serum-free RPMI-1640 with glutamine, much as 25% of total secreted protein mass) [2,34–38] and the penicillin and streptomycin as previously described [16]. Staurosporine was work of Chen et al. suggested that endogenous apoE may inhibit stored as a 0.2 mM stock in ethanol and was diluted in medium immediately apoptosis [28], we speculated that the relative resistance of before addition to cells. After 48 h the media was removed and cells assessed for caspase-3 activation using the acetyl-Asp-Glu-Val-Asp-AMC substrate [3]. macrophages to undergo apoptosis may be related to their Apoptosis was determined by measuring TUNEL (terminal deoxynucleotidyl constitutive expression of apoE [16]. The primary aim of the transferase-mediated dUTP nick-end labelling) signal using the Cell Death present study was to examine the impact of reduced apoE Detection ELISA-Plus kit (Roche Applied Science), following the manufac- expression on staurosporine-induced apoptosis. As membrane turer’s protocol. Apoptosis was also confirmed morphologically by assessing cholesterol concentration is thought to be a key regulator of nuclear condensation and fragmentation [3,16]. For mBMDM, apoptosis was induced 9-11 days after plating of the monocytes by removing the media and macrophage apoptosis [39,40] we also examined the impact that applying 500 nM staurosporine dissolved in serum-free DMEM with glutamine, reduced apoE expression has on one of the major homeostatic penicillin and streptomycin. After 2 h or 4 h the media was removed and mechanisms for regulating membrane cholesterol concentra- apoptosis was assessed by caspase-3 activation, TUNEL signal and nuclear tion, i.e. the macrophage cholesterol efflux pathway [41]. morphology, as described above. In addition in the mBMDM experiments apoptosis was also assessed by annexin-V binding to the cell surface and by analyzing internucleosomal DNA 2. Materials and methods fragmentation (DNA laddering). The annexin-V binding method detects phosphatidylserine (PS) exposure on the cell surface and was analysed using the 2.1. Cell culture apoDETECT™ ANNEXIN V-FITC KIT (Invitrogen), following the manufacturer’s instructions, whereby Annexin V-fluorescein isothiocyanate (FITC) Human monocytes were isolated from buffy coats using Ficoll-Paque [16], binds PS and propidium iodide (PI) exclusion is used to differentiate between and seeded into either 12-well plates or 96-well plates. Monocytes were allowed apoptotic and necrotic cells. Briefly, adherent cells were detached by scraping, to differentiate into hMDM by culture for up to 2 weeks in RPMI-1640 medium cells and media were then collected, centrifuged at 3000 rpm for 1 min, containing 10% (v/v) human serum supplemented with 2 mM L-glutamine, resuspended in PBS (4 °C), centrifuged, resuspend in binding buffer containing 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) at 37 °C in a Annexin V-FITC and incubated for 10 min at room temperature. Cells were then

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D.A. Elliott et al. / Biochimica et Biophysica Acta 1780 (2008) 145–153 147 centrifuged, rinsed once in binding buffer, resuspended in binding buffer AGCTTTTTCGTGGATGCCACAGGACT-3′ (reverse). All PCR reactions containing PI (1 μg/ml), transferred to a 96-well plate and analysed using a were carried out using Taq DNA Polymerase (Fermentas, Burlington, ON, fluorometric plate-reader; Ex 485 nm and Em 520 nm for FITC; Ex 544 nm and Canada). Quantitative real-time PCR was performed using a Mastercycler ep Em 620 nm for PI. realplex S and the fluorescent dye SYBR Green (Eppendorf, North Ryde, NSW, To analyse DNA fragmentation, cells were lysed in 300 μl of 0.2% (v/v) Australia). The same primer set described above was used for human apoE, and Triton-X 100, incubated on ice for 10 min, then incubated with 1.5 μl of 10 mg/ the above mentioned primers for the β-actin housekeeping gene were used for ml RNAse A for 1 h at 37 °C, followed by the addition of 12.5 μl of 10% sodium normalization. Annealing temperature was 60 °C and melting curve analysis was dodecyl sulfate (SDS) and 2 μl of 20 mg/ml proteinase K and 1 h of incubation at preformed to confirm the production of a single product in these reactions. 50 °C. DNA was then extracted using 600 μl of phenol:chloroform: isoamylalcohol (25:24:1) (Sigma) and samples were centrifuged at 13000 g for 15 min at 4 °C. DNA was then collected from the upper aqueous phase and mixed with 20 μl of 3 M sodium acetate, and precipitated by adding 400 μlof 100% ethanol and incubating at -20 °C overnight. Samples were then centrifuged at 13000 g for 15 min at 4 °C, ethanol was removed and DNA pellet was resuspended in 20 μl of TE buffer. Samples were then loaded onto a 1.0% agarose gel containing ethidium bromide and electrophoresed for 2 h at 60 volts. Cell viability was determined by measuring the level of lactate dehydrogenase (LDH) released into the media using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega), following the manufacturer’s protocol. Briefly, the assay substrate tetrazolium salt is converted to a red formazan product in proportion to the amount of LDH present, thus allowing photometric detection (absorbance at 490 nm). Total cellular LDH level is determined by lysis with Triton X-100 (0.9% v/v) and serves as the positive control. Media LDH is then calculated as a percentage of the total LDH present. It was necessary to use a different protocol for inducing apoptosis in mBMDM as prolonged incubation with lower staurosporine concentrations (as per the hMDM experiments) resulted in detachment of mBMDM from dishes and irreproducible cell recovery.

2.4. Western blotting

Macrophage apoE and β-actin was detected by Western blotting and signal intensity quantified using NIH Image J software [3]. Protein loading and transfer efficiency were monitored by Ponceau-S staining.

2.5. Cholesterol efflux and apoE secretion

Macrophages were grown in 96 well plates. Directly after siRNA treatment cells were rinsed three times with PBS and labeled with 2 μCi/ml [3H] cholesterol-labeled acLDL (50 μg/ml) [42]. Equilibrated [3H]cholesterol-labeled cells were rinsed with PBS and incubated in 100 μl RPMI-1640 media containing 0.1% BSA with or without 25 μg/ml apoA-I [43]. After 6 and 24 h, 30 μl of media was removed and analysed by scintillation counting. At 24 h, the hMDM were also collected and analysed by scintillation counting and cholesterol efflux to the medium was expressed as a percentage of total cholesterol present [43]. To assess apoE secretion into the medium, macrophages were treated with siRNA as outlined above in 96-well plates. After removal of the transfection complex, 100 μl of serum-free media was applied and 20 μl aliquots collected (from 24 h incubations) at 30, 54 and 90 h time points. Equal volumes of media were then assayed by Western blotting to determine macrophage apoE secretion [3].

2.6. Polymerase chain reaction Fig. 1. siRNA inhibition of hMDM apoE expression. Semi-quantitative RT-PCR Macrophages were harvested for total RNA using Tri Reagent (Sigma). For (A) and Western blot (B) comparing apoE mRNA and protein levels in triplicate each reverse transcription reaction, 2 μg of total RNA was reverse-transcribed from hMDM treated with either non-specific control (con) siRNA, apoE specific using Random Primers (Promega, Madison, WI, USA) and M-MLV Reverse siRNA (apoE) or transfection reagents alone (untreated), at 24 and 48 h post- Transcriptase (Promega). PCR was performed using either human or murine transfection. Levels of the control GAPDH and β-actin mRNA and protein, apoE gene-specific primers. For human: 5′-GTCGCTTTTGGGATTACCTGC- respectively, were unaffected by the treatments. Relative differences in cellular 3′ (forward) and 5′-CCGGGGTCAGTTGTTCCTC-3′ (reverse). For murine: apoE expression comparing control siRNA and apoE siRNA treated hMDM 5′-TCTGACCAGGTCCAGGAAGAG-3′ (forward) and 5′-AGCTGTTC- were semi-quantitatively calculated for mRNA (C) and protein (D), at both 24 CTCCAGCTCCTTT-3′ (reverse). Results were normalized using the house- and 48 h time points by performing optical density analysis of the gels shown in keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in “A” and “B”, respectively. The amount of apoE in each sample was standardized hMDM: 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ (forward) and 5′- for the level of GAPDH and β-actin, respectively. Values mean±S.E., from CATGTGGGCCATGAGGTCCACCAC-3′ (reverse), and β-actin in mBMDM: two independent experiments using triplicate samples. (⁎P=0.05, ⁎⁎ Pb0.01, 5′-GAATTCTGGCCACGGCTGCTTCCAGCT-3′ (forward) and 5′- ⁎⁎⁎ Pb0.0001).

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3.2. Impact of apoE knockdown on staurosporine-induced caspase-3 activity

Knockdown of hMDM apoE resulted in a 78% increase in caspase-3 activity in response to 48 h of exposure to 100 nM staurosporine, and a 61% increase with 250 nM staurosporine (Fig. 2A). However, when apoptosis was assessed using TUNEL as a final-stage marker of apoptosis, no significant difference was found between siRNA induced apoE knockdown and control conditions (Fig. 2B). In addition, using DAPI staining of fixed macrophages, apoptotic changes in nuclear morphology (condensed and fragmented nuclei) were present in fewer than 5% of hMDM and there were no significant differences when apoE expression was inhibited with siRNA (data not shown). A similar trend was observed when comparing mBMDM from apoE-/- and wild type (WT) mice. After 2 h exposure to 500 nM staurosporine, a 49% increase in caspase-3 activation was seen in the apoE-/- mBMDM relative to the WT (Fig. 3A). However, no difference was observed

Fig. 2. siRNA inhibition of hMDM apoE expression is associated with increased staurosporine-induced caspase-3 activity without significant change in TUNEL signal. hMDM treated with either control siRNA or apoE siRNA were exposed to either 100 nM or 250 nM of staurosporine (stsp) for 48 h. Induction of caspase-3 activity (A) and TUNEL signal (B) was then assessed. (⁎Pb0.05; ⁎⁎Pb0.01). Control values for both caspase-3 activity and TUNEL signal for hMDM not treated with staurosporine were not significantly different when comparing scr-siRNA and apoE-siRNA and the mean background values were thus 23.6±3.3 for caspase-3 activity and 0.65±0.12 for TUNEL signal. In the latter case, the background TUNEL signal values for the untreated hMDM have been subtracted from the values presented in “B”. Data represent mean±S.E. from single experiments representative of 3 independent experiments performed in triplicate or quadruplicate.

2.7. Statistics

All experiments were repeated at least twice in either triplicate or quadruplicate. Statistical significance was determined using a two-tailed t-test for unpaired data, with a P value b0.05 classified as significant.

3. Results

3.1. Inhibition of human monocyte derived macrophage (hMDM) apoE synthesis using siRNA

Although hMDM are known to be difficult to transfect [44– 46] we were able to significantly reduce apoE mRNA and protein expression using siRNA (Fig. 1). Semi-quantitative PCR indicated significant reductions in apoE mRNA of 31% at 24 h and 73% at 48 h (Fig. 1A and C). Quantitative real-time PCR analysis confirmed significant reduction (97±0.01%, mean±S.E.) in apoE mRNA 48 h after transfection, as determined using triplicate samples from two independent experi- Fig. 3. ApoE knockout in mBMDM is associated with an increase in ments (Pb0.0001). Western blot analysis revealed a 25% and staurosporine-induced caspase-3 activity, but no change in TUNEL signal. -/- 61% reduction in apoE protein at 24 h and 48 h, respectively Wild type and apoE mBMDM were either exposed to 500 nM staurosporine for 2 h, or left untreated. Induction of caspase-3 activity (A) and TUNEL signal (Fig. 1B and D). No further reduction in cellular apoE protein (B) was then assessed. The absence of apoE in the apoE-/- mBMDM was verified level was observed at later time-points, assessed up to 90 h (data by semi-quantitative PCR (C). (⁎Pb0.05). Data represent mean±S.E. from 2 not shown). independent experiments performed in triplicate or quadruplicate. 58 Author's personal copy

D.A. Elliott et al. / Biochimica et Biophysica Acta 1780 (2008) 145–153 149 when assessed by TUNEL (Fig. 3B) or nuclear morphology (data not shown). The absence of apoE in the mBMDM derived from apoE-/- mice was confirmed by semi-quantitative PCR (Fig. 3C). Additional markers of apoptosis were also used to assess mBMDM after 2 h and 4 h exposure to 500 nM staurosporine. Annexin-V binding was not significantly increased with staurosporine treatment (Fig. 4A) and propidium iodide staining (which detects dead cells but not live cells or apoptotic cells) was also not significantly increased (Fig. 4B). Release of LDH was also used as a marker of necrosis and the data indicate that staurosporine treatment did not induce macrophage necrosis (Fig. 4C). In addition, we could find no evidence for internucleosomal DNA fragmentation in either WT or apoE-/- mBMDM treated for 4 h with 500 nM staurosporine, although Fig. 5. siRNA inhibition of hMDM apoE secretion. Timeline detailing the steps human fibroblasts and SK-N-SH neuroblastoma cells treated involved in the hMDM cholesterol efflux assay (A). Media samples were under identical conditions showed early signs of DNA collected from control and apoE siRNA treated hMDM 30 h, 54 h and 90 h post- fragmentation although much of the genomic DNA remained transfection and analysed by Western blotting for apoE protein (B). intact (Fig. 4D). These data indicate that even though caspase-3 activity was increased in both hMDM and mBMDM, this did 3.3. Impact of apoE knockdown on apoE secretion and not correlate with markers of apoptosis. Inhibition of apoE cholesterol efflux expression in hMDM, or its complete absence in mBMDM, therefore does not significantly alter sensitivity to apoptosis Secretion of apoE by macrophages is thought to contribute to overall. cholesterol efflux and thus regulate macrophage membrane

Fig. 4. ApoE knockout in mBMDM is not associated with an increase in staurosporine-induced annexin-V binding, internucleosomal DNA fragmentation or release of cellular lactate dehydrogenase (LDH). Wild type and apoE-/- mBMDM were either exposed to 500 nM staurosporine for 2 h or 4 h, or left untreated. Annexin-V binding (A) and propidium iodide staining (B), LDH release (C) and DNA fragmentation (D) and was then assessed. In panel A and panel B data are presented as relative fluorescence where the cell free detection reagent blank (Blk) is define as “100”. “C”, control (without staurosporine); “2h” and “4h”, treated with 500 nM staurosporine for 2 h and 4 h respectively. In panel C, “Tr” represents cells treated with Triton X-100 as a positive control for LDH release. In panel D the 4 h treatment of mBMDM, human fibroblasts (Fibro.) or neuroblastoma cells (Neuro.) with 500 nM staurosporine (stsp) is indicated by a cross “+”. Data represent mean±S.E. from 2 independent experiments performed in triplicate.

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150 D.A. Elliott et al. / Biochimica et Biophysica Acta 1780 (2008) 145–153 cholesterol concentration [47]. In order to examine whether experiment total cellular [3H]-cholesterol levels were very apoE knockdown has functional consequences on cholesterol similar in both wild type and apoE-/- acLDL-loaded macro- efflux (that could potentially increase sensitivity to apoptosis phages (11112±633 versus 11135±718 CPM/well, mean±SE, over time in a pathological setting such as atherosclerosis [33]) n=12) and there was a non-significant trend for a 16% reduc- we examined the impact that siRNA inhibition of hMDM apoE tion in cholesterol efflux in the apoE-/- condition (7.6±0.4 secretion has on cholesterol efflux. versus 6.4±0.7%, mean±SE, n=12, P=0.09). Thus a sustained The time course for siRNA inhibition of apoE expression, 60% suppression or complete absence of macrophage apoE loading cells with [3H]cholesterol and assessing [3H]cholesterol secretion does not appear to have a major impact on cholesterol efflux is outlined in Fig. 5. A period of 90 h elapsed between the efflux and this pathway is therefore unlikely to alter the administration of the siRNA duplexes and media collection for sensitivity of these cells to apoptotic cell death. analysis of [3H]cholesterol efflux at the final time point. ApoE secretion was reduced by 13%, 60% and 59% at the 30, 54 and 4. Discussion 90 h time points, respectively (Fig. 5), thus confirming that the siRNA induced reduction in apoE secretion was maintained In the initial experiments reported here, we developed an throughout the duration of the experiment. We then investigated siRNA method to suppress hMDM apoE expression. Although whether inhibition of apoE secretion modulates cholesterol siRNA has been commonly used to inhibit gene expression in efflux in the presence or absence of the cholesterol acceptor vitro, it has not been widely used with hMDM. This is at least apoA-I. Fig. 6 indicates that siRNA suppression of macrophage partially due to problems associated with siRNA delivery using apoE secretion moderately inhibited cholesterol efflux (by 20%) lipid based transfection reagents (note that macrophage in the presence of BSA only (i.e. basal cholesterol efflux) but transfection has been previously achieved by electroporation, only at the 24 h time point, while a moderate inhibition of osmotic delivery, particle bombardment or after conjugation of cholesterol efflux was detected at 6 h in the presence of apoA-I DNA to receptor recognition signals [44,45,48]) and the (19%) but this did not reach statistical significance at the 24 h potential for induction of an immunological response including time point (in 2 independent experiments performed in up-regulation of cytokine synthesis (see [48] and references quadruplicate). We also examined basal cholesterol efflux cited within). The data presented here indicates that by using from both WT and apoE-/- mBMDM in order to assess whether relatively high siRNA concentrations (500 nM) for a short the complete absence of apoE has a similar effect to the 60% period (6 h) while maintaining a Lipofectamine concentration of reduction we achieved with siRNA treatment of hMDM. In this 3 μl/ml (i.e. following the manufacturer’s instructions), hMDM apoE mRNA and protein levels could be successfully reduced. The incomplete reduction in apoE protein levels observed indicates that a pool (or pools) of apoE exist that turn over very slowly, consistent with the existence of a cell surface pool of apoE that is regulated by membrane-associated proteoglycans [49]. It is also possible that in the absence of apoE synthesis, a portion of the cell surface pool may be recycled and secreted; thus accounting for the continued low level apoE secretion when apoE mRNA levels were reduced by N95% (assessed by quantitative PCR). Alternatively, inhibition of apoE degradation and intracellular apoE recycling pools may have provided a source of the residual apoE. A contribution of apoE derived from growth medium is extremely unlikely as in the studies in which apoptosis was measured, human serum was absent for 48 h prior to cellular apoE analysis. Similarly, in the experiments analyzing cholesterol efflux, human serum was not present for up to 90 h prior to assessing apoE secretion by hMDM, arguing against any significant contribution to apoE in the cell culture medium from serum. The primary aim of this study relates to the potential for apoE to modify cellular sensitivity to apoptosis. Since the majority of cell types in the body do not constitutively express significant Fig. 6. siRNA inhibition of hMDM apoE expression is associated with reduced amounts of apoE, it follows that apoE loss in itself should not basal cholesterol efflux. hMDM were treated with either control siRNA or apoE promote apoptosis. However, previous studies identified a siRNA and [3H]cholesterol efflux assessed as outlined in Fig. 5A. Cholesterol selective increase in the number of apoptotic macrophages efflux was performed in the presence of 1 mg/ml BSA alone or 1 mg/ml BSA present in the liver of apoE-/- mice compared to wild type mice plus 25 μg/ml apoA-I. Equal samples of media were collected at 6 h and 24 h, and 3 3 (i.e. in wild type animals 10% of liver macrophages were [ H]cholesterol efflux was calculated as a percentage of total [ H]cholesterol. -/- Data represents mean±S.E. from 2 independent experiments performed in TUNEL positive whereas in the apoE animals N50% of liver quadruplicate. (⁎Pb0.05). macrophages were TUNEL positive [17]); which prompted us 60 Author's personal copy

D.A. Elliott et al. / Biochimica et Biophysica Acta 1780 (2008) 145–153 151 to investigate a potential direct role for apoE in the sensitivity of that apoE may contribute to basal cholesterol efflux from macrophages to staurosporine-induced apoptosis. Inhibition of hMDM. In the case of mBMDM, we detected a non-significant hMDM apoE synthesis using siRNA significantly increased the trend for reduced cholesterol efflux from the apoE-/- macro- level of staurosporine-induced caspase-3 activity, but this did phages. The overall contribution that macrophage apoE secre- not significantly alter overall cell survival as assessed by late tion makes to total cholesterol efflux in vivo, however, where stage apoptotic markers including TUNEL positivity and altera- cholesterol acceptors such as apoA-I and HDL are present, tions in nuclear morphology. Therefore the high endogenous would be predicted to be only modest. This is supported by our level of apoE in hMDM does not appear to contribute signifi- data showing that in the presence of apoA-I, cholesterol efflux cantly to the capacity for this cell type to resist apoptosis. from apoE siRNA treated hMDM was moderately inhibited in Considering that the same trend was observed when comparing comparison to control siRNA treated cells, showing a statis- wild type and apoE-/- mBMDM, it seems unlikely that the tically significant difference at the 6 h time point but not at the residual ~40% of apoE protein that remained associated with 24 h time point. Our data indicating that sustained reduction of hMDM after siRNA treatment would be maintaining an anti- hMDM apoE secretion has minimal impact on cholesterol apoptotic function. Thus, our overall conclusion is that endog- efflux also argues against apoE-mediated regulation of choles- enous apoE does not appear to contribute directly to the capac- terol efflux as a contributing factor in modulation of cellular ity for macrophages to resist apoptosis. sensitivity to apoptosis. The observed increase in staurosporine-mediated caspase-3 In conclusion, an siRNA approach to inhibit apoE expression activity when apoE expression was inhibited with siRNA (or was developed and used to address the potential contributions of deleted in the case of the apoE-/- mBMDM) appeared to initially hMDM apoE to cholesterol efflux and to the modulation of support the proposal of Chen et al. that apoE may have an anti- staurosporine-induced caspase-3 activation and apoptosis. The apoptotic function [28]. In our work though, the increase in data reveal that apoE appears to play a minor role in hMDM caspase-3 activity did not result in increased hMDM or cholesterol efflux and that while staurosporine-induced caspase- mBMDM apoptosis. It remains possible that the modulation 3 activation was increased with apoE knockdown, this had no of caspase-3 activity associated with apoE expression level may significant impact on apoptosis overall. It is unlikely that be more relevant for non-apoptotic functions of caspase-3 residual apoE can confer an anti-apoptotic effect as similar which have been reported in several cell types (see [50] and results were obtained comparing WT and apoE-/- mBMDM. references cited therein). For example, capase-3 modulates Further work is required to elucidate the mechanism responsible activation and release of the pro-inflammatory cytokine IL-16 in for the observed modulation of caspase-3 activity by apoE and, primary human monocytes [51]. Interestingly, apoE deletion is more generally, to resolve the underlying mechanisms respon- correlated with increased levels of caspase-1, an enzyme in- sible for the association of apoE with cell survival and inhibition volved in the activation of pro-inflammatory cytokines IL-1β of apoptosis in non-macrophage cell types. and IL-18, in the hippocampus of mice fed a high cholesterol diet [50,52,53]. It therefore seems plausible that the modulation Acknowledgements of caspase-3 activity we have observed may be related to the previously described anti-inflammatory function of apoE Supported by the Australian Research Council (Grant No. [54,55]. Caspase-3 has also been identified as an important DP0557295). regulator of cytoskeletal remodelling and may play a role in other non-apoptotic cellular events such as cell cycle regulation, References migration, and differentiation in a variety of cell types [56–59]. Further studies are clearly required to address the non-apoptotic [1] R.W. 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3.2.3 Publication III

Woojin S. Kim , David A. Elliott , Maaike Kockx, Leonard Kritharides, Kerry-Anne

Rye, David A. Jans and Brett Garner. (2008) Analysis of apolipoprotein-E nuclear localization using green fluorescent protein and biotinylation approaches. The

Biochemical Journal 409: 701-9.

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I certify that I have performed more than 50% of the research work and writing contained in the following publication and that reproduction in this thesis does not breach copyright regulations.

David Anthony Elliott

www.biochemj.org

Biochem. J. (2008) 409, 701–709 (Printed in Great Britain) doi:10.1042/BJ20071261 701

Analysis of apolipoprotein E nuclear localization using green ﬂuorescent protein and biotinylation approaches Woojin S. KIM*, David A. ELLIOTT*, Maaike KOCKX†, Leonard KRITHARIDES†, Kerry-Anne RYE‡, David A. JANS§ and Brett GARNER*1 *Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia, †Centre for Vascular Research, University of New South Wales, Sydney, NSW 2052, Australia, ‡The Heart Research Institute, Sydney, NSW 2050, Australia, §Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia, and School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia

Previous results indicate that apoE (apolipoprotein E) may be apoE–GFP did not result in nuclear apoE–GFP localization in associatedwiththenucleusinspecificcell types, particularly under the recipient cells. Similarly, biotinylated apoE did not reach the stress conditions such as serum starvation. In addition, nuclear nucleus of control CHO cells or SK-N-SH neurons. In contrast, apoE localization in ovarian cancer was recently shown to be when biotinylated apoE was delivered to recipient cells as a correlated with patient survival. In order to better understand the lipidated apoE disc, apoE was detected in the nucleus, suggesting factors associated with apoE nuclear localization, we examined that the lipoprotein complex alters the intracellular degradation intracellular apoE trafficking using live-cell imaging of CHO or trafficking of apoE. Biotinylated apoE discs containing each of (Chinese-hamster ovary) cells that constitutively expressed apoE– the three common human apoE isoforms (E2, E3 and E4) were GFP (green fluorescent protein). In addition, we used biotinylated also tested for nuclear trafficking. All three apoE isoforms apoE (in a lipid-free state and as a lipidated discoidal complex) were equally detected in the nucleus. These studies provide new to track the uptake and potential nuclear targeting of exogenous evidence that apoE may be targeted to the nucleus and shed light apoE. Our results indicate that a small proportion of apoE–GFP is on factors that regulate this process. detected in the nucleus of living apoE–GFP-expressing CHO cells and that the level of apoE–GFP in the nucleus is increased with Key words: apolipoprotein E (apoE), green fluorescent protein serum starvation. Exposure of control CHO cells to exogenous (GFP), lipoprotein, live-cell imaging, nuclear targeting.

INTRODUCTION appears to facilitate clearance of apoptotic debris which would provide a pathway for re-utilization of membrane lipids [10,15]. ApoE (apolipoprotein E) is a ∼34 kDa glycoprotein that ApoE also has additional biological functions not directly related Biochemical Journal plays an important role in plasma lipoprotein metabolism and to lipid transport and these include roles in antioxidant actions, cellular lipid transport [1]. ApoE facilitates endocytosis of regulation of cell signalling, immunoregulation and in cancer lipoproteins via the LDLr (low-density lipoprotein receptor) and [1,16–18]. related LDLr family members and, in humans, there are three An intriguing and largely unexplored aspect of cellular apoE major apoE isoforms, E2, E3 and E4, which differ in their expression and function is the finding that apoE has also been cysteine/arginine residue composition at positions 112 and 158 detected in the nucleus. Earlier studies identified apoE in a nuclear [2,3]. The apoE2 isoform (when associated with type III hyper- fraction of rat liver, and our group subsequently detected small lipoproteinaemia) and the apoE4 isoform are associated with amounts of apoE associated with the nucleus of human fibroblasts increased atherosclerosis risk [3,4]. The apoE4 isoform is a risk [9,19]. More recently, apoE was detected in the nucleus of ovarian factor for late-onset Alzheimer’s disease [5,6]. The complete cancer cells [20]. Of potential importance in the studies by Chen lack of apoE expression in ApoE gene knockout (ApoE−/−)mice et al. [20] is the fact that apoE in the nucleus appeared to be results in severe atherosclerosis and neurological abnormalities, associated with better survival in ovarian cancer patients. In our including memory and learning defects [7,8]. own studies, we have found that serum starvation can lead to ApoE is constitutively expressed at high levels in macrophages, increased levels of nuclear apoE; however, since total apoE levels hepatocytes and astrocytes, and is detectable in most peripheral were also increased, it was unclear if the increase in the nuclear tissues [1]. ApoE expression may also be induced under certain pool simply reflected a proportional increase that correlated types of cellular stress. For example, apoE expression is with the total cellular apoE level, or if starvation stimulates transcriptionally up-regulated during apoptosis in neurons and apoE nuclear transport. Potentially relevant to the latter theory, fibroblasts [9,10]. Serum starvation also induces apoE expression starvation has been shown to promote the nuclear accumulation in fibroblasts and astrocytoma cell lines [9,11]. Other studies of proteins such as Hsp70 [21]. indicate that brain apoE expression may be increased as a In the present study, we used apoE–GFP (green fluorescent consequence of aging [12] and in specific types of cancer [13,14]. protein)-expressing cells and biotinylation approaches to study The role that apoE plays under these various inducible/stress potential nuclear trafficking of endogenous and exogenous apoE conditions is unclear; however, in the case of apoptosis, apoE respectively. The results indicate that a small but detectable

Abbreviations used: ApoE, apolipoprotein E; BCA, bicinchoninic acid; CCM, cell-conditioned medium; CHO, Chinese-hamster ovary; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GFP, green fluorescent protein; LDLr, low-density lipoprotein receptor; LRP, LDLr-related protein; NLS, nuclear localization sequence; NP40, Nonidet P40; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; Sm, Smith. 1 To whom correspondence should be addressed (email [email protected]). 65 c The Authors Journal compilation c 2008 Biochemical Society 702 W. S. Kim and others amount of endogenous apoE–GFP can be demonstrated in the lens (1.45 numerical aperture; Nikon). Fluorescence was detected nucleus and that nuclear apoE–GFP levels can be increased by using Semrock filter sets and an excitation/emission filter block serum starvation. Furthermore, exogenous biotinylated apoE can of 472/520 nm for GFP and 562/624 nm for Alexa Fluor® 568 also be transported to the nucleus, but only if the apoE is presented staining. to the cell as a lipoprotein complex. These findings may have implications for an intracellular function of apoE beyond that of lipoprotein transport. Western blotting Cells were rinsed with cold PBS and lysed in RIPA buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% MATERIALS AND METHODS (v/v) NP40, 0.5% deoxycholate and 0.1% SDS] containing protease and phosphatase inhibitors. BCA (bicinchoninic acid) Cell culture protein assays were performed on lysates and equal amounts Cell culture media and additives were obtained from Invitrogen of protein were resolved by SDS/PAGE (12% gels) and unless stated otherwise. The human cell lines SK-N-SH and transferred on to nitrocellulose membranes at 100 V for 30 min. foreskin fibroblasts (AG01518) were obtained from the A.T.C.C. Membranes were blocked overnight at 4 ◦C in PBS contain- (Manassas, VA, U.S.A.) and the Coriell Institute for Medical ing 5% (w/v) non-fat dried skimmed milk and 0.1% Tween Research (Camden, NJ, U.S.A.) respectively. Cells were cultured 20 and probed with rabbit polyclonal anti-apoE (1:1000 dilu- in DMEM (Dulbecco’s modified Eagle’s medium) containing tion; Dako), rabbit polyclonal anti-GFP (1:4000 dilution; 10% (v/v) FCS (fetal calf serum), 2 mM L-glutamine, penicillin Invitrogen), mouse monoclonal anti-(Sm antigen) (1;200 dilution; (100 i.u./ml) and streptomycin (100 μg/ml) at 37 ◦C in humidified Abcam), rabbit polyclonal anti-nucleolin (1:500 dilution; Santa air containing 5% CO2. The CHO (Chinese-hamster ovary) Cruz Biotechnology), mouse monoclonal anti-biotin (1:1000 dilu- apoE–GFP cell line was established as described previously tion; Sigma) and rabbit polyclonal anti-β-actin (1:2000 dilution; [22] and cultured in Ham’s F12 medium containing 10% (v/v) Dako) antibodies. Anti-β-actin was used as a loading control. FCS, 2 mM L-glutamine, penicillin (100 i.u./ml), streptomycin The membranes were washed three times in PBS containing (100 μg/ml) and geneticin (300 μg/ml). For starvation studies, 0.1% Tween 20 and then incubated with horseradish peroxidase- the cells were initially grown in the normal Ham’s F12 medium conjugated goat anti-rabbit (1:2000 dilution; Dako) or rabbit until reaching confluency, and then were switched to serum-free anti-mouse (1:2000 dilution; Dako) secondary antibodies for medium for 2 weeks, with a medium change every 3 days. 2 h. Signals were detected using ECL® (enhanced chemiluminescence) (Amersham Biosciences) and X-ray film. Live-cell imaging CHO apoE–GFP cells were cultured on coverslip-based 35 mm Isolation of nuclei diameter Petri dishes until the cells reached approx. 80% Cell nuclei were isolated using the Nuclei EZ Prep nuclei isolation confluency. They were then transferred to a live-cell imaging kit (Sigma) following the manufacturer’s instructions as detailed Nikon TE2000 inverted microscope fitted with an environmental previously [9]. Briefly, cells were rinsed with ice-cold PBS, lysed chamber/CO2 incubator (Solent Scientific, Segensworth, U.K.) with ice-cold Nuclei EZ lysis buffer and scraped up with a and Prior stage (Prior Scientific, Rockland, MA, U.S.A.). Cells bladed cell scraper. The lysate was collected, vortexed briefly ◦ ◦ were maintained at 37 C and humidified 5% CO2,andGFP and centrifuged at 500 g for 5 min at 4 C. The supernatant images were collected using a blue excitation filter block (part was removed and the nuclei resuspended in the lysis buffer and number 41001; Semrock, Rochester, NY, U.S.A.). Z-stacks centrifuged as above. The pellet was resuspended in 200 μl of ice- were routinely collected at 1 μm spacing, and for time course cold Nuclei EZ storage buffer, a BCA protein assay was performed studies intervals of 20–60 s were selected. Time course series and equal amounts of nuclear protein were resolved by PAGE. were compiled using Image Pro-Plus v6.1 (Media Cybernetics, Prior to PAGE and Western blotting, the nuclear preparations were Bethesda, MD, U.S.A.) and Nearest Neighbour Deconvolution treated with DNaseI for 1 h at 37 ◦C. It is established that β-actin was applied to Z-stacks using the Sharp Stack function. is a component of mammalian chromatin-modifying complexes [24], and therefore blotting for nuclear β-actin was used to confirm equal protein loading of nuclear lysates. Immunocytochemistry ApoE–GFP-expressing CHO cells were fixed using a combination Nuclear speckle isolation of 4% (w/v) paraformaldehyde in PBS and methanol/acetone (1:1, v/v). Briefly, cells were treated with 4% (w/v) paraform- Subnuclear structures known as interchromatin granule clusters or aldehyde for 5 min at 4 ◦C, followed by a 10 min incubation at nuclear speckles were isolated from nuclei as described previously 22 ◦C. Cells were then rinsed in PBS and treated with methanol/ [25]. Briefly, cell nuclei were resuspended in ice-cold TM5 buffer ◦ acetone (1:1, v/v) for 6 min at −20 C [23]. Cells were then [10 mM Tris/HCl (pH 7.4) and 5 mM MgCl2 (pH 7.4)] containing permeabilized with 1% (w/v) NP40 (Nonidet P40) (Sigma) and protease inhibitors, incubated with 1% (v/v) Triton X-100 and 10% (v/v) goat serum in PBS for 20 min at 22 ◦C, then treated 2 mM vanadium ribonucleoside complex for 5 min on ice and then with mouse monoclonal anti-[human Sm (Smith) antigen] Y12 centrifuged at 780 g for 5 min at 4 ◦C. The pellet was resuspended antibody (1:400 dilution; Abcam, Sapphire Bioscience, Redfern, in TM5 buffer and treated with DNaseI for 1 h at 4 ◦C, with NSW, Australia) for 1 h at 22 ◦C. After rinsing in 1% (w/v) NP40, intermittent mixing. NaCl (0.5 M final concentration) was added, cells were incubated with Alexa Fluor® 568-conjugated goat anti- incubated on ice for 5 min and centrifuged at 770 g for 5 min at (mouse IgG) (1:100 dilution; Invitrogen) for 1 h at 22 ◦C. Samples 4 ◦C. The pellet was resuspended in TM5 buffer containing 0.5 M were again rinsed three times with 1% (w/v) NP40 and PBS and NaCl and centrifuged as before, and this process was repeated then mounted in Vectashield (Vector Laboratories, Burlingame, prior to resuspension of the pellet in TM5 buffer containing 0.5 M CA, U.S.A.). Specimens were studied using a Nikon TE2000 NaCl and 5 mM dithiothreitol and incubation on ice for 5 min. The microscope and a ×60 Plan-Apochromat oil immersion objective homogenate was then passed 10 times through a needle (27 gauge) 66 c The Authors Journal compilation c 2008 Biochemical Society Nuclear localization of apolipoprotein E 703 and homogenized 20 times using a Dounce homogenizer. The for 2 h in DMEM containing 0.1% BSA to allow equilibration of homogenate was then mixed with TM5 buffer containing 0.2 M [3H]cholesterol in intracellular pools. The cells were then rinsed ◦ Cs2SO4 and centrifuged at 20800 g for 2 min at 4 C. The once in PBS and then incubated in serum-free DMEM containing supernatant was transferred to polycarbonate tubes (Beckman) 0.1% BSA with apoE3 (15 μg/ml) or apoE3 discs (15 μg/ml) and centrifuged at 60000 rev./min in a Beckman Optima TLX for 24 h. Samples of the medium were collected at specific times ultracentrifuge (TLA 100.3 rotor) for 1 h at 4 ◦C. The speckle and cleared of any cellular debris by centrifugation at 1000 g for pellet was resuspended in 30 μl TM5 buffer and stored at −20 ◦C. 5minat22◦C. The cells were lysed with 0.1 M NaOH and the radioactivity in the medium samples and cell lysates was measured by scintillation counting. Cholesterol efflux to the medium was Isolation and characterization of secreted apoE–GFP calculated as a percentage of total radioactivity in the cell lysates ApoE–GFP-expressing CHO cells were grown in Ham’s F12 and medium. medium containing serum and antibiotics as above. Once confluent, cells were washed twice with fresh Ham’s F12 medium without serum or antibiotics and cultured in the same Statistical analysis medium for a further 24 h. The CCM (cell-conditioned medium) Experiments were routinely performed in triplicate and repeated containing secreted apoE–GFP was collected and centrifuged at at least twice. Where indicated, results are means −+ S.E.M. and 1000 g for 5 min at 22 ◦C to remove any cellular debris. The statistical significance was determined using the Student’s t test, CCM was then concentrated using Centricon YM-50 membrane with P < 0.05 considered significant. filters (Millipore) following the manufacturer’s instructions. The concentrated medium was analysed by SDS/PAGE (12% gels) and gels were stained with Coomassie Brilliant Blue solution RESULTS [0.1% Coomassie Brilliant Blue G250, 40%(v/v) methanol and Nuclear localization of apoE–GFP in CHO cells 10% (v/v) acetic acid] for 2 h and destained with methanol/acetic acid/water (4:1:5, by vol.). Previous studies indicated that apoE can be trafficked to the nucleus [9,19,20]. We sought to confirm and extend these observations using CHO cells that had been stably transfected ApoE synthesis and biotinylation to express apoE–GFP (isoform E3) under the control of a CMV Recombinant human apoE (E2, E3 and E4 isoforms) were (cytomegalovirus) promoter [22]. Live-cell microscopy indicated prepared from Escherichia coli as described previously [26,27]. that there was strong expression of apoE–GFP in the majority of ApoE discs containing apoE, POPC (1-palmitoyl-2-oleoyl cells (Figure 1A), with apoE–GFP predominantly present in the phosphatidylcholine) and cholesterol were prepared using the cytosol, apparently within secretory vesicles and in association cholate dialysis method and characterized as described by Kim et with the endoplasmic reticulum and Golgi compartments al. [28]. The apoE discs had an average diameter of 17.0 nm (Figure 1A). A small proportion of apoE–GFP was also detected as judged by gel-filtration chromatography [28]. The POPC/ within the nucleus. Using multiple Z-plane focus positions and cholesterol/apolipoprotein molar ratio of the apoE discs was live cells, it was clear that numerous speckle-like structures ∼0.3– approx. 105:11:1. The disc size, apoE conformation and 1 μm in diameter were present throughout the nucleus (Figures 1A lipid composition resembled nascent apoE discs secreted from and 1B). With time course experiments and single cell analysis, astrocytes [29]. ApoE and apoE discs were biotinylated as nuclear and cytosolic pools could be clearly discriminated and described previously [30]. Briefly, 0.2 mg of apoE and apoE disc apoE–GFP was also detected in association with cytoskeletal protein were treated with 1.5 mg of EZ-Link Sulfo–NHS (N- structures that, on the basis of previous work [32,33], hydroxysuccinimido)–Biotin (Pierce) in 1 ml PBS for 4 h at 4 ◦C are very likely to include microtubules (see Supplementary and then dialysed using a Slide-A-Lyzer dialysis cassette [10 kDa Figure 1 at http://www.BiochemJ.org/bj/409/bj4090701add.htm). MWCO (molecular mass cut-off); Pierce] in PBS overnight at Occasionally, small apoE–GFP cytosolic vesicles were observed 4 ◦C with four buffer changes. To study the nuclear trafficking to travel along the path of the apoE–GFP-positive cytoskeletal of exogenous biotinylated apoE or apoE discs, we adapted a structures (see Supplementary Figure 1). method described previously [31]. SK-N-SH cells were treated Confocal laser-scanning microscopy confirmed that apoE–GFP with 20 μg/ml biotinylated lipid-free apoE or biotinylated apoE was present in the nucleus in a punctate pattern (Figure 1C). discs for up to 24 h, rinsed twice with fresh DMEM, and Starvation induced by serum withdrawal has been shown incubated for a further 2 h in fresh medium prior to a final previously to induce growth arrest and increase fibroblast apoE rinse with PBS. Cells were then lysed directly or subjected to expression in whole-cell lysates and nuclear extracts [9,11]. the nuclear isolation protocol described above. As a biotin-only To assess whether apoE–GFP nuclear targeting was modulated control, cells were treated for 24 h with 250 μg/ml biotin, which by starvation, apoE–GFP-transfected CHO cells were initially is greatly in excess of the concentration of free biotin that would grown in complete medium and then cultured in medium lacking be predicted to remain after dialysis. This control condition serum for 2 weeks. As a control, cells grown only in complete was added to ensure that any biotinylated signal detected by medium were also prepared. As expected, serum starvation of the Western blotting was not the result of non-specific labelling CHO apoE–GFP cell line resulted in growth arrest (results not of cellular/nuclear proteins that was caused by traces of biotin shown), with live-cell microscopy indicating a dramatic change remaining after the dialysis step. in the appearance of apoE–GFP in the cytosol which appeared to coalesce to form large intracytosolic structures (Figure 1D). The appearance of apoE–GFP in the nucleus was also altered under Cholesterol efflux assay serum starvation, with a more diffuse signal detected in addition to Cellular cholesterol efflux was used as an index of apoE or apoE the punctate appearance observed in cells cultured in the presence disc structural integrity as described previously [28]. Briefly, of serum (Figure 1D). SK-N-SH cells were labelled with 2 μCi/ml [3H]cholesterol In order to determine whether the absolute levels of apoE– (Amersham Biosciences) for 24 h, rinsed with PBS and incubated GFP were affected by serum starvation, cellular and nuclear 67 c The Authors Journal compilation c 2008 Biochemical Society 704 W. S. Kim and others

Figure 1 Detection of apoE–GFP in the nucleus

(A, B) CHO apoE–GFP cells were grown to approx. 80% confluence and examined using a Nikon TE2000 live-cell imaging system equipped with a ×60 oil objective lens. The focal plane generating the clearest distinction of the boundary of the nuclear envelope was captured initially (A) and a second Z-plane focus position 2 μm above the initial plane was also captured (B). Note that the cytosolic apoE–GFP signal can be distinguished from the nuclear signal when comparing (A)and(B). (C) An optical section was taken through the middle of the nucleus using confocal laser scanning microscopy of formalin-fixed CHO apoE–GFP cells to confirm the presence of nuclear apoE–GFP. (D) CHO apoE–GFP cells were cultured in serum-free medium for 2 weeks (starved) and cellular apoE–GFP examined using the live-cell imaging system. The inset shows detail of the nucleus from the cell at the bottom left of the panel. Note that the exposure time settings in (D)were lower than in (A)and(B) as a result of the high signal intensity of the coalesced apoE–GFP vesicles. Scale bars = 10 μm. proteins were isolated from the serum-starved and control cells, localization of the speckle marker with apoE–GFP (Figure 3A). In analysed by Western blotting and quantified using NIH (National additional experiments, the nuclear speckle fraction was isolated Institutes of Health) ImageJ software (Figure 2). A 52 kDa protein from CHO apoE–GFP nuclei by ultracentrifugation [25], and the corresponding to apoE–GFP was highly expressed in cell lysates, nuclear and speckle proteins were analysed by Western blotting and protein levels were similar under the serum-starved and the (Figure 3B). The speckle-specific protein, the Sm antigen, was control conditions (Figure 2). In contrast, nuclear apoE–GFP detected in the nucleus and highly enriched in the speckle fraction levels were increased with starvation (Figure 2). as expected (Figure 3B). In contrast, apoE–GFP was clearly The mechanism of apoE entry into the nucleus is unlikely detected in the nuclear fraction, but present at only low levels to be through simple diffusion through the nuclear pores, as in the speckle fraction and was, therefore, not enriched in nuclear apoE–GFP at 52 kDa is probably too large to enter by this route speckles (Figure 3B). The nuclear and speckle fractions were [34]. In addition, the increase in nuclear apoE–GFP localization also probed for nucleolin, which should not be present in nuclear under starvation conditions, which is not simply the result of in- speckles. As predicted, nucleolin was present in the nuclear frac- creased expression levels, implies an active mechanism of nuclear tion but not detected in the nuclear speckle fraction, indicating accumulation. that the speckle fraction was free of contaminating proteins. These results indicate that despite the speckle-like distribution of apoE–GFP in the nucleus, interchromatin granule clusters/nuclear Nuclear apoE–GFP is not associated with interchromatin granule speckles are not the primary location of nuclear apoE–GFP. clusters/nuclear speckles The punctate appearance of nuclear apoE–GFP and the general absence of signal in the nucleolus (see enlarged insert, Fig- Trafficking of exogenous apoE to the nucleus ure 1D) led us to speculate that apoE may be associated with To determine whether secreted apoE–GFP could be targeted to the interchromatin granule clusters/nuclear speckles [25,35]. Nuclear nucleus, serum-free growth medium from CHO apoE–GFP cells speckles are subnuclear structures enriched in pre-messenger was collected, concentrated and semi-purified using Centricon RNA splicing factors including snRNPs (small nuclear ribonu- filters before being incubated with non-GFP-expressing recipient cleoproteins). Using the Y12 antibody to detect Sm antigen as cells. Western blot analysis of the cell culture medium from a marker for nuclear speckles [36], we observed very little co- CHO apoE–GFP cells indicated the presence of the expected 68 c The Authors Journal compilation c 2008 Biochemical Society Nuclear localization of apolipoprotein E 705

Figure 3 Examination of apoE–GFP subnuclear localization

(A) CHO apoE–GFP cells were grown under standard culture conditions until cells reached ∼80% confluency and subsequently fixed and immunostained for the speckle-specific protein Sm antigen using the Y12 antibody and Alexa Fluor® 568-conjugated secondary antibody. The GFP signal (green) is overlayed with the Y12 signal (red), indicating very little co-localization of apoE with nuclear speckles in three representative nuclei. (B) Interchromatin granule clusters/nuclear speckles were isolated from CHO apoE–GFP nuclei by ultracentrifugation, and the nuclear and speckle proteins were analysed by Western blotting using the Y12 antibody against the speckle-specific protein Sm antigen (Smith antigen), anti-GFP antibody and anti-nucleolin antibody.

apoE–GFP-expressing CHO cells or SK-N-SH neurons for up to 24 h. The cells were then rinsed and pulsed for 2 h with growth medium devoid of apoE–GFP, and the presence of apoE–GFP in cell lysates and isolated nuclear fractions was examined by Western blotting. When the apoE–GFP protein concentrate was applied exogenously to control CHO cells, apoE–GFP was not detected in either cellular or nuclear fractions (Figure 5A). In similar experiments, apoE–GFP was also applied exogenously to SK-N-SH cells and, once again, the apoE–GFP protein was not detected (Figure 5B). Similar results were also obtained when cells were treated for only 1 h with apoE–GFP (results not shown). Since the structure Figure 2 Effects of serum starvation on apoE–GFP expression of apoE–GFP is not identical to native apoE and it is not part of a lipoprotein complex, it is possible that apoE–GFP may not CHO apoE–GFP cells were cultured in serum-free medium for 2 weeks (Starved) and were compared with cells grown in complete medium (Control). Cellular and nuclear protein lysates interact optimally with endocytic receptors. The lack of detection were prepared and analysed by Western blotting using anti-apoE (A) and anti-GFP (B) antibodies. of apoE–GFP in association with control CHO cells and SK-N- Total cellular β-actin or nuclear β-actin were used to confirm equal protein loading of the cellular SH neurons could therefore be the result of inefficient uptake of and nuclear preparations respectively. (C) The mean signal intensity of the Western blot derived apoE–GFP. However, earlier studies have shown that apoE–GFP from three independent experiments was quantified by densitometric analysis using ImageJ can be taken up by human fetal brain cell cultures by a process software (y-axis) and reveals a significant increase in apoE–GFP levels in the starved nuclear which was presumed to be receptor-mediated endocytosis [37]. fraction, but not in the whole cellular fraction (n = 3; **, P < 0.01). Con, control. To address the lack of apoE–GFP signal in the SK-N-SH neurons, we conducted an additional control experiment to assess whether 52 kDa apoE–GFP protein (Figure 4A). After filter concentration apoE expression could be up-regulated by serum starvation and and purification, the 52 kDa apoE–GFP protein accounted for if this was associated with an increase in nuclear apoE (i.e. to en- ∼70% of proteins detected on Coomassie-stained SDS/PAGE sure the potential for nuclear trafficking of apoE exists in gels (Figure 4B). In addition, ∼90% of the apoE-positive material this cell type). The results from this experiment indicated was present in the 52 kDa form, indicating that the secreted that apoE was barely detectable by Western blot in SK-N- apoE–GFP remained intact and could be concentrated by approx. SH cells under standard culture conditions, but was induced 30-fold (Figure 4B). The concentrated and purified apoE–GFP with starvation, and this was associated with a parallel increase was then diluted in growth medium and incubated with non- in nuclear apoE (Figure 5C). This result was very similar to 69 c The Authors Journal compilation c 2008 Biochemical Society 706 W. S. Kim and others

Figure 4 Purification and analysis of secreted apoE–GFP Figure 5 Analysis of cellular uptake of exogenous apoE–GFP CHO apoE–GFP cells were cultured in Ham’s F12 medium without serum or antibiotics and the To assess whether extracellular apoE–GFP could be targeted to the nucleus, the concentrated CCM containing secreted apoE–GFP was collected. (A) The CCM was then concentrated using CCM containing apoE–GFP was applied to non-apoE–GFP-expressing CHO cells (A)and Centricon YM-50 membrane filters and was analysed by Western blotting using anti-apoE and SK-N-SH neurons (B) for 24 h and chased for 2 h with medium devoid of apoE–GFP. As a anti-GFP antibodies. (B) To assess the purity and amount of apoE–GFP present, the concentrated control, untreated cells were also included. The presence of apoE–GFP in cell lysates and CCM was analysed by Coomassie staining and Western blotting using anti-apoE antibody. The isolated nuclear fractions was analysed by Western blotting using anti-GFP antibody. The effect concentration of apoE–GFP in the concentrated CCM was estimated to be 0.1 μg/ml. of serum starvation on endogenous apoE expression was also analysed in SK-N-SH neurons (C) and fibroblasts (D). The cells were cultured in serum-free medium for 2 weeks (Starved) and observations made using human fibroblasts (Figure 5D), which were compared with cells grown in complete medium (Control). Cellular and nuclear protein were used as a positive control for the experimental system, and lysates were prepared and analysed by Western blotting using anti-apoE antibody. Blotting for β-actin was used as a loading control in all cases. thus confirmed our previous results [9]. We therefore conclude that under the experimental conditions described, apoE–GFP is possibly endocytosed and degraded before it has an opportunity and apoE discs were applied to SK-N-SH neurons. After a 24 h in- to reach detectable levels in the nucleus or other intracellular cubation with biotinylated apoE or apoE discs, followed by a 2 h compartments. chase with standard DMEM and washing with PBS, cell lysates were analysed by Western blotting. The apoE–biotin protein was detected only in cells that were treated with biotinylated apoE discs, not in untreated cells or in cells treated with Analysis of biotin-labelled apoE lipid-free biotinylated apoE (Figure 6C). This demonstrates that To avoid possible issues associated with the bulky GFP tag exogenously added apoE can accumulate in cells and furthermore interfering with binding, internalization and degradation, we used that the lipidated form appears to be either stabilized or at least biotin as an alternative molecule to label and track exogenous partly trafficked to a non-destructive intracellular compartment. apoE (isoform E3). Biotin is a small molecule that covalently binds lysine residues (of which there are 12 in human apoE). Two forms of recombinant apoE were labelled with biotin: Nuclear localization of biotinylated apoE lipid-free apoE and apoE discs (containing phospholipids and As we demonstrated that biotinylated apoE discs can accumulate cholesterol). The apoE discs resemble nascent brain lipoproteins in SK-N-SH neurons, additional experiments were conducted and are known to play a role in the regulation of neuronal to assess whether biotinylated apoE could be detected in the cholesterol transport [28]. The biotinylated apoE preparations nucleus. As described above, SK-N-SH cells were treated with were extensively dialysed to remove unbound biotin and then biotinylated apoE discs, chased, washed, and cellular and nuclear analysed by Western blotting. The molecular mass of the proteins were analysed by Western blotting. Figure 7 indicates biotinylated apoE protein increased from 34 kDa to 38 kDa, that biotinylated apoE was detected both in whole-cell lysates reflecting the addition of biotin bound to multiple lysine residues and in isolated nuclei. In the untreated control conditions, native (Figure 6A). The structural integrity and associated function of apoE was below the limit of detection in these experiments and, lipid-free apoE and apoE discs was confirmed in cholesterol as expected, biotinylated apoE was not present. efflux assays, where both forms of apoE were able to stimulate Because the apoE genotype is a determinant of apoE func- cholesterol efflux from SK-N-SH neurons (Figure 6B), consistent tion and a risk factor for cardiovascular disease and Alzheimer’s with previous results [28]. To test whether exogenously added disease [1,5,38], we assessed the nuclear trafficking of bio- apoE could be taken up by cells, the biotinylated lipid-free apoE tinylated apoE discs containing each of the three common human 70 c The Authors Journal compilation c 2008 Biochemical Society Nuclear localization of apolipoprotein E 707

Figure 7 Nuclear localization of biotinylated apoE discs

(A) To assess whether biotinylated apoE (isoform E3) derived from exogenous apoE discs can be targeted to the nucleus, biotinylated apoE discs were applied to SK-N-SH neurons for 24 h and chased for 2 h with medium devoid of apoE. As a control, untreated cells were also included. Cellular and nuclear proteins were analysed by Western blotting using anti-apoE and anti-biotin antibodies. (B) The nuclear trafﬁcking of biotinylated apoE discs containing the apoE E2, E3 and E4 isoforms was also assessed as in (A), using anti-apoE antibody. For both (A)and(B), blotting for β-actin was performed as a loading control.

apoE are not well understood. Intracellular apoE regulates the routing of internalized lipoprotein remnants, and is involved in Figure 6 Analysis of biotin-labelled apoE the assembly and secretion of VLDL (very-low-density lipo- Lipid-free human apoE3 and apoE3 discs (containing phospholipid and cholesterol) were protein) [39–41]. In addition, the endocytosis of apoE–lipoprotein labelled with biotin and dialysed to remove unincorporated biotin. (A) The labelled products complex initiates signal transduction [17,42]. Whether apoE tar- before and after dialysis were analysed by Western blotting using anti-apoE antibody. As geting to the nucleus underscores yet another function of apoE controls, untreated lipid-free apoE (free apoE) and apoE discs (apoE disc) were also included. remains unknown. (B) Cholesterol efflux induced by lipid-free apoE and apoE discs was also assessed. SK-N-SH neurons were labelled with [3H]cholesterol for 24 h and then incubated with 0.1% BSA (as a The exact pathway by which apoE, a protein normally destined control), 15 μg/ml lipid-free apoE or apoE discs. Radioactivity in the medium samples and cell for secretion, is able to reach and enter the nucleus remains lysates was measured by scintillation counting and the percentage cholesterol efflux calculated unknown. Nucleocytoplasmic trafficking requires that proteins (y-axis). Experiments were performed in triplicate (n = 3; **, P < 0.01). (C) To test whether cross the nuclear membrane via the nuclear pore complexes. Small exogenously added apoE could be taken up by cells, the biotin-labelled lipid-free apoE and proteins (less than 40–50 kDa) can passively diffuse through apoE discs were applied to SK-N-SH neurons for 24 h and chased for 2 h with medium devoid nuclear pore complexes unassisted, whereas relatively large of apoE. As controls, untreated cells and cells treated with biotin only were included. The cell > lysates were analysed by Western blotting using anti-biotin antibody. Blotting for β-actin was proteins ( 50 kDa) require an active transport pathway largely performed as a loading control. mediated by importins [34,43]. Our results indicated that apoE– GFP at 52 kDa could be detected in the nucleus, which suggested that an active transport pathway may be involved, rather than a apoE isoforms (E2, E3 and E4). SK-N-SH cells were treated diffusion-based mechanism. In humans, apoE exists as a glycoprotein containing a single with the three biotinylated apoE discs as described above, chased, 194 washed, and cellular and nuclear proteins were analysed by O-linked oligosaccharide at Thr [44]. It is not known if apoE Western blotting. All three apoE isoforms were equally detected glycosylation can modulate its trafficking, but it is possible that the in both whole-cell lysates and in isolated nuclei (Figure 7B). hydrophilic glycan moiety may have some impact; for example, during transmembrane movement. It is therefore possible that the recombinant human apoE used in some of our experiments may DISCUSSION behave differently from native glycosylated apoE. Nucleocytoplasmic trafficking usually requires the presence of Although the role of apoE as a secretory extracellular protein has NLSs (nuclear localization sequences). NLSs are short stretches been extensively studied, the possible functions of intracellular of basic amino acids existing as a single cluster (monopartite) or 71 c The Authors Journal compilation c 2008 Biochemical Society 708 W. S. Kim and others as two clusters separated by a spacer region (bipartite) [45]. ApoE 3 Rall, Jr, S. C., Weisgraber, K. H., Innerarity, T. L. and Mahley, R. W. (1982) Structural basis does not appear to contain the classical monopartite or bipartite for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic NLS, however it does contain two polybasic domains that fulfil the subjects. Proc. Natl. Acad. Sci. U.S. A. 79, 4696–4700 criterion for potential ‘weak’ NLSs, as defined by the presence of 4 Kypreos, K. E., Li, X., van Dijk, K. W., Havekes, L. M. and Zannis, V. I. (2003) Molecular four to five basic residues in a hexapeptide [46,47]. Whether these mechanisms of type III hyperlipoproteinemia: the contribution of the carboxy-terminal domain of ApoE can account for the dyslipidemia that is associated with the E2/E2 weak NLSs are functional remains to be determined. It should phenotype. Biochemistry 42, 9841–9853 also be noted that specific proteins may translocate to the nucleus 5 Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, without having an apparent NLS. One example is the growth factor G. W., Roses, A. D., Haines, J. L. and Pericak-Vance, M. A. (1993) Gene dose of midkine, which is involved in neuronal survival and differentiation apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. [48]. Midkine binds to LRP (LDLr-related protein) on the cell Science 261, 921–923 surface and, once internalized, utilizes nucleolin as a nuclear 6 Aleshkov, S., Abraham, C. R. and Zannis, V. I. (1997) Interaction of nascent ApoE2, targeting chaperone [48]. Our results indicate that extracellular ApoE3, and ApoE4 isoforms expressed in mammalian cells with amyloid peptide β apoE can be trafficked to the nucleus when presented to the cell (1–40). Relevance to Alzheimer’s disease. Biochemistry 36, 10571–10580 in the form of a lipidated disc, suggesting a pathway that shares 7 Plump, A. S., Smith, J. D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J. G., Rubin, features in common with the midkine nuclear-targeting pathway E. M. and Breslow, J. L. (1992) Severe hypercholesterolemia and atherosclerosis in could therefore be involved. As noted previously, apoE binding apolipoprotein E- deficient mice created by homologous recombination in ES cells. Cell 71, 343–353 to nucleolin or LRP and heparan sulfate proteoglycans on the cell 8 Gordon, I., Genis, I., Grauer, E., Sehayek, E. and Michaelson, D. M. (1996) Biochemical surface may result in apoE being trafficked to the nucleus in a and cognitive studies of apolipoprotein-E-deficient mice. Mol. Chem. Neuropathol. 28, manner analogous to midkine, fibroblast growth factor and other 97–103 heparin-binding proteins (see [9] and references therein). 9 Quinn, C. M., Kagedal, K., Terman, A., Stroikin, U., Brunk, U. T., Jessup, W. and Garner, B. Previous work indicated that small amounts of apoE can be (2004) Induction of fibroblast apolipoprotein E expression during apoptosis, detected in the cytosol in association with microtubules [32]. Our starvation-induced growth arrest and mitosis. Biochem. J. 378, 753–761 observations derived from the living CHO apoE–GFP cell line (see 10 Elliott, D. A., Kim, W. S., Jans, D. A. and Garner, B. (2007) Apoptosis induces neuronal Supplementary Figure 1) concur with this previously published apolipoprotein-E synthesis and localization in apoptotic bodies. Neurosci. Lett. 416, work. Interestingly, certain microtubule-associated proteins can 206–210 be transported to the nucleus via a dynein-dependent pathway [49– 11 Do Carmo, S., Seguin, D., Milne, R. and Rassart, E. (2002) Modulation of apolipoprotein 51]. It is possible therefore that apoE–GFP may be trafficked to the D and apolipoprotein E mRNA expression by growth arrest and identification of key elements in the promoter. J. Biol. Chem. 277, 5514–5523 nucleus via microtubules. The alternative possibility, that apoE– 12 Terao, A., Apte-Deshpande, A., Dousman, L., Morairty, S., Eynon, B. P., Kilduff, T. S. and GFP may be initially secreted from CHO apoE–GFP cells and Freund, Y. R. (2002) Immune response gene expression increases in the aging murine then interact with cell-surface molecules that facilitate or mediate hippocampus. J. Neuroimmunol. 132, 99–112 its transport to the nucleus, seems unlikely, as exogenously added 13 Hough, C. D., Sherman-Baust, C. A., Pizer, E. S., Montz, F. J., Im, D. D., Rosenshein, apoE–GFP did not result in the appearance of apoE–GFP in the N. B., Cho, K. R., Riggins, G. J. and Morin, P. J. (2000) Large-scale serial analysis of nucleus. gene expression reveals genes differentially expressed in ovarian cancer. Cancer Res. 60, Our current results also indicate that nuclear apoE–GFP levels 6281–6287 were increased under serum starvation conditions. Previous 14 Yokoyama, Y., Kuramitsu, Y., Takashima, M., Iizuka, N., Terai, S., Oka, M., Nakamura, K., work has shown that nucleocytoplasmic trafficking of proteins Okita, K. and Sakaida, I. (2006) Protein level of apolipoprotein E increased in human is dependent on certain stress conditions. Interestingly, starv- hepatocellular carcinoma. Int. J. Oncol. 28, 625–631 ation can induce nucleocytoplasmic trafficking and promote accu- 15 Grainger, D. J., Reckless, J. and McKilligin, E. (2004) Apolipoprotein E modulates clearance of apoptotic bodies in vitro and in vivo, resulting in a systemic proinflammatory mulation of specific proteins in the nucleus [21,52]. It appears state in apolipoprotein E-deficient mice. J. Immunol. 173, 6366–6375 that the accumulation of specific proteins in the nucleus may 16 Mahley, R. W. and Rall, Jr, S. C. (2000) Apolipoprotein E: far more than a lipid transport be an adaptive survival response to changes in the extracellular protein. Annu. Rev. Genomics Hum. Genet. 1, 507–537 environment [52]. It remains to be determined whether targeting 17 Strittmatter, W. J. (2001) Apolipoprotein E and Alzheimer’s disease: signal transduction of apoE to the nucleus represents a generalized cell survival mechanisms. Biochem. Soc. Symp. 67, 101–109 response. 18 Thilakawardhana, S., Everett, D. M., Murdock, P. R., Dingwall, C. and Owen, J. S. (2005) In conclusion, we have shown that endogenous apoE–GFP Quantification of apolipoprotein E receptors in human brain-derived cell lines by real-time is detected in the nucleus and that the level of nuclear apoE– polymerase chain reaction. Neurobiol. Aging 26, 813–823 GFP is increased with serum starvation. Furthermore, exogenous 19 Panin, L. E., Russkikh, G. S. and Polyakov, L. M. (2000) Detection of apolipoprotein A-I, biotinylated apoE can also be transported to the nucleus if apoE B, and E immunoreactivity in the nuclei of various rat tissue cells. Biochemistry (Moscow) 65, 1419–1423 is presented to the cell as a lipoprotein complex. These studies 20 Chen, Y. C., Pohl, G., Wang, T. L., Morin, P. J., Risberg, B., Kristensen, G. B., Yu, A., provide new evidence that apoE may be targeted to the nucleus Davidson, B. and Shih Ie, M. (2005) Apolipoprotein E is required for cell proliferation and and shed light on factors that regulate this process. survival in ovarian cancer. Cancer Res. 65, 331–337 21 Chughtai, Z. S., Rassadi, R., Matusiewicz, N. and Stochaj, U. (2001) Starvation promotes This work was support by the Australian Research Council (Grant No. DP0557295). B.G. is nuclear accumulation of the hsp70 Ssa4p in yeast cells. J. Biol. Chem. 276, supported by an R.D. Wright Fellowship from the Australian National Health and Medical 20261–20266 Research Council. We are grateful to Professor Karl Weisgraber (Gladstone Institute of 22 Kockx, M., Guo, D. 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Received 13 September 2007; accepted 26 October 2007 Published as BJ Immediate Publication 26 October 2007, doi:10.1042/BJ20071261

73 c The Authors Journal compilation c 2008 Biochemical Society

3.2.4 Publication IV

4. David A. Elliott, Kayan Tsoi, Sandra Holinkova, Sharon L. Chan, Woojin S. Kim,

Declaration

I certify that I have performed the majority of the research work and writing contained in the following publication and that reproduction in this thesis does not breach copyright regulations.

David Anthony Elliott

Isoform-specific proteolysis of apolipoprotein-E in the brain

David A. Elliott a, Kayan Tsoi a, Sandra Holinkova a, Sharon L. Chan a, Woojin S. Kim

a,b, Glenda M. Halliday a,b, Kerry-Anne Rye c and Brett Garner a,b,*

a Prince of Wales Medical Research Institute, Randwick NSW 2031, Australia; b

School of Medical Sciences, Faculty of Medicine, University of New South Wales,

Sydney NSW 2052, Australia; c The Heart Research Institute, Sydney NSW 2050,

Australia

* Corresponding author: Dr Brett Garner, Prince of Wales Medical Research Institute,

Sydney, NSW 2031, Australia, Tel.: +61-2-93991024, Fax.: +61-2-93991005, E-mail: [email protected]

75 D. A. Elliott et al.

Abstract

Apolipoprotein-E (apoE) plays important roles in neurobiology and the apoE4 isoform increases risk for Alzheimer’s disease (AD). ApoE peptides are biologically active and may be produced in the brain. It is unclear if apoE proteolysis is dependent on isoform or AD status and this was addressed here. Hippocampus, frontal cortex, occipital lobe and cerebellum samples were homogenized into fractions that were soluble in Tris-buffered saline (TBS), Triton X-100 or guanidine hydrochloride and analysed for apoE fragmentation by Western blotting. Approximately 20% of apoE3 was detected as fragments and this was predominantly in TBS-soluble fractions. ApoE3 fragmentation was significantly greater than apoE4 fragmentation in all areas of the brain examined and this was not related to AD status. Cathepsin-D treatment generated apoE fragments that were similar to those detected in brain, however, no apoE isoform- specific differences in cathepsin-D proteolytic susceptibility were detected. ApoE3 was also present as disulphide-linked dimers in human and rabbit brain. This indicates apoE fragmentation in human brain is dependent on apoE isoform but not AD status.

Keywords: apoE, apoE-fragmentation, Alzheimer’s disease, human-brain, cathepsin-D

76 D. A. Elliott et al.

1. Introduction

Apolipoprotein-E (apoE) is a ~34 kDa glycoprotein that plays a crucial role in lipid transport in the peripheral circulation and in the central nervous system (CNS) (Ladu et al., 2000; Mahley, 1988). In humans, apoE exists as three major isoforms apoE2, apoE3 and apoE4 which differ in their Cys/Arg composition at positions 112 and 158. ApoE2 contains Cys112, Cys158; apoE3 contains Cys112, Arg158; and apoE4 contains Arg112,

Arg158 (Rall et al., 1982). ApoE4 is a major genetic risk factor for late-onset

Alzheimer’s disease (AD) whereby possession of one or two copies of the 4 allele confers a ~5 or ~10 fold increase, respectively, in AD risk (Corder et al., 1993;

Strittmatter et al., 1993). In contrast, the 2 allele is associated with decreased AD risk.

ApoE isolated from cerebrospinal fluid (CSF) is present in the form of both spherical and discoidal lipoprotein complexes (Fagan et al., 1999; LaDu et al., 1998; Pitas et al.,

1987). Astrocytes are thought to be the primary source of apoE in the brain, although microglia and neurons may also contribute to the CNS apoE pool under certain circumstances (Boschert et al., 1999; Elliott et al., 2007; LaDu, et al., 1998; Mahley,

1988; Metzger et al., 1996; Xu et al., 1999; Xu et al., 2000).

ApoE has several proposed functions beyond lipid transport including roles in immunoregulation, oxidative stress, stabilization of neuronal microtubules, nerve regeneration and apoptosis (Arai et al., 1999; Elliott, et al., 2007; Mahley et al., 1996;

Mahley and Rall, 2000; Miyata and Smith, 1996; Strittmatter et al., 1994). ApoE is also associated with amyloid plaques that are a characteristic of AD and substantial evidence indicates apoE plays a role in amyloid-beta (A) peptide clearance (Beffert et al., 1999; Bell et al., 2007; Jordan et al., 1998). Recent studies demonstrate that

77 D. A. Elliott et al. lipidated apoE promotes the extracellular degradation of A by insulin degrading enzyme (IDE) and also confirm that apoE targets A for intracellular degradation in microglia (Jiang et al., 2008). Despite intense research into the biological function of apoE, the precise mechanism by which the apoE4 isoform increases AD risk remains to be fully elucidated. Many differences between apoE3 and apoE4 structure and function have been reported that are potentially relevant to AD and these include the findings that: domain interaction mediated by a salt bridge between Arg61 and Glu255 in apoE4 reduces lipid-binding capacity (Xu et al., 2004), lipidated apoE4 has a lower affinity for

A (LaDu et al., 1994; Tokuda et al., 2000), apoE4 is less efficient at stabilizing microtubules (Strittmatter, et al., 1994), apoE4 exhibits weaker antioxidant activity

(Miyata and Smith, 1996), and apoE4 is structurally less stable (Clement-Collin et al.,

2006; Morrow et al., 2000) when compared to apoE3. In addition, a significant proportion apoE3 (and apoE2) is present in plasma and CSF as a disulfide-linked homodimer and as an apoE-apoA-II heterodimer (LaDu et al., 1997; Montine et al.,

1998; Rebeck et al., 1998; Weisgraber and Shinto, 1991). This may be important as apoE4 lacks Cys and cannot form disulphide-linked dimers and the available data suggests that heterodimeric forms of apoE bind A with higher avidity than monomeric apoE and that this attenuates A neurotoxicty (Yamauchi et al., 2000; Yamauchi et al.,

1999).

It is also recognised that apoE undergoes proteolytic cleavage in the brain to form truncated fragments; some of which preferentially associate with neurofibrillary tangles

(NFT) and amyloid plaques (Aizawa et al., 1997; Cho et al., 2001; Harris et al., 2003;

Huang et al., 2001). Although the identity of the key proteolytic enzyme(s) generating apoE fragments in vivo is still unknown, previous work points toward a chymotrypsin-

78 D. A. Elliott et al. like serine protease and/or an aspartic protease such as cathepsin-D (CatD) as potential candidates (Harris, et al., 2003; Marques et al., 2004; Zhou et al., 2006). Because apoE is susceptible to cleavage by thrombin, which is also expressed in the brain, this could represent another plausible pathway for the formation of apoE fragments in vivo

(Aizawa, et al., 1997; Arai et al., 2006; Castano et al., 1995; Mhatre et al., 2004;

Smirnova et al., 1997; Tolar et al., 1997).

The generation of apoE fragments in the brain is very likely to be physiologically significant as several studies have demonstrated that truncated apoE (and apoE-mimetic peptides) exert potent bioactive properties that regulate neuronal signalling (Gay et al.,

2007; Hoe et al., 2005) and (depending on the fragments analysed) may promote neurodegeneration (Chang et al., 2005; Clay et al., 1995; Crutcher et al., 1994; Huang, et al., 2001; Tolar et al., 1999; Tolar, et al., 1997; Wellnitz et al., 2005) or stimulate neuroprotective and anti-inflammatory pathways (Aono et al., 2003; Laskowitz et al.,

2001; Li et al., 2006; Lynch et al., 2003; Lynch et al., 2005; Singh et al., 2008). It is therefore important to examine the extent to which apoE fragmentation occurs in the human brain and whether the level of proteolysis as well as the fragmentation profile is associated with apoE4 geneotype and/or AD.

In this investigation we conducted a comprehensive analysis of apoE fragmentation in the human brain and examined the influence of apoE4 isoform and AD disease status on apoE proteolysis.

2. Materials and methods

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2.1. Human brain tissue

Brain tissue samples were obtained through the Australian Brain Donor Program with ethics approval from the University of New South Wales Human Research Ethics

Committee (approval No. HREC03322). Cortical neuritic plaques and neurofibrillary tangles were assessed according to current international standards in order to pathologically confirm the diagnosis of AD post-mortem (Mirra et al., 1991; NIA,

1997). The tissue samples were supplied as two separate cohorts (Table 1). Samples from Cohort 1 were supplied as archived tissue homogenates that were stored -80˚C.

Sample from Cohort 2 were supplied as frozen tissue and homogenized by us.

2.2. Human tissue preparation

Samples in Cohort 2 were taken from the hippocampus, cerebellum and both the white and gray matter from the frontal cortex and stored at -80°C. The homogenisation protocol was based on an established method (Hirsch-Reinshagen et al., 2005).

Between 60-90 mg of brain tissue was homogenized with a pre-chilled 1 mL glass dounce homogenizer, using 15 volumes of ice-cold TBS (pH 7.4) containing protease and phosphatase inhibitors (Calbiochem, San Diego, CA). After centrifugation at

16,000 g for 25 minutes at 4°C the TBS-soluble supernatant fraction was collected. The pellet was then washed with 50 μL of TBS and centrifuged again for 5 min, the supernatant was then discarded and pellet was resuspended in 15 volumes of TBS containing protease and phosphatase inhibitors and 1% (w/v) Triton X-100 (TBS-X) by pipetting up and down. Samples were then mixed by rotation for 30 min at 4°C, followed by centrifugation at 16,000 g for 25 min at 4°C. The TBS-X soluble

80 D. A. Elliott et al. supernatant fraction was collected, the pellets were then re-centrifuged for 5 min to remove residual TBS-X. Pellets were then dislodged by vortex mixing in the presence of 400 μL of 5M guanidine HCl (pH 8.0) (gHCl) containing protease and phosphatase inhibitors, then resolubilised by rotation for 4 h at 22°C. Samples in Cohort 1 were processed in a similar method with the exception that 5% (w/v) SDS was used to prepare the detergent-soluble fraction and 8M urea with 8% (w/v) SDS was used to solubilise the detergent-insoluble pellet. There were slight variations in the regions of the brain that were available for sampling in the two cohorts (Table 1).

An adult male Watanabe rabbit was euthanased via cardiac puncture using 5ml

Lethabarb (1 ml per 2kg body weight, Virbac, Sydney, Australia) and the brain surgically removed to dry ice and processed immediately in order to eliminate post- mortem delay. Approximately 100 mg of tissue was removed from the cerebral cortex, all visible vasculature was removed and the sample was rinsed three times in ice-cold

PBS. The sample was then homogenized following the protocol detailed above for the human Cohort 2 samples.

2.3. ApoE genotyping

Genomic DNA was extracted from brain tissue and APOE amplified by PCR. Briefly, each reaction (50 μL) contained 200 nM of each primer (Invitrogen, Carlsbad, CA) 5’-

TCCAAGGAGCTGCAGGCGGCGCA-3’ (forward) and 5’-

ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3’ (reverse), 2 mM dNTPs, 2 mM

MgCl2, 2 U Taq polymerase (PCR reagents supplied by Promega, Madison, WI) and

400 ng DNA, all combined in nuclease free H2O. Amplification was carried out with 38

81 D. A. Elliott et al. cycles of denaturation (95 ºC, 30 sec), annealing (60 ºC, 30 sec) and extension (70 ºC,

30 sec). The 244 bp PCR product was purified using the QIAquick PCR purification kit

(Qiagen, Venlo, Netherlands), following the manufacturer’s protocol, and eluted in 40

μL H2O. Endonuclease restriction digests (25 μL) were performed on 15 μL of eluted

DNA using either AflIII (5 U) or HaeII (20 U) in the presence of BSA (100 μg/mL) and the supplied buffer (New England Biolabs, Ipswich, MA) at 37 ºC for 16 h. The 3 allele is resistant to both enzymes while 4 is cleaved by AflIII (producing a 190 bp product) and 2 is cleaved by HaeII (producing a 191 bp product) assessed using ethidium bromide stained 8% polyacrylamide gels.

2.4. SK-N-SH neuroblastoma cell culture

Cell culture media and additives were from Invitrogen (Melbourne, Australia).

Human neuronal SK-N-SH cells were routinely grown in DMEM, 10% (v/v) fetal calf serum (FCS), 2mM glutamine, and 100 IU/ml penicillin and 100 μg/ml streptomycin.

2 Cultures were grown in 75 cm flasks at 37°C in 5% CO2 and plated into 6-well plates for use in experiments. To induce apoE expression, SK-N-SH cells were cultured under serum starved conditions (5 days of culture without media replenishment) and harvested in cell lysis buffer (10 mM Tris-HCl, 10 mM Na2PO4/NaHPO4, pH 7.5, 130 mM NaCl, 1% Triton-X-100, 10 mM NaPPi) as described previously (Elliott, et al.,

2007). SK-N-SH have been shown to be apoE3/3 homozygous (Dupont-Wallois et al.,

1997).

2.5. Western blotting

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Bicinchoninic acid protein assays were performed on brain homogenate samples and equal amounts of protein were separated on 12 % SDS-PAGE gels and transferred onto 0.45 μm nitrocellulose membranes at 100 volts for 30 min. Membranes were

Ponceau stained and scanned before blocking overnight at 4°C in PBS containing 5%

(w/v) non-fat dry milk. The membranes were then probed with the relevant antibodies at 22˚C for 1 h to reveal the bands of interest. Concentrations of antibodies were: rabbit polyclonal anti-human apoE 1/1000 (Dako, Glostrup, Denmark), goat polyclonal anti-human apoE 1/5000) (Calbiochem), rabbit polyclonal anti -actin

1/2000 (Sigma, St. Louis, MO, Cat No. A5060), mouse monoclonal anti-human apoE

21-F3-D2 1/1000 (Biogenesis, Poole, UK). The membranes were washed three times in PBS containing 0.1% (w/v) Tween-20 and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (Dako, 1/2000), rabbit anti-goat (Dako, 1/2500) or rabbit anti-mouse (Dako, 1/1000) secondary antibody for 1 h. The proteins of interest were detected using enhanced chemiluminescence (ECL, Amersham

Biosciences) and X-ray film and signal intensity was quantified using NIH ImageJ software.

2.6. A quantification by ELISA

The concentration of A1-40 in gHCl brain homogenate samples was determined using a BetaMark x-40 ELISA kit (Cat No. SIG-38950, Covance, Emeryville, CA) following the manufacturer’s instructions. Samples were diluted 1:2500 in 55 mM

NaHCO3 (pH 9.0) and all standards and samples were assayed in duplicate.

2.7. Preparation of recombinant apoE and apoE-discs

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Human APOE cDNA was generously provided by Professor Karl Weisgraber

(Gladstone Institutes, UCSF). Recombinant human apoE3 and apoE4 were prepared from Escherichia coli as previously described (Morrow et al., 1999). ApoE discs that resemble astrocyte-derived apoE lipoproteins (LaDu, et al., 1998) were prepared containing recombinant apoE, 1-palmitoyl-2-oleylphosphatidyl choline (POPC) and cholesterol using the cholate dialysis method and characterised as described previously

(Kim et al., 2007; Rye et al., 2006). ApoE discs had an average diameter of 17.0 nm as judged by gel filtration chromatography (Rye, et al., 2006). In the experiments addressing potential differences related to the apoE3 and apoE4 isoforms, the POPC / cholesterol / apoE molar ratios were: apoE3, 56.6:6.3:1.0; apoE4, 42.9:6.4:1.0.

2.8. Enzymatic treatment of brain homogenates, apoE and apoE-discs

Thrombin digestion was performed by incubating brain homogenates (15μg of protein) prepared in the absence of protease inhibitors with 3U of thrombin (Sigma, St.

Louis, MO) in PBS at 37°C for 16 hours. Two control conditions were also analysed: cell lysates either stored at -80°C for the incubation period or incubated with thrombin that was heat-inactivated at 95°C for 15 minutes. Thrombin digestion was also performed using 70 ng of human apoE, purified from plasma (Biodesign, Saco, ME), to assess whether monoclonal antibody 21-F3-D2 (Biogenesis) interacted with the N- terminal domain or C-terminal domain (see Supplemental Data). ApoE was also digested by CatD using conditions based on an established method (Zhou, et al., 2006).

Recombinant human apoE3 and apoE4 (250 nM, i.e. 8.46 ng / μL), either lipid free or as lipidated discs (see above), was incubated with purified human CatD (127 nM, i.e.

84 D. A. Elliott et al.

5.4 ng / μL, Calbiochem, CA, USA) in 0.1 M acetate buffer, pH 4.5, and routinely incubated at 37°C for 0, 2, 8, and 16 h. A sample incubated for 16 h in the absence of

CatD was used as the control condition.

2.9. Statistical analysis

Unless stated otherwise, experiments were performed in triplicate and repeated at least three times. Data are presented as means with S.E. shown by error bars.

Differences were considered significant where P < 0.05 as determined by the 2-tailed

Student’s t-test for unpaired data.

3. Results

3.1. Detection of apoE fragments in TBS-soluble fractions of brain homogenates

Previous studies have demonstrated that apoE proteolytic fragments are present in brain homogenates from both control and AD brains (Cho, et al., 2001; Huang, et al.,

2001; Marques, et al., 2004; Zhang et al., 2001; Zhou, et al., 2006). However, only limited data are available regarding the regions of the brain in which apoE fragmentation may occur and whether apoE fragmentation is associated with AD or apoE genotype per se. In order to address this we analysed apoE fragmentation in the TBS-soluble fraction of homogenized hippocampus from five apoE3/3 control samples and five apoE4/4 AD cases (Cohort 1, see Table 1). Western blot analysis using a rabbit polyclonal antibody revealed multiple apoE fragments of ~ 15 to 30 kDa in the apoE3/3

85 D. A. Elliott et al. control cases whereas there was a single fragment of ~ 25 kDa detected in most of the apoE4/4 AD cases (Fig. 1A). Although the levels of fragmentation varied within each of the groups analysed, the data indicate increased fragmentation in the apoE3 samples.

The apoE fragmentation patterns observed are in close agreement with patterns that have been reported previously using samples of human frontal cortex and temporal lobe gray matter (Zhang, et al., 2001; Zhou, et al., 2006). The fragments detected in both the apoE3/3 control samples and apoE4/4 AD cases were not detectable using an apoE C- terminal monoclonal antibody (Fig. 1B), suggesting that the fragments are from the N- terminal and thus derived from C-terminal truncation. This result is in general agreement with previous data indicating that C-terminal monoclonal antibodies do not detect the quantitatively major human brain apoE fragments (Huang, et al., 2001; Zhang, et al.,

2001). It should be noted though that a ~15 kDa fragment (possibly corresponding to the ~15 kDa fragment we have detected using rabbit polyclonal antibody) was previously shown to be selectively recognised by apoE C-terminal monoclonal antibody 3H1 indicating that proteolysis to generate brain apoE fragments may also occur within the N-terminal domain to some degree. Western blotting for apoE3/3 fragments was also conducted using a different polyclonal antibody (raised in goat) and this revealed an essentially identical pattern of apoE fragments (see Supplemental data). Probing with the secondary antibodies alone did not generate a signal (data not shown). This indicates that the detection of apoE fragments above is not a peculiarity associated with the antibody we have used.

3.2. ApoE fragmentation in different brain regions

86 D. A. Elliott et al.

We next examined tissue homogenates derived from the occipital lobe and the frontal cortex for the presence of apoE fragmentation. The samples used for this analysis were all derived from the same brains used for the analysis of hippocampus shown in Figure

1A. Representative samples of apoE3/3 control and apoE4/4 AD tissues indicate that apoE fragmentation is more pronounced in the control samples and that this is independent of the brain region analysed (Fig. 1C). Overall, the levels of apoE fragments detected in the TBS-soluble fractions of occipital lobe, hippocampus and frontal cortex were significantly increased by 1.9, 2.7 and 3-fold, respectively, in the apoE3/3 control samples compared to the apoE4/4 AD samples (Fig. 1D).

3.3. ApoE fragmentation in soluble and insoluble homogenate fractions

The data above indicate that brain apoE fragmentation is more extensive in apoE3/3 control as compared to apoE4/4 AD samples. From this data it is not clear if the reduction in fragmentation in the latter group is due to the apoE4 genotype or due to

AD. To address this we analysed additional samples (from Cohort 2) including hippocampal tissues from apoE3/3 AD cases. The data indicate that multiple apoE fragments are also detected in the apoE3/3 AD cases (Fig 2A). One possible explanation for the reduced apoE fragmentation detected in the apoE4 samples could be related to an enhanced deposition of apoE fragments in amyloid plaques or within fractions of the brain homogenates that are not soluble in TBS. The SDS-soluble and urea-soluble fractions of the Cohort 1 homogenate samples were therefore also assessed for apoE fragments however, fragments were not consistently detected in either of these fractions

87 D. A. Elliott et al.

(data not shown). We therefore used Triton X-100 (TX-100) and guandine hydrochloride (gHCl) to solublize the TBS-insoluble pellets in the Cohort 2 samples

(see Experimental Procedures) and analyzed these fractions for apoE fragments. The data indicate that apoE fragments were scarcely detectable in any of the TX-100- soluble fractions while some of the fragments were apparent in the gHCl fractions (Fig.

2B-C). The detection of apoE fragments in the gHCl-soluble fractions was variable but it was noted that at least in the hippocampal samples, fragments were detected in the apoE4/4 AD cases (Fig. 2C). Interestingly, a triplet of apoE fragments at ~12 to 15 kDa was detected in the apoE4/4 gHCl-soluble fractions of hippocampus and this shows striking similarities with fragments previously characterized in AD brain (Zhou, et al.,

2006). While one or more of these fragments may in fact be apoE C-terminal fragments

(as previously suggested (Zhang, et al., 2001)), derived from amyloid plaque, the major findings from the work presented here is the identification of the more extensive apoE fragmentation in TBS-soluble fractions of all apoE3/3 samples; independent of AD diagnosis.

3.4. ApoE fragmentation in soluble and insoluble homogenate fractions from different brain regions

In order to asses if apoE fragments could also be detected in TX-100-soluble and gHCl-soluble fractions of tissue homogenates derived from regions in addition to the hippocampus, we went on to analyse frontal cortex and cerebellum from Cohort 2. In addition, it was previously suggested that apoE fragmentation may occur to a greater extent in white matter than gray matter (Zhang, et al., 2001) so this was specifically

88 D. A. Elliott et al. assessed in the frontal cortex samples. ApoE fragmentation in the frontal cortex gray matter was clearly detected in all apoE3/3 samples independent of AD diagnosis, whereas the apoE4/4 AD samples contained much lower levels of fragments overall with a quantitatively minor band detected at ~ 25 kDa (Fig. 3A). This is in close agreement with the data derived from the Cohort 1 TBS-soluble fractions (Fig. 1). A similar pattern was observed in the TX-100-soluble fraction from frontal cortex (Fig

3B) whereas apoE fragments were variably detected in the gHCl-soluble fraction of frontal cortex gray matter (Fig 3C). Very similar apoE fragmentation patterns were detected in the corresponding frontal cortex white matter samples (Fig. 3D-F). There was no evidence to suggest that apoE fragmentation was increased in white matter as compared to gray matter. Because the TBS-soluble frontal cortex gray matter samples from Cohort 1 and Cohort 2 were generated using the same method, data for quantification of apoE fragmentation was pooled and the results indicate that the level of apoE fragments in the TBS-soluble fraction of apoE3/3 control and AD samples was significantly higher than in the apoE4/4 samples (Fig 3G).

The same experimental approach was also used to examine cerebellum samples and this also revealed that apoE fragmentation was more pronounced in the apoE3/3 samples (see Supplemental data). In order to confirm the pathological diagnosis of AD in the Cohort 2 samples, the levels of A1-40 in the gHCl fractions of the hippocampus, frontal cortex gray matter and cerebellum were measured be ELISA. The data indicated that all AD cases contained high levels of A deposition and in the cases with the highest overall A levels, A was also deposited in the cerebellum (Fig 3H).

89 D. A. Elliott et al.

Using these samples as a qualitative guide, there was no clear relationship between the levels of apoE fragments detected in the gHCl fractions of any of the tissues and the amount of A detected.

3.5. Comparison of brain apoE fragments with thrombin and cathepsin-D cleaved apoE

The data above showed that apoE fragments were consistently detected in TBS- soluble fractions of human brain tissues and that this was present to a significantly greater degree in apoE3/3 samples. Because both thrombin and CatD are present in the brain and implicated in the generation of apoE fragments (Arai, et al., 2006; Mhatre, et al., 2004; Zhou, et al., 2006), we next compared the brain apoE fragmentation pattern with apoE fragments derived from thrombin and CatD treatment.

Two thrombin cleavage sites are present in the apoE protein, a major site at

Arg191/Ala192 and a minor site at Arg215/Ala216 (Wetterau et al., 1988). Thrombin cleavage of apoE generates two major fragments of 22 kDa and 10-12 kDa. To asses the possibility that one or more of the apoE fragments detected in human brain may be due to thrombin cleavage, a control apoE3 brain sample was incubated with thrombin and this resulted in the formation two prominent fragments at ~22 kDa and ~17 kDa (Fig.

4A). The formation of the 22 kDa band is entirely consistent with the predicted apoE

N-terminal domain fragment whereas the 17 kDa may represent a further degradation product of the 22 kDa fragment (i.e. generated by endogenous proteases that act upon

90 D. A. Elliott et al. the thrombin-generated N-terminal domain). Ponceau staining of the membranes indicated that there were no gross changes to the major proteins detected in the regions of the apoE fragments which suggests that the immunostaining is not simply due to non-specific staining of newly formed thrombin fragments that are not derived from apoE. A careful comparison of the western blot patterns of apoE thrombin fragments with the endogenous brain apoE fragments (Fig. 4A) indicated that they were not identical and that several of the endogenous fragments were also susceptible to thrombin cleavage (indicating that the fragments contain the one or both of the abovementioned thrombin cut sites). Since the endogenous brain apoE fragments did not appear to be the same as apoE thrombin fragments, we next focused on the lysosomal protease CatD.

Using a similar experimental protocol (modified to pH 4.5 to mimic lysosomal conditions), we used recombinant apoE (r-apoE) as a substrate to generate CatD cleavage fragments. Although there may be some differences in peptide patterns when comparing the r-apoE with brain-derived apoE (e.g. due to glycosylation at Thr194), comparison of the CatD proteolytic apoE fragments human indicated that the major r- apoE fragment produced at ~24 kDa aligned with one of the brain apoE fragments (Fig.

4B). This is consistent with previous data indicating that CatD is involved in apoE fragmentation in the human brain (Zhou, et al., 2006). CatD is known to play a role in apoptosis under many conditions (Kagedal et al., 2001; Kagedal et al., 2001; Quinn et al., 2004; Roberg et al., 2002) and we have shown previously that apoE is produced in apoptotic SK-N-SH neurons (Elliott, et al., 2007). Interestingly, a ~24 kDa apoE

91 D. A. Elliott et al. fragment was also detected in cell lysate from apoptotic human SK-N-SH neuroblastoma cells (Fig. 4C).

3.6. Proteolysis of apoE3 and apoE4 by cathepsin-D in vitro

Based on our findings that at least one of the major apoE fragments found in the brain shared similarities with fragments derived form CatD-treated r-apoE3, we next examined the possibility that differences in CatD proteolysis of apoE3 and apoE4 may explain the isoform-specific fragmentation patterns we observed in human brain samples. CatD proteolysis of r-apoE3 and r-apoE4 over a 16 h time course generated several fragments predominantly in the 24 to 30 kDa range (Fig 5A and B). Although there was a tendency for r-apoE4 to be more resistant to proteolysis, this trend was not consistently observed and when the data from 3 independent experiments were combined, no significant difference in either the rate of degradation or the size of the fragments was detected (Fig 5). Similar experiments were conducted using apoE discs, which more closely resemble CNS lipoproteins, and once again we could not detect a significant difference in sensitivity to CatD-mediated proteolysis when the apoE3 and apoE4 isoforms were compared (Fig 5C-D). The data did indicate that fragmentation of apoE incorporated in discs resulted in the generation of several bands below ~24 kDa, however this was also isoform-independent overall. This suggests that some of the fragments formed initially may be protected to a small degree from further CatD proteolysis by the lipid environment. While the data provided here and in previous reports (Cataldo and Nixon, 1990; Zhou, et al., 2006) are consistent with a role for

92 D. A. Elliott et al.

CatD in human brain apoE fragmentation, it does not appear that apoE isoform modulates this proteolytic pathway. This therefore cannot explain the isoform-specific differences in apoE fragmentation pattern we have detected in the human brain.

3.7. ApoE3 forms disulfide-linked dimers in human brain

It is established that apoE3 forms disulfide-linked homodimers and apoE3-apoA-II heterodimers in human plasma and CSF (Rebeck, et al., 1998; Weisgraber and Shinto,

1991). As apoE4 lacks Cys it cannot form these bonds. Whether apoE3 exists in a dimeric state in human brain is unknown and we therefore focused on this issue in a final series of experiments. Western blot analysis of brain homogenate derived from the frontal cortex of a control apoE3/3 subject indicated a clear apoE3 homodimer when the sample was run under non-reducing conditions (Fig 6A). The homodimer was detected at ~95 kDa (as opposed to the predicted ~68 kDa) which is consistent with previous data (Rebeck, et al., 1998; Weisgraber and Shinto, 1991). The apoE3 homodimer was removed after incubation with thrombin, as predicted, further confirming that the ~95 kDa band is due to apoE homodimer and not non-specific binding of the antibodies used. Incubation in the presence of heat-inactivated thrombin resulted in a partial loss of the apoE dimer which suggests that endogenous proteases may also degrade apoE

(Fig 6A). A band of relatively lower intensity was also observed ~43 kDa in the non- incubated apoE3 control condition, which may represent the apoE-apoA-II heterodimer previously observed in plasma and CSF (Rebeck, et al., 1998; Weisgraber and Shinto,

1991). An alternative possibility is that this band may represent a disulfide-linked apoE

93 D. A. Elliott et al.

N-terminal domain homodimer with an predicted MW of ~44 kDa. If so, this band would theoretically be resistant to thrombin cleavage, however this was not observed

(Fig 6A). While this suggests that the ~43 kDa band is not a disulfide-linked apoE N- terminal domain homodimer, one caveat is that endogenous proteases may also degrade the ~43 kDa form of apoE. The data also indicate that the apoE3 homodimer and heterodimer were both present in apoE3 AD brain and, as predicted, dimers were not detected in apoE4 AD brain (Fig 6B-C). Since the post-mortem delay for the specimen shown in Figure 6B is only 1 h, it is unlikely that apoE3 dimers form artificially.

3.8. ApoE3 forms disulfide-linked dimers in human SK-N-SH neurons and rabbit brain

In order to confirm that apoE3 can be detected in a dimeric state under phsyiological conditions, we analysed apoE derived from the human SK-N-SH neuroblastoma cell line which has previously been shown to up-regulate apoE production under serum starved conditions (Elliott, et al., 2007) and to express the apoE3/3 genotype (Caillet-Boudin et al., 1998). Analysis of SK-N-SH cell lyaste under non-reducing conditions revealed the presence of both the ~95 kDa apoE homodimer and the ~43 kDa heterodimer (Fig. 7A).

As this western blot was probed using C-terminal specific monoclonal antibody, the data also show that the ~43 kDa band is not a disulfide-linked N-terminal homodimer.

Rabbits are one of the few non-human species known to contain an apoE Cys112 residue (LaDu, et al., 1997). Rabbit brain was processed immediately at the time of death to eliminate post-mortem delay and all measures were taken to prevent serum and

94 D. A. Elliott et al.

CSF contamination (see Experimental Procedures). An apoE band ~95 kDa was also detected in rabbit brain when samples were run in the non-reduced state (Fig 7B), again indicating that apoE containing Cys112 does form a homodimer in the brain.

4. Discussion

The data presented here demonstrate two inherent differences between the apoE3 and apoE4 isoforms in the human brain. First, fragmentation of apoE3 is more extensive than apoE4 and this is independent of AD status, A load and brain region. We analysed areas of the brain that are known to be severely affected by AD, such as the hippocampus and frontal cortex, as well as areas that are relatively spared, such as the occipital lobe and cerebellum, and the data show that apoE3 fragmentation occurs in all regions in both control and AD brains. Therefore, apoE3 fragmentation is not associated with AD. Second, we identified apoE3 disulfide-linked dimers in human brain and showed that similar dimers are also detected in human neuroblastoma cells expressing apoE3 and in freshly prepared rabbit brain. This suggests that apoE3 dimerisation is a physiologically relevant process in the human brain; as it is also in human CSF and plasma (Rebeck, et al., 1998; Weisgraber and Shinto, 1991). Although the functional consequences of these differences are yet to be elucidated, the data suggest that the formation of stable apoE3 fragments and apoE3 disulphide-linked dimers are normal physiological processes in the human brain.

Our data, along with previous work (Marques, et al., 2004; Zhou, et al., 2006), suggests that CatD may play a role in apoE proteolysis in the brain. CatD is a lysosomal enzyme, however, there is also evidence of extracellular and cytoplasmic

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CatD localisation, particularly with brain ageing (Cataldo and Nixon, 1990; Cataldo et al., 1991; Jung et al., 1999). It is conceivable that apoE3 fragmentation is not strictly a process of lysosomal apoE destruction. Interestingly, studies in CatD-deficient mice suggest that CatD plays a vital role in limited proteolysis of biologically active proteins as opposed to bulk degradation in lysosomes (Saftig et al., 1995). This provides a plausible role for CatD in the generation of soluble apoE fragments that may be biological active.

It remains possible that other proteases may also be involved in apoE3 fragmentation or that additional in vivo factors, such as apoE lipidation state or more subtle differences in apoE conformation, may differentially regulate the proteolysis of the apoE3 and apoE4 isoforms. An in vitro study has provided evidence consistent with the latter theory by demonstrating that apoE4 is more resistant to proteolysis than apoE3 due to restricted access of proteases to the C-terminal region comprising amino acid residues 230-260 (Barbier et al., 2006). Differences in intracellular trafficking of apoE may also play a role in the regulation of proteolysis, for example, previous studies have shown that when exogenous apoE3 or apoE4 is added to human neuron cultures, more apoE4 localises to lysosomes (DeKroon and Armati, 2001). An alternative explanation for the increased levels of apoE3 fragments detected in comparison to apoE4 may be related to the structural stability of the apoE N-terminal domain. Related to this, studies of the truncated apoE N-terminal domain demonstrate that the apoE4 fragment is more susceptible to denaturation than the apoE3 fragment (Acharya et al., 2002; Clement-

Collin, et al., 2006; Morrow, et al., 2000).

96 D. A. Elliott et al.

Previous studies indicate that apoE fragments from the C-terminal rather than the N- terminal are more likely to be associated with amyloid plaques (Aizawa, et al., 1997;

Cho, et al., 2001). Consistent with this, we detected three fragments of apoE at ~12 to

15 kDa in the gHCl-soluble fractions of hippocampus in AD cases with apoE4 genotype that bare a striking similarity to apoE C-terminal fragments previously detected in the human brain (Zhou, et al., 2006). The deposition of apoE fragments in insoluble complexes / plaque however does not account for the differences in apoE fragmentation seen in the TBS-soluble fractions as in other regions (e.g. frontal cortex gray matter, frontal cortex white matter and cerebellum) there were major differences in the amounts of apoE fragments in TBS-soluble fractions but not in the corresponding gHCl-soluble fractions.

Our data raise the possibility that apoE3 fragments may be biologically active. In vitro studies have investigated the bioactive properties of apoE and apoE-derived peptides, with results varying depending on the apoE fragment and cell model used. In brief, several studies have demonstrated that apoE interacts with cell surface receptors mediating a range of effects including down-regulation of microglial activation, suppression of NMDA induced excitotoxicity and the activation of anti-apoptotic cell signalling pathways with most of the effects though to be mediated by the receptor binding domain (RBD) of apoE (Aono, et al., 2003; Hayashi et al., 2007; Laskowitz, et al., 2001). ApoE peptides encompassing this region, as well as mimetic peptides containing a tandem repeat of this region, have been shown to exhibit even greater activity than the full length apoE (Aono, et al., 2003; Laskowitz, et al., 2001; Li, et al.,

2006; Singh, et al., 2008). Some studies have also reported that apoE derived peptides and the N-terminal domain possess neurotoxic properties (Crutcher, et al., 1994; Tolar,

97 D. A. Elliott et al. et al., 1999; Tolar, et al., 1997). These studies emphasise that apoE fragments are biologically active and imply that apoE3 fragments present in the human brain may interact with cell surface receptors in an analogous manner. These fragments may also interact with a range of soluble apoE receptors present in the CNS (Rebeck et al.,

2006).

Previous studies have demonstrated that the ~22 kDa N-terminal domain exhibits enhanced LDL receptor binding in the presence of lipid and may maintain some degree of lipid binding ability (Innerarity et al., 1984; Lu et al., 2000; Weers et al., 2003;

Weers et al., 2001). Interestingly, the N-terminal domain of apoE is also capable of binding the A peptide (Aleshkov et al., 1997; Golabek et al., 2000). It is possible that the apoE3 fragments we have detected in the human brain may also bind lipids and play a role in the A degradation pathways described recently (Jiang, et al., 2008).

This study demonstrates for the first time, to our knowledge, that apoE3 disulfide- linked dimers are present in the human brain. In vitro studies have demonstrated that the apoE3 homodimer is present in media from cultured astrocytes (Sun et al., 1998) and our data demonstrate that under certain circumstances apoE dimers are also present in neuroblastoma cell lysate. Previous studies have demonstrated that apoE3 homodimers and apoE3-apoA-II heterodimers have diminished LDL receptor binding activity, 20% and 30%, respectively, in comparison to apoE3 monomer (Innerarity et al., 1978; Weisgraber and Shinto, 1991). Interestingly, in vitro studies have also shown that the apoE-apoA-II dimer is more effective than apoE monomer in binding soluble

A1-42 and inhibiting its internalisation by neurons (Yamauchi, et al., 2000;

Yamauchi, et al., 1999). It is therefore plausible that the apoE3 dimers we have

98 D. A. Elliott et al. detected in human brain have distinct biological properties that differ from apoE monomer.

In conclusion, we have identified apoE fragments in the human brain that are increased by the apoE3 genotype but are not associated with AD. In addition, we show for the first time that apoE3 is present in a dimerised form in the human brain. Based on these observations and previously published work, we predict that these fragments and dimers may represent novel biologically active forms of apoE that highlight fundamental differences between apoE3 and apoE4 in the human brain.

Disclosure statement

The authors declare no conflict of interest.

Acknowledgements

Melissa Broe, Karen Murphy and Dr John Rawlinson are thanked for their expertise in the collection and preparation of brain tissues. Human brain tissue samples were received from the Australian Brain Donor Program, Prince of Wales Medical Research

Institute Tissue Resource Centre, which is supported by the Australian National Health and Medical Research Council (NHMRC). We are very grateful for helpful advice provided by Prof Keith Crutcher and Dr Weidong Zhou. This work was supported by the Australian Research Council (Discovery Project ID. DP0557295). GH and BG are

99 D. A. Elliott et al. supported by Fellowships from the NHMRC.

Abbreviations

A, amyloid-; Ab, antibody; AD, Alzheimer's disease; apoE, apolipoprotein-E; CatD, cathepsin-D; CNS, central nervous system; CSF, cerebrospinal fluid; CON, control;

FC, frontal cortex; gHCl, guanidine hydrochloride; HI-Thr, heat-inactivated thrombin;

Hippo, hippocampus; IDE, insulin degrading enzyme; NEP, neprilysin; NFT, neurofibrillary tangles; LBD, lipid binding domain; NR, non-reduced; Occip, occipital lobe; PBS, phosphate buffered saline; POPC, 1-palmitoyl-2-oleylphosphatidyl choline;

R, reduced; RBD, receptor binding domain; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TBS, tris buffered saline; TBS-X, TBS containing

Triton X-100; Thr, thrombin

100 D. A. Elliott et al.

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113 D. A. Elliott et al.

FIGURE LEGENDS

Fig. 1. ApoE fragmentation in TBS-soluble brain homogenates from control apoE3 and

AD apoE4 donors. Western blot analysis of brain apoE fragmentation in control apoE3

“C” and AD apoE4 “AD” homozyogtes. Comparison of hippocampal samples using rabbit anti-apoE polyclonal Ab (A). Analysis of hippocampal samples using anti-apoE

C-terminal specific monoclonal Ab (B). Samples from three different brain regions; occipital lobe “Occip”, hippocampus “Hippo” and frontal cortex “Front” were compared using rabbit anti-apoE polyclonal Ab, representative western blot shown

(C). Optical density analysis was performed to quantify the percentage of apoE present as fragmentation products in control apoE3 and AD apoE4 samples (D). Equal protein loading was verified by -actin detection on stripped and re-probed western blots (A, C). Data in “D” are means (n=5) with S.E. represented by error bars. *P<0.05.

Fig. 2. ApoE fragmentation in different fractions of hippocampal homogenate. Western blotting was performed using goat anti-apoE polyclonal Ab. Control apoE3, AD apoE4 and AD apoE3 homozygote samples are compared in the TBS-soluble (A), TBS-

Triton-X100-soluble (B) and gHCl-soluble (C) fractions. Equal protein loading was verified by both Ponceau protein staining and by -actin detection on stripped and re- probed western blots.

Fig. 3. ApoE fragmentation are in both gray and white matter of frontal cortex homogenate. Western blotting was performed using goat anti-apoE polyclonal Ab.

Control apoE3, AD apoE4 and AD apoE3 homozygote samples are compared using

114 D. A. Elliott et al. gray matter (A-C) and white matter (D-F) from frontal cortex TBS-soluble (A,D), TBS-

Triton-X100-soluble (B,E) and gHCl-soluble (C,F) fractions. Equal protein loading was verified by -actin detection on stripped and re-probed western blots. Optical density analysis was performed to quantify the percentage of apoE present as fragmentation products in control apoE3 (n=7), AD apoE4 (n=7) and AD apoE3 (n=5) TBS-soluble frontal cortex gray matter samples (G). Data in “G” are means with S.E. represented by error bars. #, P<0.05 compared to AD 3/3/; **, P<0.001 compared to C 3/3. A1-40 concentration in the gHCl-soluble fractions of the Cohort 2 (see Table 1) hippocampus

(black bars), frontal cortex gray matter (gray bars) and cerebellum (white bars) samples were determined by ELISA (H).

Fig. 4. Proteolytic cleavage of apoE3 in vitro. Cleavage of apoE3 by thrombin was assessed in a control apoE3 hippocampal TBS-soluble sample (A). Three incubation conditions were used; absence of thrombin, presence of active thrombin ‘Th’ and presence of heat-inactivated thrombin ‘hi-Th’. Levels of total protein in the regions of interest were confirmed by Ponceau staining. ApoE fragmentation pattern observed in a control apoE3 hippocampal TBS-soluble sample is compared with that obtained in vitro by treating recombinant apoE3 with CatD; arrow indicates a prominent fragment at ~24 kDa (B). Fragmentation of apoE in apoptotic SK-N-SH cell lysate was assessed using rabbit anti-apoE polyclonal Ab (C).

Fig. 5. Proteolytic cleavage of r-apoE and apoE discs by CatD in vitro. Recombinant apoE3 and apoE4 (r-apoE) (A, B) and lipidated discs (C) were treated with CatD as described in “Experimental Procedures”. Samples were collected after 0h, 2h, 8h and

16h incubation and analysed by western blot using rabbit anti-apoE polyclonal Ab. A

115 D. A. Elliott et al.

16h “no enzyme” control is also shown for r-apoE (A,B). Optical density analysis was used to quantify the degradation of the major ~34 kDa apoE band (solid lines) and production of apoE fragments (broken lines) over the time course of the experiment.

ApoE3, black circles; apoE4, white circles. Data are mean values from 3 and 4 independent experiments, for r-apoE and apoE-discs, respectively, each performed in duplicate, with S.E. represented by error bars.

Fig. 6. ApoE3 dimers are present in human hippocampal homognates. The presence of disulphide-linked dimers of apoE were detected by analysing samples under both non- reducing “NR” and reducing “R” SDS-PAGE conditions. The susceptibility of dimers to thrombin cleavage was tested using three different conditions; storage at -80°C

“Con”, incubation at 37°C in the presence of thrombin “Thr” or heat-inactivated thrombin “hi-Thr”. Hippocampal TBS-soluble fractions from control apoE3 (A), AD apoE3 (B) and AD apoE4 (C) homozygote samples were analysed. Western blotting was performed using goat anti-apoE polyclonal Ab.

Fig. 7. ApoE3 dimers are present in SK-N-SH cell lysate and TBS-soluble rabbit brain homogenate. SK-N-SH cell lysate was analysed under non-reducing “NR” and reducing “R” SDS-PAGE conditions and apoE was detected using anti-apoE C- terminal monoclonal Ab (A). TBS-soluble rabbit brain homogenate was analysed under

NR and R conditions and apoE detected using goat anti-apoE polyclonal Ab (B). Two bands marked with asterisks are believed to be due to non-specific cross-reaction with the proteins indicated by Ponceau staining (B).

116 D. A. Elliott et al.

TABLES

apoE age at brain regions case # diagnosis sex PMI genotype death analysed

AD 1 1 AD F E4/E4 68 44 H, FG, O

AD 2 1 AD F E4/E4 75 80 H, FG, O

AD 3 1 AD F E4/E4 78 24 H, FG, O

AD 4 1 AD M E4/E4 67 60 H, FG, O

AD 5 1 AD F E4/E4 84 74 H, FG, O

AD 6 1 AD F E3/E3 79 4 FG, O

AD 7 1 AD M E3/E3 60 2 FG, O

AD 8 1 AD M E3/E3 75 1 FG, O

AD 9 2 AD M E4/E4 74 5 H, FG, FW, CB

AD 10 2 AD M E4/E4 83 25 H, FG, FW, CB

AD 11 2 AD M E3/E3 70 35 H, FG, FW, CB

AD 12 2 AD F E3/E3 94 7 H, FG, FW, CB

C 1 1 normal F E3/E3 73 60 H, FG, O

C 2 1 normal F E3/E3 83 24 H, FG, O

C 3 1 normal F E3/E3 77 36 H, FG, O

C 4 1 normal M E3/E3 79 60 H, FG, O

C 5 1 normal M E3/E3 82 43 H, FG, O

C 6 2 normal F E3/E3 93 21 H, FG, FW, CB

C 7 2 normal F E3/E3 85 23 H, FG, FW, CB

Table 1. Brain donor information, APOE genotype and tissues available for analysis

(AD, Alzheimer’s disease; C, control; PMI, post-mortem interval (h); H,

Hippocampus; FG, frontal cortex gray matter; FW, frontal cortex white matter; CB, cerebellum; O, occipital lobe. 1 Cohort 1 or 2 shown by superscript)

117 Elliott Fig 1

A C AD C AD C AD C AD C AD 3/3 4/4 3/3 4/4 3/3 4/4 3/3 4/4 3/3 4/4

34 25

-actin

B C AD C AD C Occip. Hippo. Front. 3/3 4/4 3/3 4/4 C AD C AD C AD 34 3/3 4/4 3/3 4/4 3/3 4/4 25 34 25 15

-actin D

10 * * * * *

% apoEfragments 0 * C AD C AD C AD 3/3 4/4 3/3 4/4 3/3 4/4

Occip. Hippo. Front.

118 Elliott Fig 2

A Tris Hippocampus

C C AD AD AD AD Ponceau 170 3/3 3/3 4/4 4/4 3/3 3/3 130 100 70 55 40 35 25

10 -actin

B TX-100 Hippocampus

C C AD AD AD AD Ponceau 170 3/3 3/3 4/4 4/4 3/3 3/3 130 100 70 55 40 35 25

15 10 -actin

C gHCl Hippocampus

C C AD AD AD AD Ponceau 170 3/3 3/3 4/4 4/4 3/3 3/3 130 100 70 55 40 35 25

15 10 -actin

119 Elliott Fig 3

A Tris Gray matter D Tris White matter C AD AD CADAD C AD AD CADAD 3/3 4/4 3/3 3/3 4/4 3/3 3/3 4/4 3/3 3/3 4/4 3/3

34 34 25 25

15 15

-actin -actin

B TX-100 Gray matter E TX-100 White matter C AD AD CADAD C AD AD CADAD 3/3 4/4 3/3 3/3 4/4 3/3 3/3 4/4 3/3 3/3 4/4 3/3

34 34 25 25

15 15

-actin -actin

C gHCl Gray matter F gHCl White matter C AD AD CADAD C AD AD CADAD 3/3 4/4 3/3 3/3 4/4 3/3 3/3 4/4 3/3 3/3 4/4 3/3

34 34 25 25

15 15

-actin -actin

G 30 H 600

20 400 ** # 10 200 ng / mg protein A % apoEfragments 0 0 C AD AD C AD AD 3/3 4/4 3/3 3/3 4/4 3/3

120 Elliott Fig 4

A C C C 3/3 3/3 3/3 Th hi-Th Ponceau

34 25

B C rE3 C Ra 3/3 CatD SK 34 34 25 25

15 15

121 Elliott Fig 5

A r-apoE3 Con 0h 2h 8h 16h 24h 16h

34 26

B r-apoE4 Con 0h 2h 8h 16h 24h 16h

34 26

C apoE3 - disc apoE4 - disc 0h 2h 8h 16h 0h 2h 8h 16h

34 26

D r-apoE apoE - disc 100 75 50 25 0 ApoE optical density 084 81216 40 1216 Time (h)

122 Elliott Fig 6

A apoE3/3 Con Thr hi-Thr NR R NR R NR R 95 72 55 43 34 26

B apoE3/3 AD Con Thr hi-Thr NR R NR R NR R 95 72 55 43 34 26

C apoE4/4 AD Con Thr hi-Thr NR R NR R NR R 95 72 55 43 34 26

123 Elliott Fig 7

A SK-N-SH 3/3 NR R

95 72 55 43 34 26

B Rabbit brain apoE Ponceau NR R NR R 95 72 55 **** 43 34 26 * * * * 17

124 ELLIOTT ET AL. SUPPLEMENTAL DATA

Supplemental Figure Legends

Supplemental Figure 1.

Analysis of apoE fragmentation in hippocampal from a TBS-soluble fraction of control apoE3/E3 donor using both rabbit anti-apoE polyclonal Ab (Ra) and goat anti- apoE polyclonal Ab (Go). The same fragments are detected using either Ab (A).

Demonstration that monoclonal Ab 21-F3-D2 binds to apoE C-terminal only. Purified apoE3 was incubated with either active (+) or heat-inactivated (-) thrombin and analysed using either rabbit anti-apoE polyclonal Ab or 21-F3-D2 monoclonal Ab

(Biogenesis). The pAb detected both the ~22 kDa and ~10 kDa fragments while the mAb only detected the C-terminal ~10 kDa fragment (B).

Supplemental Figure 2.

ApoE fragmentation in different fractions of cerebellum homogenate. Western blotting was performed using goat anti-apoE polyclonal Ab. Control apoE3, AD apoE4 and AD apoE3 homozygote samples are compared in the TBS-soluble (A),

TBS-Triton-X100-soluble (B) and gHCl-soluble (C) fractions. Equal protein loading was verified by both Ponceau protein staining and by -actin detection on stripped and re-probed western blots.

125 Elliott Supplemental Fig 1

A Ra Go 34 25

126 Elliott Supplemental Fig 2

A Tris Cerebellum

C C AD AD AD AD Ponceau 170 3/3 3/3 4/4 4/4 3/3 3/3 130 95 72 55 43 34 26

11 -actin

B TX-100 Cerebellum

C C AD AD AD AD Ponceau 170 3/3 3/3 4/4 4/4 3/3 3/3 130 95 72 55 43 34 26

17 11 -actin

C gHCl Cerebellum

C C AD AD AD AD Ponceau 170 3/3 3/3 4/4 4/4 3/3 3/3 130 95 72 55 43 34 26

-actin

127 4. GENERAL DISCUSSION

This thesis has focused on exploring novel biological properties and functions of apoE.

4.1 POSSIBLE ROLES OF apoE IN APOPTOSIS:

4.1.1 Cell survival

Previous studies have identified a link between apoptosis and increased apoE expression in fibroblasts, macrophages and ovarian carcinoma cells (Quinn et al. 2004,

Tedla et al. 2004, Chen et al. 2005). The possibility that apoE may protect cells from apoptosis is supported by studies from Chen et al., (Chen et al. 2005) showing that inhibiting apoE expression in ovarian fibroblasts using siRNA made them undergo cell cycle arrest and apoptosis. In support of this, information available in the SAGE database reveals that apoE expression is increased in several types of cancer (Hough et al. 2000) (Chen et al. 2005) (Yokoyama et al. 2006). A major goal of this thesis was to further investigate the impact apoE may have on cellular sensitivity to apoptosis. Since the majority of cell types in the body do not constitutively express significant amounts of apoE, it follows that apoE loss in itself should not promote apoptosis. Therefore macrophages were chosen for this study as they constitutively express large amounts of apoE and a previous study by Grainger et al., (Grainger et al. 2004) has revealed that there is an increase in the number of apoptotic macrophages in the liver of apoE-/- mice.

The inhibition of apoE expression in both human and murine macrophages resulted in a significant increase in caspase-3 activation in response to staurosporine treatment, however, this did not significantly alter overall cell survival as assessed by late stage apoptotic markers. Thus my overall conclusion is that endogenous apoE does not appear to confer a direct anti-apoptotic effect in macrophages. Also, it is conceivable that the upregulation of apoE observed during induction of apoptosis in SK-N-SH cells

128 may be associated with a pro-survival/anti-apoptotic response, however, this remains unproven.

It is possible that the attenuation of caspase-3 activation, observed in the macrophage studies, may reflect a subtle anti-apoptotic property of apoE that is insufficient to promote cell survival in the experimental model I have used, but may be more pronounced in different settings such as the ovarian carcinoma cells studied by Chen et al., (Chen et al. 2005). Alternatively, the modulation of caspase-3 activity associated with apoE expression level may be more relevant to the non-apoptotic functions of caspase-3 which have been reported in several cell types (Nhan et al. 2006). For example, caspase-3 modulates activation and release of the pro-inflammatory cytokine

Il-16 in primary human monocytes (Elssner et al. 2004), thus playing a role more akin to the ‘cytokine activator’ members of the caspase family. Interestingly, apoE deletion is correlated with increased levels of caspase-1, a caspase involved in the activation of pro-inflammatory cytokines Il-1 and Il-18, in the hippocampus of mice fed a high cholesterol diet (Nhan et al. 2006, Rahman et al. 2005, Yuan et al. 1993). It therefore seems plausible that the modulation of caspase-3 activity I have observed may be related to the previously described anti-inflammatory function of apoE (Ali et al. 2005,

Tenger & Zhou 2003).

Caspase-3 has also been identified as an important regulator of cytoskeletal remodelling and may play a role in other non-apoptotic cellular events such as cell cycle regulation, migration and differentiation in a variety of different cell types (Schwerk & Schulze-

Osthoff 2003, McLaughlin et al. 2003, McLaughlin 2004, Acarin et al. 2007). Further studies are clearly required to address the non-apoptotic roles of caspase-3. Also, it is

129 worth highlighting that the Ac-DEVD-AMC substrate used to measure caspase-3 activity in these studies is also capable of being cleaved by caspase-7, caspase-10 and, to a lesser extent, caspase-6 (reviewed in (Cohen 1997)). Thus caspases in addition to caspase-3 may also be activated to a greater extent during apoptosis in the absence of apoE and therefore may contribute to the increased signal detected using the Ac-

DEVD-AMC assay.

4.1.2 Clearance of apoptotic debris

The association of apoE with apoptotsis was further extended in this thesis by the discovery that apoE expression is significantly increased during apoptosis in a neuronal cell type. Furthermore, I observed that apoE was enriched in the neuronal apoptotic bodies, consistent with a potential role of apoE in the facilitation of apoptotic body clearance by phagocytic cells (Grainger et al. 2004). The possibility that this may occur in the CNS is supported by the fact that the apoE binding receptor LRP is present on microglial cells and has previously been shown to mediate engulfment of apoptotic bodies associated with another apolipoprotein, apoJ (Bartl et al. 2001, Gardai et al.

2005). The uptake of apoE enriched apoptotic bodies by LRP expressed on the microglial cells could therefore represent a novel pathway to clear neuronal apoptotic bodies and limit inflammation associated with AD (Maderna & Godson 2003); (Wyss-

Coray 2006) and other forms of neurological insult where neuronal apoE expression is induced (Aoki et al. 2003, Boschert et al. 1999) (Xu et al. 2006). Caspase-3 mediated apoptosis also occurs during development where it plays an important role in ‘pruning’ redundant neural circuits (Yuan & Yankner 2000) and the expression of both apoE and

LRP8 are correlated during this process (Kim, Wong, Weickert, Bahn and Garner unpublished data).

130

In the context of AD, apoE accumulation in amyloid plaque and neurofibrillary tangles may be a consequence of caspase-dependent neurodegeneration which shares features in common with the apoptotic pathway. The over-expression of apoE in the AD brain

(Thomas et al. 2003) is consistent with this idea; although apoE4-A complexes may also induce neuronal apoptosis via an LRP-dependent process involving lysosomal rupture and caspase activation (Ji et al. 2006).

4.2 NUCLEAR TRAFFICKING OF apoE:

Our study showed that endogenously synthesized apoE-GFP is translocated to the nucleus and that this process is enhanced during serum starvation. We also showed that exogenously added apoE can be localized to the nucleus but only when it is presented to the cell in association with a lipid complex. The possibility that apoE-GFP may be initially secreted from CHO apoE-GFP cells and then interact with cell-surface molecules that facilitate or mediate its transport to the nucleus, seems unlikely, as exogenously added apoE-GFP did not result in the appearance of apoE-GFP in the nucleus.

My specific focus within this aspect of the thesis involved elucidating the sub-nuclear localization of apoE. Several sub-nuclear structures, referred to as nuclear bodies (NB), have previously been identified and are associated with roles including transcriptional regulation, mRNA splicing and apoptosis signaling (Mintz et al. 1999, Cremer &

Cremer 2001, Lamond & Spector 2003, Krieghoff-Henning & Hofmann 2008). ApoE was found to localise within the nucleus in a distinctly punctuate manner, therefore, it

131 became of interest to determine whether apoE co-localised within any previously established NBs. I demonstrated that apoE does not primarily co-localise with interchromatin granule clusters/nuclear speckles or nucleoli. The possibility that apoE co-localizes with promyelocytic leukemia (PML) NBs was not investigated, however, this could be an interesting future experiment considering that PML NBs play a role in senescence (Krieghoff-Henning & Hofmann 2008) and apoE nuclear localization is increased during senescence (Quinn et al. 2004).

The exact function of apoE in the nucleus is currently not clear. However, it seems likely that it may be serving a protective role considering it is increased during times of cellular stress, including serum-starvation and senescence. Analogous to this, the nuclear accumulation of other proteins has been recognized as an adaptive survival response to changes in the extracellular environment (Hood & Silver 1999). It is tempting to speculate that this finding may represent further evidence that apoE plays a poorly understood role in cell survival, particularly in light of the work by Chen et al., and my data demonstrating an ability to attenuate caspase-3 activity. Further work is clearly needed to understand the significance of apoE in the nucleus and, more broadly, in cell survival.

4.3 STRUCTURAL DIFFERENCES BETWEEN apoE ISOFORMS

IN THE HUMAN BRAIN:

This thesis has identified two distinct differences between the apoE3 and apoE4 isoforms in the human brain. A greater proportion of apoE3 is present as fragments and apoE3 is present in disulfide-linked dimers.

132

4.3.1 Fragmentation of apoE3 in the human brain

My work showed that the level of TBS-soluble apoE fragments was increased in the apoE3 brain, compared to apoE4, and that this is independent of AD status, A load and brain region. Areas of the brain that are known to be severely affected by AD, such as the hippocampus and frontal cortex, were analysed along with areas that are relatively spared, such as the occipital lobe and cerebellum, and the data show that apoE3 fragmentation occurs in all regions in both control and AD brains. Therefore, apoE3 fragmentation was not associated with AD in the present study. The possibility of apoE4 fragmentation in the control brain could not be addressed due to the unavailability of age-matched 4 control donors. However, this possibility seems unlikely given the comprehensive data from apoE3 brains showing that apoE processing appears to be independent from AD pathology.

The range of different sized apoE fragments observed in my studies is very similar to those observed previously by another group of investigators (Zhang 2001); (Zhou et al.

2006), although, Zhang et al., (Zhang 2001) came to the conclusion that there were more fragments in the 4/4 brain. Quantitative data from Zhang et al., however, was based on data obtained from an ELISA based technique as opposed to western blot analysis, a factor which may explain this discrepancy. Interestingly, the majority of the apoE fragmentation data presented in this thesis conflicts with another previous study where the apoE4 isoform and AD were found to be associated with increased apoE fragmentation (Huang et al. 2001). While I did observe increased fragments in the gHCl fractions from the hippocampus in AD cases with 4/4 genotype, consistent with

Huang et al., (Huang et al. 2001) this trend was not consistently observed in all brain

133 regions examined, unlike in the TBS soluble fraction where apoE fragments were consistently more abundant in 3/3 brains. When hippocampal tissue from AD brains was homogenized directly in a detergent-rich buffer, to ensure very rapid processing and thus reduce the likelihood of the fragments being generated artificially (data not shown), fragments were more abundant in the apoE3 samples, similar to data from the

TBS soluble fraction. This further supports my overall conclusion and suggests that the discrepancy between my data and Huang et al., (Huang et al. 2001) is unlikely to be due to differences in methodological technique. Interestingly, when Western blot data of apoE fragmentation in the human brain is taken from four independent studies, the overall pattern of fragmentation appears to be quite similar (Figure 10). One exception is that in the publication by Brecht et al. (Brecht et al. 2004) the quantitatively major fragment appears to migrate close to their 30 kDa marker. The reason for this discrepancy is unclear but could potentially be due to issues related to the identity or labeling of the molecular weight standards or to variables in the electrophoretic methods.

The sequential extraction of brain tissue into three fractions was used in this study as it enabled me to determine whether the apoE fragments were present in a soluble environment (TBS soluble), membrane associated (TBS-Triton X-100 soluble) or contained within an insoluble aggregate such as amyloid plaques (gHCl). Previous studies have reported the presence of apoE fragments in amyloid plaques (Aizawa et al.

1997, Cho et al. 2001), however, my data demonstrates that the majority of apoE fragments are present in soluble regions of the brain, independent from aggregated amyloid plaques, which supports the possibility that these fragments may be playing an active biological role. Consistent with this theory, the ~22 kDa N-terminal fragment of

134

Figure 10.

ApoE fragmentation patterns reported in four independent studies. Western blotting of human brain homogenates was performed as described in each of the publications listed (the figures from which the data are extracted are given in parentheses). Overall apoE fragmentation patterns are quite similar in the various publications. A prominent fragment is identified at ~ 25 kDa. In the publication by Brecht et al. the major fragment appears to migrate close to their 30 kDa marker. The reason for this discrepancy is unclear but may be due to issues related to the identity or labeling of the molecular weight standards or to variables in the electrophoretic methods.

135 apoE can be recycled within intracellular compartments and secreted in a manner similar to the full length apoE protein (Farkas et al. 2004).

4.3.2 Mechanism of apoE proteolysis in the brain

My data, along with previous work (Marques et al. 2004, Zhou et al. 2006), suggests that CatD may play a role in apoE proteolysis in the brain. CatD is a lysosomal enzyme, however, there is also evidence of extracellular and cytoplasmic CatD localisation, particularly with brain ageing (Cataldo & Nixon 1990, Cataldo et al. 1991, Jung et al.

1999). It is conceivable that apoE3 fragmentation is not strictly a process of lysosomal apoE destruction. Interestingly, studies in CatD-deficient mice suggest that CatD plays a vital role in limited proteolysis of biologically active proteins as opposed to bulk degradation in lysosomes (Saftig et al. 1995). This provides a plausible role for CatD in the generation of soluble apoE fragments that may be biologically active.

The major apoE fragment formed in vivo has an apparent molecular weight of 25 kDa and it is important to consider how this relative selectivity may be achieved. It is established that CatD preferentially cleaves C-terminally to amino acids with aromatic side chains (i.e. Phe, Trp and Tyr) (Minarowska et al. 2008). Human apoE thus contains

13 predicted preferential cut sites for CatD. It is also important to consider how the structure of apoE may influence susceptibility to protease action. Of particular relevance, the structure of apoE contains a hinge domain between the N-terminal (4- helix bundle, amino acids 1-191) and C-terminal (amino acids 219-299) domains and it is predicted that enzymatic cleavage in the hinge is favourable as this region is exposed

(refer to Figure 1). This is supported by the preferential formation of a 22 kDa thrombin fragment that cuts in the apoE hinge domain. Interestingly, only one of the 13

136 preferred CatD cut sites exists in the apoE hinge at Trp210. The predicted mass of the apoE peptide spanning amino acids 1-210 is 24 kDa. Since this potential peptide would be expected to contain an occupied O-glycosylation site at Thr194, the actual molecular weight is predicted to be closer to 25 kDa (e.g. based on sialylated mucin-type core 1 structures that are present on human apoE (Zanni et al. 1989)). The size of this predicted apoE fragment fits well with the major TBS-soluble apoE3 fragment I have detected in the human brain.

It remains possible that other proteases may also be involved in apoE3 fragmentation.

Studies by Harris et al., have suggested the possibility that a chymotrypsin-like serine protease may be involved and other studies have established that MMP-14 is both present in the brain and can cleave apoE to create fragments (Park et al. 2008,

Candelario-Jalil et al. 2008, Toft-Hansen et al. 2007). Differences in apoE structure and conformation (eg. apoE4 domain interaction) may differentially regulate the proteolysis of the apoE3 and apoE4 isoforms. An in vitro study has provided evidence consistent with this theory by demonstrating that apoE4 is more resistant to proteolysis than apoE3 due to restricted access of proteases to the C-terminal region comprising amino acid residues 230-260 (Barbier et al. 2006). Also, differences in fragmentation may be related to differences in the structural stability of the apoE3 and apoE4 N-terminal domains. Studies of the truncated apoE N-terminal domain demonstrate that the apoE4 fragment is more susceptible to denaturation than the apoE3 fragment (Acharya et al.

2002, Clement-Collin et al. 2006, Morrow et al. 2000). The apoE4 fragment is also more prone to developing a partially unfolded molten globule formation, resulting in the exposure of proteolytic cleavage sites within the fragment which could conceivably

137 make the apoE4 fragment more susceptible to complete degradation in vivo (Morrow et al. 2002).

Alternatively, differences in intracellular trafficking of apoE may also play a role in the regulation of proteolysis, for example, previous studies have shown that when exogenous apoE3 or apoE4 is added to human neuron cultures, more apoE4 localises to lysosomes (DeKroon & Armati 2001). Furthermore, transgenic mice studies have revealed that newly synthesized apoE4, in comparison to apoE3, undergoes accelerated intracellular degradation in the brain (Riddell et al. 2008).

4.3.3 Biological function of apoE fragments?

My studies show that apoE fragments are soluble and this raises the possibility that they may be biologically active. In vitro studies have investigated the bioactive properties of apoE and apoE-derived peptides, with results varying depending on the apoE fragment and cell model used. Previous studies have demonstrated that apoE interacts with cell surface receptors mediating a range of effects including down-regulation of microglial activation, suppression of NMDA induced excitotoxicity and the activation of anti- apoptotic cell signalling pathways with most of the effects thought to be mediated by the receptor binding domain (RBD) of apoE (Aono et al. 2003, Hayashi et al. 2007,

Laskowitz et al. 2001). ApoE peptides encompassing this region, as well as mimetic peptides containing a tandem repeat of this region, have been shown to exhibit even greater activity than the full length apoE (Aono et al. 2003, Laskowitz et al. 2001, Li et al. 2006, Singh et al. 2008). It has not yet been elucidated if the apoE fragments identified in vivo encompass the RBD, however, if this were the case then there would be an increased quantity of apoE RBD in the apoE3 brain which could, hypothetically,

138 be responsible for mediating isoform differences in function. Some studies have also reported that apoE derived peptides and the N-terminal domain possess neurotoxic properties (Crutcher et al. 1994, Tolar et al. 1999, Tolar et al. 1997). These studies emphasise that apoE fragments may be biologically active and imply that apoE3 fragments present in the human brain may interact with cell surface receptors in an analogous manner. These fragments may also interact with a range of soluble apoE receptors present in the CNS (Rebeck et al. 2006).

Future work aimed at determining the biological activity of these fragments could be achieved by firstly isolating and sequencing the human brain apoE3 proteolytic fragments. Recombinant apoE peptides corresponding to the fragments produced in vivo could then be synthesized and used to investigate their interaction with neuronal

LDL family receptors and their potential biological activities.

4.3.4 ApoE disulfide linked dimers

My research demonstrates for the first time that apoE3 disulfide-linked dimers are present in the human brain. In vitro studies have demonstrated that the apoE3 homodimer is present in media from cultured astrocytes (Sun et al. 1998) and I demonstrate that apoE3 dimers are also present in neuroblastoma cell lysate and freshly prepared rabbit brain. Previous studies have demonstrated that apoE3 homodimers and apoE3-apoA-II heterodimers have diminished LDL receptor binding activity, 20% and

30%, respectively, in comparison to apoE3 monomer (Innerarity et al. 1978,

Weisgraber & Shinto 1991). Interestingly, in vitro studies have also shown that the apoE-apoA-II dimer is more effective than apoE monomer in binding soluble A1-42 and inhibiting its internalisation by neurons (Yamauchi et al. 1999, Yamauchi et al.

139

2000). While the apoE-apoE homodimer is more effective than the monomer at binding

A1-40 (Aleshkov et al. 1997). It is therefore plausible that the apoE3 dimers we have detected in human brain have distinct biological properties that differ from apoE monomer, however, additional research will be required to determine if this modulates the apoE associated pathways of A degradation and clearance (Jiang et al. 2008,

Deane et al. 2008, Beffert et al. 1999a).

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5. MAJOR CONCLUSIONS

In conclusion, this thesis has generated several novel findings:

- ApoE expression is up-regulated during apoptosis in a neuronal cell type and becomes enriched in neuronal apoptotic bodies.

- The reduction/deletion of apoE in macrophages does not alter susceptibility to staurosporine-induced apoptosis, however, it does significantly increase caspase-3 activation.

- The nuclear accumulation of apoE is increased during serum starvation. Also, apoE localizes within an intra-nuclear compartment that is predominantly independent from nucleoli and interchromatin granule clusters/speckles.

- Fragmentation of apoE in the human brain is strongly associated with the apoE3 isoform and occurs independently from AD and brain region examined.

- A portion of apoE exists as disulphide-linked dimers in the brains of apoE3 carriers,

SKNSH neuronal cells and freshly prepared rabbit brain.

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