THE MECHANISM OF APOLIPOPROTEIN E
IN THE PROTEOLYTIC DEGRADATION OF Aβ
By
CHUNG-YING DANIEL LEE
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Thesis Advisor: GARY E. LANDRETH
Department of Neurosciences
CASE WESTERN RESERVE UNIVERSITY
May, 2012
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Chung-Ying Lee
candidate for the Ph.D. degree *.
(signed) Dr. Evan Deneris (chair of the committee)
Dr. Gary Landreth
Dr. Bruce Lamb
Dr. Jonathan Smith
(date) January 27, 2012
*We also certify that written approval has been obtained for any
proprietary material contained therein.
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TABLE OF CONTENTS
List of figures ...... 5
Acknowledgement ...... 7
Abstract ...... 9
Chapter 1 Introduction ...... 11
Epidemiology and genetics of AD ...... 13
Aβ production and the amyloid cascade hypothesis ...... 15
Apolipoprotein E ...... 18
Microglia ...... 27
Aβ clearance mechanisms ...... 29
Soluble Aβ clearance ...... 30
Proteolysis of soluble Aβ ...... 31
The role of apoE in soluble Aβ clearance ...... 33
Fibrillar Aβ clearance ...... 34
Microglial Aβ receptor complex ...... 35
Role of inflammation in fibrillar Aβ clearance ...... 37
Antibody- and complement-mediated clearance of fibrillar Aβ ...... 39
Cholesterol and Alheimer’s Disease ...... 41
Cholesterol in the CNS ...... 42
Cholesterol synthesis ...... 43
Cholesterol uptake and intracellular trafficking ...... 44
Cellular cholesterol homeostasis ...... 45
Cholesterol excretion from the CNS ...... 47
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Cholesterol and Aβ production ...... 48
Cholesterol homeostasis-related genes and AD ...... 50
ATP-Cassette Transporter A1 ...... 51
Liver X Receptors ...... 52
Niemann-Pick Type C Proteins ...... 54
Conclusion and research objectives ...... 55
Figures ...... 58
Chapter 2 Apolipoprotein E Promotes β-amyloid Trafficking and Degradation by
Modulating Microglial Cholesterol Levels ...... 63
Abstract ...... 64
Introduction ...... 65
Materials and methods ...... 68
Results ...... 76
Facilitation of Aβ degradation is a common feature of HDL apolipoproteins ...... 76
Facilitation of Aβ degradation relies on the cholesterol efflux function of
apolipoproteins ...... 77
Degradation of Aβ is influenced by the cellular cholesterol levels ...... 79
The transcription of Aβ degrading enzymes is not regulated by cholesterol levels.. 80
Cholesterol levels regulate Aβ transport ...... 81
Cholesterol regulates the trafficking of Aβ-containing endosomes via the recruitment
of Rab7 ...... 82
Discussion ...... 84
Figures ...... 91
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CHAPTER 3 ApoE Acts as a chaperone to promote IDE-mediated Aβ degradation105
Abstract ...... 106
Introduction ...... 107
Materials and Methods ...... 110
Results ...... 115
IDE-mediated Aβ degradation is facilitated in the presence of apoE ...... 115
ApoE acts as a chaperone to facilitate Aβ degradation by IDE ...... 116
Lipidation status of apoE regulates its ability to promote IDE-mediated Aβ
degradation ...... 118
ApoE isoforms differentially facilitate Aβ degradation by IDE ...... 120
Aβ12-28 promotes Aβ degradation by IDE through enhancing allosteric reaction 121
Discussion ...... 122
Figures ...... 129
Chapter 4 Discussion ...... 138
Effect of cholesterol modulation on Aβ levels in vivo ...... 142
Cholesterol derivatives and ACAT inhibitors in Aβ degradation ...... 145
Resident and infiltrating microglia in the clearance of Aβ ...... 147
Clearance of Aβ by other glial cells ...... 151
Anti-inflammatory properties of apoE ...... 152
Conclusion ...... 153
Figures ...... 155
Chapter 5 Literatures cited ...... 159
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LIST OF FIGURES
Figure 1.1: APP mutations associated with early-onset Alzheimer’s disease ...... 59
Figure 1.2: Proteolytic processing pathways of APP ...... 60
Figure 1.3: Model of the structure of lipid-free apoE and the formation of an α-helical hairpin conformation in apoE-HDL-like particles...... 62
Figure 2.1: Facilitation of Aβ degradation is a common feature of apolipoproteins ...... 91
Figure 2.2: Facilitation of Aβ degradation relies on the cholesterol efflux function of
apolipoproteins ...... 92
Figure 2.3: Blocking cholesterol efflux impairs the ability of ApoE to enhance Aβ
degradation ...... 95
Figure 2.4: Reducing cellular cholesterol levels promotes Aβ degradation ...... 96
Figure 2.5: Increasing cellular cholesterol levels impairs Aβ degradation ...... 97
Figure 2.6: Direct manipulation of cellular cholesterol levels regulates Aβ degradation in
the absence of apoE ...... 98
Figure 2.7: Transcription of Aβ degradation enzymes is not modulated by cholesterol....99
Figure 2.8: ApoE has minimal effects on the activity of Aβ degradation enzymes ...... 100
Figure 2.9: Cholesterol levels regulate the trafficking of Aβ-containing endosomes ...... 101
Figure 2.10: Cholesterol regulates the recycling of Rab7 ...... 103
Figure 3.1: IDE-mediated extracellular degradation of Aβ is facilitated in the presence of
ApoE ...... 129
Figure 3.2: ApoE is not an activator of IDE ...... 130
Figure 3.3: Kinetic analysis supported that apoE mighty act as a chaperone to facilitate
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Aβ degradation by IDE ...... 131
Figure 3.4: Lipidation status of apoE regulates its ability to promote IDE-mediated Aβ
degradation ...... 132
Figure 3.5: ApoE isoforms differentially facilitate Aβ degradation by IDE ...... 134
Figure 3.6: Aβ12-28 promotes Aβ degradation by IDE through enhancing allosteric
reaction ...... 136
Figure 4.1: The mechanism of apoE in facilitating intracellular Aβ degradation by
microglia ...... 155
Figure 4.2: Inhibition of ACAT activity does not regulate the transcription of Aβ degradation enzymes ...... 156
Figure 4.3: Inhibition of ACAT activity facilitates Aβ degradation ...... 157
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ACKNOWLEDGEMENT
This work cannot be accomplished without the help of many people and I would
like to thank all those who have helped and carried me along the way. First and foremost,
I would like to thank my thesis advisor and mentor Dr. Gary Landreth. I have always felt
very lucky to be a member in Gary’s lab. His commitment to his students is exceptional.
He always encourages me to explore my curiosity and also provides timely guidance to
keep me on the right path. His passion for science and eagerness for knowledge
profoundly influenced me.
I would like to thank Drs. Evan Deneris, Bruce Lamb and Jonathan Smith for their valuable insight and guidance as members of my thesis committee. Their enthusiasm and support of this project made committee meeting always enjoyable and inspiring.
Many thanks go to the past and current members of the Landreth lab for their friendship and assistance. I especially want to thank Colleen Karlo, Brandy Wilkinson,
Qingguang Jiang, Shweta Mandrekar and Paige Cramer not only for every intellectual discussion we have had, but also for your always being on my side in every “I hate science” moment. They are not just labmates but my best friends.
Studying abroad is not as exciting and enjoyable as it sounds. Sometimes, it is full of nostalgia and stress. Thus, I would also like to give special thanks my Taiwanese
-7- friends in Cleveland for their company and encouragement. They made the daily life joyful. They are my family in the States.
None of this has been possible without the love and support from my family. A special thank you goes to my parents and brother, without whom I would not be able to pursue my dream. To my wife, Stephanie Liu, I forever love and appreciate. Your unwavering love and unconditional support carried me through the ups and downs of these years and made me where I am today. I love you and thank you!
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The Mechanism of Apolipoprotein E in the Proteolytic Degradation of Aβ
Abstract
by
CHUNG-YING DANIEL LEE
Alzheimer’s disease is a neurodegenerative disease, which results from the imbalance of the generation and the clearance of β-amyloid (Aβ) peptides.
Apolipoprotein E (apoE) facilitates Aβ degradation in a dose- and isoform-dependent
manner. However, the mechanisms underlying these activities remain unclear.
In microglia, apoE promotes Aβ degradation through eliciting cholesterol efflux.
Inhibition of cholesterol efflux blocks the apoE-facilitated Aβ degradation, while eliciting
cholesterol efflux by a mimetic peptide enhances it. These findings indicate that the
cholesterol efflux property of apoE is both sufficient and necessary for promoting Aβ
degradation. Regulation of Aβ degradation could be achieved by solely manipulating
cellular cholesterol levels, suggesting that apoE-enhanced Aβ degradation is a
consequence of reduced cellular cholesterol levels through cholesterol efflux. Although
the transcription and activity of all known Aβ degrading enzymes were not changed, the
trafficking of Aβ is regulated by cholesterol. We further showed that cholesterol regulates
the recycling of Rab7, which recruits the dynein motor complex onto endosomes.
Cholesterol efflux elicited by apoE lowers cellular cholesterol levels and consequently
promotes efficient recycling of Rab7, which leads to faster Aβ trafficking to lysosomes.
As a result, the degradation of Aβ is enhanced. These findings highlight a direct role of
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cholesterol in regulating microglial degradation of Aβ.
The extracellular degradation of Aβ is carried out principally by insulin degrading
enzyme (IDE). In contrast to the intracellular degradation, apoE facilitates IDE-mediated
Aβ degradation through its direct interaction with Aβ. Enzyme kinetic analysis shows that
the Km of IDE for Aβ degradation in the presence of apoE was reduced, while the Vmax remained unchanged. These results suggest that apoE promotes the interaction/recognition of IDE and Aβ. Manipulating apoE’s affinity for Aβ regulates its ability to promote Aβ degradation. ApoE2 shows the highest affinity for Aβ and greatest efficiency in facilitating Aβ degradation, while apoE4 has low Aβ-binding affinity and is insufficient in promoting Aβ degradation. Together, these data support the chaperone function of apoE in facilitating Aβ degradation by IDE.
Our study advances the knowledge of the mechanisms of apoE in facilitating Aβ degradation, which provides insight for developing effective therapies for AD.
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CHAPTER 1
INTRODUCTION
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Alzheimer’s disease (AD) is a devastating neurodegenerative disease. It is the
leading cause of dementia in the elderly, affecting more than 40% of people over age of
85 (Cummings and Cole, 2002). Currently, approximately 5.4 million individuals in the
United States are affected by this disease and it is estimated that by 2050, the number of
people with AD will be tripled to a projected total of 11 to 16 million. However, there is
currently no effective treatment for the disease. The available therapies nowadays only
ameliorate symptoms associated with the disease. These drugs act to compensate for the
loss of neurons or synaptic connections by preventing the degradation of the
neurotransmitter acetylcholine to maintain its levels at the synapses. Nevertheless, these
treatments are only effective for a short period of time and the degeneration of neurons
ultimately mitigates the effect of the drugs. No effective long-term treatment in
preventing or arresting the progression of AD has yet been discovered. Thus, the large
number of AD cases is an immense burden to our healthcare system. The aggregate payments for health care, long-term care and hospice for patients with AD and other dementias are $183 billion in 2011 and are projected to increase to $1.1 trillion in 2050
(http://www.alz.org/downloads/Facts_Figures_2011.pdf). There is an urgent need in the
field to develop an effective therapy for AD. Based on the demographic distribution of
the risk for AD, it has been argued that even delaying the onset of the disease by only 5
years could greatly reduce the number of affected individuals by nearly 50%.
Alzheimer’s disease is named after Dr. Alois Alzheimer, who first described this disease in 1906. His patient, Auguste D., developed memory deficits and progressive loss
of cognitive functions in her middle ages, which we refer to as “presenile dementia”
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nowadays. The postmortem analysis of her brain revealed amyloid plaques and
neurofibrillary tangles, which are now identified as the distinctive pathological hallmarks
of AD. Amyloid plaques are extracellular aggregation of the β-amyloid (Aβ) peptides, generated by sequential proteolytic cleavage of the amyloid precursor protein (APP)
(Hardy and Selkoe, 2002). The plaques are generally distributed throughout the cerebral cortex and hippocampus, where massive loss of neurons and synapses are also observed in the late stages AD. Aβ deposits are also commonly found in the walls of the cerebral vasculature, which are known as the cerebral amyloid angiopathy (CAA) and are linked
to the compromised blood-brain barrier (BBB) function. Neurofibrillary tangles (NTFs)
are intraneuronal accumulation of hyperphosphorylated forms of the microtubule-
associated protein tau (Hutton and McGowan, 2004). NTFs disorganize the microtubule
cytoskeleton and are thought to contribute to the dystrophic neurites and dysfunction of
neural circuits. Although the etiology of AD remains unclear, cumulative evidence
suggests that accumulation of Aβ could be causative for AD pathogenesis. The balance
between production and efficient clearance of Aβ from the brain is essential to maintain
the health of the brain. By understanding the metabolism of Aβ shall we be able to
develop effective treatment for AD. This thesis will focus on the mechanisms of the
degradation of Aβ and the factors that facilitate it.
EPIDEMIOLOGY AND GENETICS OF AD
The majority of AD cases are sporadic and late-onset (LOAD), in which
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individuals develop the symptoms of AD after age 65. Early-onset cases of AD (EOAD)
are uncommon and account for 6-7% of all cases. In these cases, individuals develop AD
before age 65, sometimes as young as age 30 arises due to inheritance of genes associated
with the disease. Advance of age is the greatest risk factor for AD. It is estimated that the prevalence is around 1.5% at age 65 years and doubles every 4 years to reach about 43% at age 85 years or older (Cummings and Cole, 2002; Plassman et al., 2007). AD is not a normal part of aging (Katzman, 1993). Epidemiological studies tell us that autosomal dominant, familial forms of this disease (FAD) are very rare (less than 1%). By contrast, susceptibility of LOAD shows less-obvious or no apparent familial aggregation (hence it is sometimes called “sporadic” AD) and is likely to be governed by an array of common risk alleles across a number of different genes. Notably, aside from the earlier age of onset, the symptoms and pathology of FAD are similar to the noninherital, sporadic form of AD.
Because of the rare cases of FAD, it is particularly interesting given the high incidence of developing AD-like neuropathological changes that is inevitably observed in the older Down’s syndrome patients, who have an extra copy of all or part of chromosome 21. It was also observed that when compared to controls, there is a significantly higher ratio of AD affected individuals derived from families with a greater incidence of Down’s syndrome. Take together, these observations led to the suggestion that duplication of a gene on chromosome 21 might link to the etiology of AD (Glenner and Wong, 1984). In 1987, 80 years after Dr. Alzheimer documented the pathological characteristics of this presenile dementia, a gene locus on chromosome 21 linking to AD
-14- was finally mapped (St George-Hyslop et al., 1987). In the mean time, the sequence of the 4-kDa Aβ peptide, the central compound of senile plaques, was identified and mapped to the same gene located on chromosome 21 of George-Hyslop’s study (Tanzi et al., 1987). The product of this gene was later named amyloid precursor protein (APP) as the full length precursor of Aβ. Shortly after the discovery of the APP gene, several mutations on APP in the individuals from the families exhibiting a high incidence of AD were discovered (Fig. 1.1) (Tanzi and Bertram, 2005). Although these mutations are fully genetically penetrant, it was soon realized that APP mutations do not account for all FAD cases. In 1995, presenilin 1 (PSEN1) on chromosome 14 and presenilin 2 (PSEN2) on chromosome 1 were reported as novel genes linked to FAD (Levy-Lahad et al., 1995;
Rogaev et al., 1995; Sherrington et al., 1995). The presenilins are eight-pass transmembrane proteins that function as a part of the γ-secretase intramembrane protease complex to cleave several proteins, including APP. To date, 32 autosomal-dominant mutations in APP, 185 in PSEN1 and 13 in PSEN2 have been identified
(http://www.molgen.ua.ac.be/ADMutations/) (Tanzi and Bertram, 2005).
Aβ PRODUCTION AND THE AMYLOID CASCADE HYPOTHESIS
Aβ is generated through sequential proteolytic cleavage of APP by β- and γ- secretases (Hardy and Selkoe, 2002). APP is a ubiquitously expressed type I transmembrane protein. It contains a large ectodomain with sites for N-glycosylation, a single membrane-spanning helix and a short C-terminal domain (Jacobsen and Iverfeldt,
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2009). APP matures through the constitutive secretory pathway, where it is post-
translationaly modified by addition of both N- and O-linked sulfate and carbohydrate
moieties before being transported to the plasma membrane. Proteolytic cleavage of APP occurs in the endolytic vesicles and on the plasma membrane through one of two pathways (Fig. 1.2). In the non-pathogenic pathway, APP is first cleaved by α-secretase, a member of the ADAM family, which cleaves APP between Lys16 and Leu17 of the canonical Aβ sequence and generates a soluble APP ectodomain α, known as sAPPα, and a membrane-bound C-terminal fragment, C83 or CTFα (Lichtenthaler, 2011). The CTFα is subsequently cleaved by γ-secretase, leading to the secreted p3-peptide and the APP intracellular domain (AICD). As a result, there is no Aβ generated from this pathway, thus it is non-pathogenic. APP can also be cleaved though the canonical Aβ generating pathway. The initial cleavage is done by β-secretase or β-site APP cleaving enzyme
(BACE1), a membrane bound aspartyl protease (Vassar et al., 1999; Rossner et al., 2006).
This cleavage results in the shedding of sAPPβ and the formation of a membrane-bound
C-terminal fragment, C99 or CTFβ. γ-secretase then cleaves the C99 fragment to release
Aβ and AICD. The γ-secretase cleavage is not precise, generating Aβ from 39 to 43 amino acids long. The predominant forms of Aβ in the AD brain are 40 and 42 amino acids in length, which polymerize into a variety of multimeric Aβ species and subsequently fibrillize, aggregate and are deposited within the parenchyma of the brain.
Because of the extra two C-terminal amino acid residues, Aβ1-42 is more hydrophobic
than Aβ1-40 and has a greater propensity to polymerize and forms fibrils (Walsh et al.,
1997; Kirkitadze et al., 2001). In a normal individual, the majority of Aβ produced is
Aβ1-40, with only 5-15% is Aβ1-42 (Younkin, 1998). In the case of many familial APP
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mutations, including London and Indiana mutations, as well as the mutations in PSEN1
and PSEN2, the γ-secretase cleavage is shifted to favor the generation of Aβ1-42, thereby fostering fibrillization and deposition of Aβ (Mann et al., 1996; Maruyama et al., 1996;
Mann et al., 1997). By contrast, the Swedish mutation of APP favors the cleavage by
BACE1 over α-secretase, thus resulting increased production of CTFβ, and subsequently
leading to elevated level of all Aβ species in the brain (Citron et al., 1992; Felsenstein et
al., 1994).
The amyloid hypothesis posits that the deposition of Aβ is the causative agent of
AD pathogenesis, and that the neurofibrillary tangles, neuronal loss and eventually
dementia follow as a direct result of this deposition (Hardy and Higgins, 1992). Several
lines of evidence from genetic studies of familial forms of AD support the pivotal role of
Aβ in the pathogenesis of AD; (i) Trisomy 21 (Down’s syndrome), which has an extra
copy of the APP gene located chromosome 21, leads invariably to neuropathology of AD
(Olson and Shaw, 1969); (ii) mutations at or near the cleavage sites of β- and γ-secretase
on the APP gene (Goate et al., 1991; Wisniewski et al., 1991b; Hardy, 1992; Hendriks et
al., 1992; Mullan et al., 1992) or (iii) the mutations of γ-secretase constituents, PSEN1
and PSEN2, result in increase production of Aβ1-42 and consequently lead to early onset
of AD (Citron et al., 1992; Cai et al., 1993; Suzuki et al., 1994). Inheriting any of these
genetic mutations inevitably leads to the development of AD. Although the familial
forms of AD account for less than 1% of total AD patients, genetic analysis of heritable cases provided important insights into the pathogenesis of this disease. Transgenic mouse models of AD that develop Aβ plaques as they age have provided a valuable tool to test
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the hypothesis. Introducing human disease-related transgenes into mice recapitulates the
amyloid pathology of AD as well as the cognitive impairment, but not neuronal loss
(Games et al., 1995; Dodart et al., 2002). Administration of pharmacological inhibitors of
β- or γ-secretase in these mice reduces amyloid deposition and ameliorates cognitive dysfunction (Sinha et al., 1999; Dovey et al., 2001; Comery et al., 2005). Aβ is secreted from neurons as a consequence of normal synaptic transmission (Kamenetz et al., 2003;
Cirrito et al., 2005). Since Aβ peptides are normally generated at high levels in the brain
(approximately 8% per hour) and are cleared at an equivalent rate in both humans and mice (Bateman et al., 2006), even moderately increased production or decreased clearance of Aβ eventually leads to an overall elevation of its steady state levels and ultimately the enhanced deposition in the brain promoting AD pathogenesis.
APOLIPOPROTEIN E
In addition to the genes directly involving Aβ production, genome-wide linkage studies on late-onset, sporadic AD have provided strong evidence for the existence of additional genes associated with AD. Among them, the polymorphism of the
apolipoprotein E gene (APOE) is identified as the major genetic risk factor for sporadic
AD (Strittmatter et al., 1993a; Kim et al., 2009). There are several single nucleotide
polymorphisms (SNPs) distributed across the human APOE gene (Nickerson et al., 2000).
The three most common SNPs give rise to the changes in amino acid residues at position
112 and 158 and are expressed as the three major isoforms: apoE2 (Cys112, Cys158),
-18- apoE3 (Cys112, Arg158) and apoE4 (Arg112, Arg158), among which apoE3 is the most common allele in the population. Namba and colleagues discovered that the immunoreactivity for apoE was colocalized unexpectedly with amyloid plaques and neurofibrillary tangles from the brain sections of AD patients (Namba et al., 1991). Direct binding of apoE and Aβ was then confirmed in vitro soon after (Strittmatter et al., 1993a;
Strittmatter et al., 1993b). Since then, numerous genetic studies have concluded that possession of the apoE4 allele is correlated with a higher risk of AD (Corder et al., 1993;
Saunders et al., 1993; Strittmatter et al., 1993a; Farrer et al., 1997; Bertram et al., 2007).
In the sporadic patients with clinical symptoms of AD but no known family history, the frequency of the apoE4 allele was found to be over 40%, compared to only 16% for age- matched controls (Roses, 1996). Inheriting one apoE4 allele confers 2- to 3-fold increased risk for AD, and people with two apoE4 alleles have 12-fold elevated risk to develop AD compared to individuals with no apoE4 alleles. In contrast, the E2 allele is associated with a lower risk for AD (Corder et al., 1994). It is worth noting that the allelic effect of apoE on the rate of cognitive decline following dementia onset is not as consistent as its influence on the risk and age-of-onset for AD (Dal Forno et al., 1996;
Growdon et al., 1996; Caselli et al., 1999; Caselli et al., 2004).
ApoE is the predominant apolipoprotein of the high density lipoprotein (HDL) involved in the redistribution of cholesterol and phospholipids within the brain (Mahley et al., 2006a). Cholesterol trafficking is necessary to maintain the structural and functional integrity of synapses and membranes (Mahley, 1988). ApoE is expressed in several organs, with the highest expression in the liver, followed by the brain. In the brain,
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it is produced locally, mainly by astrocytes and to some extent by microglia (Hauser et al.,
2011). Neurons can also produce apoE under certain conditions, but at a much lower
level than astrocytes (Xu et al., 1996; Mahley et al., 2006b; Xu et al., 2006). Owing to the
BBB, exchange between peripheral and brain-derived apoE does not occur. ApoE, consisting of 299 amino acid residues, has an independently folded amino (N)-terminal and carboxy (C)-terminal domains, which are joined together by a flexible hinge region
(Hatters et al., 2006). ApoE functions as an acceptor for lipid and cholesterol to form
HDL particles. The principal lipid-binding elements of apoE lie in residues 244-272 of
the C-terminal domain, while the LDL receptor-binding regions are located in residues
136-150 of helix 4 in the N-terminal domain and the Arg172 of the hinge region.
Association with lipid is required for apoE to bind with high affinity to the LDL receptor
(LDLR) and its related receptor proteins (Morrow et al., 2000). Nascent apoE is lipidated by ATP-binding cassette transporter A1 (ABCA1) to form discoidal HDL discs, which is then further lipidated by ABCG1, ABCG4 and lecithin:cholesterol acyltransferase
(LCAT) to from spherical HDL particles (Grehan et al., 2001; Wahrle et al., 2004;
Hirsch-Reinshagen et al., 2009). Brain-derived apoE-HDL particles differ from peripheral apoE-containing lipoproteins in that they are comprised mostly of phospholipid and unesterified cholesterol, and the nascent particles are discoidal in shape
(Fagan et al., 1999; Ladu et al., 2000). A key function of apoE is to maintain the cholesterol homeostasis, including cholesterol transport and clearance in the brain. Since mammalian cells are not capable of breaking down the ring structure of cholesterol, reverse cholesterol transport (RCT) is a critical pathway for removing excessive cellular cholesterol. By interacting with ABC transporters and class B, type I scavenger receptor
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(SR-BI), apoE induces the efflux of unesterified cholesterol from the membrane to the
HDL. The acquired cholesterol can then be esterified by LCAT and packed into the
hydrophobic core of the lipoprotein (Mahley et al., 2006a; Hirsch-Reinshagen et al.,
2009).
Conversely, apoE-HDL can also deliver cholesterol to the cells when needed.
This process is mediated through the LDL receptor-mediated endocytosis (Simons and
Ikonen, 2000). ApoE is reported to be a high affinity ligand for a group of receptors
known as the LDLR family. LDLR and LDLR related protein 1 (LRP1) are the main
apoE-HDL metabolic receptors in the brain (Bu et al., 2006). After endocytosis,
cholesterol is released from the lipoproteins and redistributed to the cell membrane, while
apoE is either degraded or resecreted to the extracellular milieu (Rensen et al., 2000).
Uptake of cholesterol from lipoproteins is important to support synaptogenesis, dendritic
formation and maintenance of synaptic connections (Anderson et al., 1998; Fagan et al.,
1998).
Crystal structure studies showed that apoE is comprised of two independently
folded domains that can be isolated after treatment of the protein with thrombin (Fig. 1.3)
(Hatters et al., 2006). The 22 kDa N-terminal domain (residues 1–191) adopts a hydrophilic globular conformation. By contrast, the 10 kDa C-terminal domain (residues
216–299) has a high lipid-binding affinity, and is responsible for the homo-aggregation
of apoE in the absence of lipid. It has been demonstrated that the receptor binding region
of apoE is between residues 136-150. However, for the high affinity binding to the LDL
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receptors, apoE must be associated with lipid (Dergunov et al., 2000; LaDu et al., 2006).
The lipidation status of apoE also governs its intrinsic stability and metabolism (Hirsch-
Reinshagen et al., 2004; Wahrle et al., 2004; Hatters et al., 2006). ApoE particles secreted
by Abca1-deficient astrocytes are smaller and contain remarkably decreased cholesterol content than particles from wildtype mice. Although the expression of apoE in astrocytes is independent of the Abca1 genotype, a great reduction (>80% decrease) in its levels was observed in the brain of Abca1-/- mice. These results highlight that the importance of the
lipidation for apoE stability, indicating that poorly lipidated apoE is rapidly metabolized
in the CNS (Hirsch-Reinshagen et al., 2004; Wahrle et al., 2004). It has also been shown that the polymorphisms at the amino acid residue 112 and 158 of apoE isoforms profoundly alter their tertiary structures, which lead to the difference in the cholesterol efflux and lipidation properties of the isoforms (E2 > E3 > E4) (Michikawa et al., 2000;
Hatters et al., 2005, 2006; Minagawa et al., 2009). Notably, Riddell and colleagues reported that the static apoE levels in the brain, CSF and plasma are APOE genotype- dependently regulated (ε2/2 > ε3/3 > ε4/4) in the target replacement mice. These differences arise resulted from enhanced degradation and the reduced half-life of newly synthesized apoE4 compared to apoE2 and apoE3, suggesting that the low static levels of apoE4 may result from its poor lipidation.
ApoE has been shown to co-localize with cerebral Aβ deposits in AD patients
(Namba et al., 1991) and bind fibrillar Aβ (fAβ) found in brain extracts (Naslund et al.,
1995; Yang et al., 1997). The direct interaction between apoE and soluble Aβ (sAβ) in vitro has been extensively studied (Strittmatter et al., 1993b; LaDu et al., 1994; LaDu et
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al., 1995; Zhou et al., 1996; Aleshkov et al., 1997; LaDu et al., 1997; Yang et al., 1997;
Tokuda et al., 2000). Initial in vitro studies showed that lipid-free apoE formed SDS- stable complexes with sAβ, and that apoE4 had the same or higher affinity to sAβ than apoE3 (Strittmatter et al., 1993a; Sanan et al., 1994). However, lipid-associated and lipid- free apoE interact differently with other molecules, including Aβ (LaDu et al., 1994;
LaDu et al., 1998). Ladu and colleagues reported that delipidation and denaturation, which are the standard operations for purifying apolipoproteins dramatically reduced the binding of Aβ to apoE (LaDu et al., 1995). In the CNS, apoE is always found as the lipoprotein, associated with lipids and cholesterol. Later studies using physiological
relevant lipidated apoE particles demonstrated an opposite result, with apoE2 and apoE3
bound to sAβ with much higher affinity than apoE4 did (apoE2 > apoE3 >> apoE4)
(Strittmatter et al., 1993a; LaDu et al., 1994; Sanan et al., 1994; Aleshkov et al., 1997;
Yang et al., 1997; Tokuda et al., 2000). These data suggested that the Aβ binding affinity
of apoE is isoform-dependent and is governed by apoE lipidation status (Tokuda et al.,
2000). Prolonged incubation of Aβ with apoE led to the formation of rather stable and
high molecular weight complexes. Interestingly, while all three apoE isoforms are
capable of promoting Aβ fibrillization in vitro, apoE4 was shown to have the highest
efficiency and apoE2 has the least (Ma et al., 1994; Wisniewski et al., 1994; Castano et
al., 1995). These results, however, correlate inversely to the binding affinity of apoE isoforms to Aβ. This controversy could be partially explained by the interaction between apoE and intermediate Aβ aggregates. Stratman and colleagues (2005) reported that lipidated apoE4 bound to the intermediate sized species of Aβ with 2-3 fold greater affinity than either apoE2 or apoE3. Non-lipidated apoE isoforms did not show this
-23- isoform-specific difference (Taylor et al., 2003). Thus, associating with apoE might stabilize the intermediate aggregates of Aβ, known as protofibrils, and promote further fibrillization, which eventually forms the amyloid plaques. A synthetic peptide consisting with the residues 12-28 of Aβ, which contains the binding site for apoE, has been shown to competitively inhibit the binding of apoE and Aβ (Strittmatter et al., 1993a;
Strittmatter et al., 1993b; Sadowski et al., 2004). Application of this peptide in vivo resulted in the decreased plaque formation in a mouse model of AD (Sadowski et al.,
2004). These results support the hypothesis that apoE induces β-pleated sheet formation of Aβ (Wisniewski and Frangione, 1992; Carter, 2005).
Knocking out Apoe gene in the PDAPP mice, a mouse model of AD, resulted in almost completely elimination of the cortical deposition of fibrillar, thioflavine S-positive
Aβ, while the diffuse Aβ deposits remained (Bales et al., 1997; Bales et al., 1999). These results are striking, but somewhat expected, because apoE is shown to promote Aβ fibrilization. However, transgenically expressed human apoE isoforms (E2, E3 or E4) in
PDAPP mice with the murine Apoe-knockout background markedly delayed the age- dependent increases in Aβ levels and its deposition (Holtzman et al., 1999; Holtzman et al., 2000b; Fagan et al., 2002). Comparing AD pathology in PDAPP mice expressing human or murine apoE, the expression of human apoE remained beneficial even though the protein levels of apoE were similar among all genotypes. This effect is also isoform- dependent with mice carrying Apoe2 or Apoe3 having more robust suppression of total
Aβ levels and deposition than the Apoe4 carriers after the age of 12 mo (Fagan et al.,
2002). These data seemingly contradict their previous findings, since expression of
-24-
murine apoE enhanced plaque deposition. This controversy could be explained by the difference between murine and human apoE. For example, in the hippocampus of young
(3-6 mo) PDAPP mice, despite total Aβ was comparable among genotype groups, the percentage of soluble Aβ (TBS-extractable) in mice expressing mouse apoE was significantly lower (~76%) than those expressing human apoE3 or apoE4 or no apoE
(Fagan et al., 2002). Thus, at certain concentration of Aβ, murine apoE may promote aggregation and fibrillogenesis of Aβ, while human apoE inhibits it. The other possibility is that human apoE may promote clearance of Aβ more efficiently than mouse apoE as proposed by Holtzman (Holtzman et al., 1999; Fagan et al., 2002). And it may also be isoform-dependent, which will be discussed later. Although the mechanism subserving the apoE-dependent modulation of Aβ deposition remains unclear, these data strongly support a critical role of apoE in promoting Aβ fibrillization and metabolism. In support of these observations, Holtzman and colleagues reported a similar apoE-dependent deposition of amyloid with a dramatic reduction of neuritic dystrophy in Tg2576 mice, another mouse model of AD. The data suggest that apoE is critical for the development of amyloid deposition of Aβ and its association with the dystrophy of neurites (Holtzman et al., 2000a). The effects of apoE on APP processing and Aβ production have also been studied in cell culture systems. While some studies suggested that apoE4 may induce Aβ production through LRP1-mediated endocytosis and processing of APP in a neuroblastoma cell line (Ye et al., 2005), there is no convincing data supporting the influence of apoE on the production of Aβ in vivo (Bales et al., 1999; Holtzman et al.,
1999; Kim et al., 2009).
-25-
An elegant study conducted by DeMattos et al. closely examined the PDAPP
transgenic AD mouse models with a deficiency in either or both apoE and apoJ
(DeMattos et al., 2004). ApoJ, also known as clusterin (gene name, Clu), is another
apolipoprotein in the brain. They reported that although the fibrillar, thioflavine S- positive amyloid plaques were much less pronounced in the Apoe knockout mice, as previously reported (Bales et al., 1997; Bales et al., 1999), the diffuse, Aβ-
immunoreactive plagues and soluble Aβ (sAβ) were present at similar levels in these
mice. Similar results were observed in the Clu knockout mice. Aβ levels in the
cerebrospinal fluid (CSF), but not plasma, were elevated in the absence of apoE,
suggesting the role of apoE in the clearance of Aβ. Further examination of Apoe and Clu
double knockout mice revealed dramatic elevation of Aβ plaques, hippocampal soluble
and insoluble Aβ and CSF Aβ levels, while APP expression and processing were not
changed. These data support the effect of apolipoproteins on Aβ metabolism by a process
that is independent of Aβ synthesis, and is possibly by regulating its clearance. Moreover,
Paul and colleagues utilized an ex vivo experiment to monitor the clearance of deposited
Aβ in brain slices of PDAPP mice by astrocytes. They reported that the astrocytic
degradation and clearance of deposited Aβ was impaired when using Apoe-deficient
astrocytes, supporting the possible role of apoE on the clearance of fibrillar Aβ (fAβ)
(Koistinaho et al., 2004). We recently demonstrated that both intracellular and
extracellular proteolysis of sAβ by microglia was enhanced in the presence of apoE
(Jiang et al., 2008). Similar to the observation of ex vivo fAβ clearance, loss of apoE
resulted in intracellular accumulation of Aβ, indicating that apoE is essential for efficient
degradation of sAβ by microglia. Supplying exogenous apoE or inducing apoE
-26- expression by GW3965, an agonist of liver X receptor (LXR), effectively enhanced the degradation of sAβ by microglia in vitro. Administrating of GW3965 to Tg2576 mice revealed reduced Aβ levels and amyloid plaque burden in the brain. Importantly, the facilitation of sAβ degradation relied upon the apoE isoforms (E2 > E3 > E4) and their lipidation status. Recent studies examined the apoE isoform effects on AD pathogenesis in a mouse model of AD (PDAPP/TRE), in which human apoE isoforms are expressed under the control of the endogenous mouse Apoe promoter. The results demonstrated that the clearance of Aβ depends on the isoform of apoE expressed (Bales et al., 2009;
Castellano et al., 2011). Furthermore, deficiency of Abca1 resulted in less efficient degradation of sAβ by microglia. This result may explain the observation from Abca1- knockout APP transgenic mice which exhibited elevation of Aβ levels and plaque loads without significant effects on Aβ production (Hirsch-Reinshagen et al., 2005; Koldamova et al., 2005a; Wahrle et al., 2005). These studies support the role of apoE in promoting
Aβ degradation; however, the molecular mechanisms underlying these events remain unknown and will be explored in the thesis.
MICROGLIA
Microglia are the brain’s tissue macrophages, and account for approximately 5% of the total cell population in the cerebral cortex of mice, but their abundance differs significantly between brain areas (Lawson et al., 1990; Block et al., 2007). Microglia are the primary immune effector cells in the CNS. They originate from peripherally-derived
-27-
myeloid lineage progenitors and invade the CNS during embryogenesis, before the
maturation of blood-brain barrier (BBB). In the mature brain microglia undergo self renewal by proliferation in situ (Ajami et al., 2007). However, whether peripheral myeloid cells can infiltrate the diseased brain remains controversial and will be discussed in the next section (Nakajima and Kohsaka, 2001; Priller et al., 2001; Hess et al., 2004;
Ransohoff and Perry, 2009). Microglia are uniformly distributed in the brain at a density of about 6/mm3 (Nimmerjahn et al., 2005) and constantly survey their immediate
environment for pathogens, foreign material and apoptotic cells (Streit et al., 2004). It has
been estimated that the resting microglia are able to completely screen the whole brain parenchyma once every few hours by consistently extending and retracting their processes (Nimmerjahn et al., 2005). Upon injury, microglia rapidly extend processes to the site of injury, then migrate to the lesion sites, recognize the pathogen, ramify, and mount an immune response in response to the stimulus (Ransohoff and Perry, 2009). In the brains of both AD patients and mouse disease models, microglia are found closely associated with the amyloid plaques and exhibit an ‘activated’ proinflammatory phenotype (Perlmutter et al., 1990; Frautschy et al., 1998). Microglia were initially postulated to play a role in the formation of amyloid deposition in the brain (Wisniewski and Frangione, 1992), but were subsequently shown not to do so. However, examination by electron microscopy of amyloid plaques showed that microglia are able to engulf Aβ with their processes and Aβ is observed in endosome-like cellular compartments
(Frackowiak et al., 1992). Later in vitro studies using radioisotope or fluorescent labeled
Aβ (Paresce et al., 1996) in combination with direct injection of fibrillar Aβ in to rat brains (Pluta et al., 1999) further demonstrated the ability of microglia to internalize Aβ.
-28-
The number and size of microglia increase in proportion to the size of plaques (Wegiel et
al., 2001; Wegiel et al., 2003; Wegiel et al., 2004) to regulate plaque dynamics
(Perlmutter et al., 1990; Wisniewski and Frangione, 1992; Bolmont et al., 2008; Meyer-
Luehmann et al., 2008; Yan et al., 2009). Recently, using in vivo imaging techniques,
local resident microglia were visualized to rapidly extend their processes and migrate
toward new plaques within 1-2 days of their appearance (Bolmont et al., 2008; Meyer-
Luehmann et al., 2008). Internalization of systemically injected amyloid-binding dye at
the vicinity of plaques provided direct evidence of the uptake of Aβ by microglia
(Bolmont et al., 2008).
Aβ CLEARANCE MECHANISMS
There are two principal mechanisms for removal of Aβ from the brain: efflux of
intact soluble Aβ (sAβ) to the peripheral circulation, and proteolytic degradation of both
soluble and fibrillar forms of Aβ (fAβ). The efflux of sAβ can occur through a number of
different routes, including efflux across the blood brain barrier (BBB) into the circulation mediated by LRP1 (low density lipoprotein receptor-related protein 1), the bulk flow of interstitial fluid (ISF)/cerebrospinal fluid (CSF) into the lymphatic system, and transport via the P-glycoprotein (PgP) efflux pump across the BBB. The efflux of Aβ through these mechanisms has been postulated to be facilitated or inhibited by its binding to chaperone proteins such as apoE, apoJ, α2-macroglobulin, transthyretin and albumin (Zlokovic et al.,
1996; Narita et al., 1997; Bell et al., 2007; Deane and Zlokovic, 2007). The efflux
-29- mechanisms have recently been thoroughly discussed by Deane et al. (2009) and the reader is directed to this excellent review.
SOLUBLE Aβ CLEARANCE
A variety of cell types have been reported to take up sAβ. One of the most well described pathways is LDL receptor-related protein 1 (LRP1)-mediated transcytosis, through which brain capillary endothelial cells export sAβ across the BBB to the peripheral circulation without significant degradation during transport (Deane et al.,
2004). LRP1 is a well described ApoE receptor and is highly expressed in the CNS
(Zerbinatti and Bu, 2005). It binds Aβ in complex with ApoE at nanomolar concentrations (Urmoneit et al., 1997; Jordan et al., 1998). Injecting antibodies against
LDL receptor–related protein-1 (LRP-1) or its inhibitor, receptor-associated protein (RAP) into brain parenchyma substantially reduces the export of Aβ to the periphery (Shibata et al., 2000). Compared to wild-type mice, Aβ efflux is also significantly reduced in Apoe knockout mice, indicating that the association of Aβ with ApoE might be required for
LRP1-mediated transcytosis of Aβ (Shibata et al., 2000). Although microglia also express
LRP1, the LRP1-mediated endocytosis is not the major pathway for sAβ uptake by microglia since antagonizing LDLR-related protein 1 (LRP1) by receptor associated protein (RAP) had no effect on internalization of sAβ (Marzolo et al., 2000; Mandrekar et al., 2009). We have demonstrated that inhibition of the fAβ receptor components, including scavenger receptor class A, class B, CD36 and CD47 did not impair the
-30-
internalization of sAβ (Mandrekar et al., 2009) and receptor-mediated endocytosis was
not the major pathway which microglia utilize to take up sAβ (Chung et al. 1999;
Mandrekar et al. 2009). Further studies demonstrated that microglia internalize sAβ
through constitutive, nonsaturable, fluid phase macropinocytosis, which requires both
actin and microtubules. Internalized sAβ is rapidly delivered to the lysosomes via the late
endolytic pathway (Mandrekar et al., 2009).
PROTEOLYSIS OF SOLUBLE Aβ
Although fAβ is largely resistant to proteolytic degradation, sAβ has been shown
to be sensitive to many proteases, including neprilysin (NEP), insulin degrading enzyme
(IDE), endothelin converting enzyme 1 (ECE1), angiotension converting enzyme (ACE),
plasmin, matrix metalloprotease 9 (MMP9) and presequence peptidase (PreP) (Soto and
Castano, 1996; Mukherjee and Hersh, 2002; Falkevall et al., 2006). Among these
proteases, NEP and IDE are the principle intracellular and extracellular enzymes for Aβ
degradation by microglia and other cell types, respectively (Kurochkin and Goto, 1994;
Iwata et al., 2000; Mukherjee and Hersh, 2002; Jiang et al., 2008). NEP is a type II
transmembrane protein with catalytic domain facing the lumen/extracellular spaces
(Malito et al., 2008). It was first identified as the major Aβ degrading enzyme using
biochemical methods (Iwata et al., 2000). Deletion of the Nep gene or inhibition of NEP activity with the metalloprotease inhibitor phosphoramidon were shown to increase Aβ levels in mouse models of AD (Iwata et al., 2001; Eckman et al., 2006; Farris et al., 2007).
Conversely, transgenic mice with NEP overexpression (Leissring et al., 2003) or ex vivo delivery of Nep gene by injecting transgenic fibroblast cells into the ventricles (Hemming
-31-
et al., 2007) resulted in reduced soluble Aβ levels and plaque burden. In addition, the decrease of NEP levels upon aging in mice and humans implicates a role of NEP in Aβ catabolism and suggests that regulation of NEP levels or activity might be a potential therapeutic approach (Iwata et al., 2002; Russo et al., 2005).
IDE is a zinc metalloprotease and can be secreted or associate with the cell surface depending on the cell type. Microglia are found to secrete IDE, yet hippocampal neurons only possess membrane associated IDE (Malito et al., 2008). IDE binds insulin with high affinity (~100 nM) and degrades insulin into fragments, which links it to the type 2 diabetes (Sladek et al., 2007). In addition to insulin, sAβ has been reported to be the canonical substrate of IDE (Iwata et al., 2000). It is interesting to note that patients with type 2 diabetes have an increased risk of Alzheimers disease (Qiu and Folstein,
2006). Elevation of insulin levels led to the increase of Aβ in the cerebrospinal fluid since
IDE has higher affinity for insulin than Aβ (Taubes, 2003). However, the link between these two substrates of IDE needs further investigation. IDE has been reported to participate in Aβ metabolism in vivo as genetically inactivation of the Ide gene in mice resulted in elevated levels of Aβ in the brain and this effect was dependent on the gene dosage of Ide (Farris et al., 2003; Miller et al., 2003). In mice heterozygous for the Ide gene, IDE activity was decreased to ~50%, whereas sAβ levels in brain homogenates were increased to intermediate levels between wildtype mice and homozygous Ide knockout mice. A recent study conducted by Hickman et al. reported that the levels of
IDE, NEP and MMP9 were dramatically reduced in older mice with the concomitant of the upregulation of proinflammatory cytokines (Hickman et al., 2008). Thus, it is possible
-32-
that inflammation in the CNS during normal aging leads to the reduction of Aβ degrading
enzymes and consequently results in increase Aβ levels and amyloid pathogenesis.
THE ROLE OF APOE IN SOLUBLE Aβ CLEARANCE
It has only recently been appreciated that ApoE plays a critical role in the normal
proteolytic clearance of sAβ from the brain. In the presence of ApoE, both intracellular
degradation by microglia and extracellular degradation by microglial conditioned media
of sAβ were induced (Jiang et al., 2008). Primary microglia derived from Abca1-/- mice
degraded sAβ less efficiently than wildtype microglia did, indicating that the ability of
ApoE to facilitate clearance is dependent upon the lipidation status of ApoE. The
lipidation status of ApoE is an important functional parameter, governing its
conformation (Fisher and Ryan, 1999), intrinsic stability (Hirsch-Reinshagen et al., 2004;
Wahrle et al., 2004), interactions with membrane receptors (Dergunov et al., 2000; LaDu
et al., 2006), and most importantly, its binding affinity for Aβ (Tokuda et al., 2000).
Knocking out Abca1 resulted in significantly higher amyloid burden in four different
mouse models of AD (Hirsch-Reinshagen et al., 2005; Koldamova et al., 2005a; Wahrle
et al., 2005). In these mice, soluble ApoE levels were diminished by 75-85%. Conversely, overexpression of ABCA1 in the brain of the PDAPP mice reduced amyloid deposition
(Wahrle et al., 2008). Although the soluble ApoE levels were also decreased in the
transgenic mice, the insoluble ApoE levels doubled, accompanied with overall higher
lipidation status. These results suggest that the gene dosage of Abca1 influences Aβ clearance through its effects on ApoE lipidation, although an effect on amyloidogenesis by ApoE could not be completely ruled out. The degree of ApoE lipidation is not only
-33-
governed by the expression level of ABCA1 but also depends on the allele of ApoE.
Variation in amino acids at position 112 and 158 of human apoE isoforms confers
differences in cholesterol efflux efficiency, lipidation status and receptor binding affinity
(Mahley et al., 2006a; Mahley et al., 2006b). ApoE2 and E3 are more highly lipidated
than the E4 isoform, and E4 is much less efficient in promoting sAβ proteolysis both within microglia and in the extracellular mileau (Jiang et al., 2008). Examination of
PDAPP mice carrying target-replaced human APOE genes both young (3 month old) and old (18 month old) mice showed ApoE isoform-dependent accumulation of soluble and insoluble Aβ levels and plaque burden (E4 >> E3 > E2), supporting the conclusion that the lipidation status of ApoE is critical for the clearance of Aβ (Wahrle et al., 2004;
Hirsch-Reinshagen et al., 2005; Wahrle et al., 2005; Jiang et al., 2008; Bales et al., 2009).
A recent study using microdialysis to directly monitor the production and clearance of Aβ in human APOE target-replaced PDAPP mice further supports the differential regulation of Aβ clearance by apoE isoforms (Castellano et al., 2011). The rate of Aβ production does not vary between human apoE isoforms.
FIBRILLAR Aβ CLEARANCE
A growing body of studies has demonstrated that Aβ interacts with immune cells through both innate and antibody-mediated adaptive immune responses. As the resident immune cells in the brain, microglia are professional phagocytes and can internalize fAβ
-34-
through this mechanism. Engulfment of fAβ by microglia through receptor-mediated
phagocytosis and its targeting to the endosome-lysosomal pathway has been investigated
in detail (Paresce et al., 1996; Frautschy et al., 1998; D'Andrea et al., 2004;
Koenigsknecht and Landreth, 2004). However, proteolytic degradation of fAβ is
controversial. Microglia are able to engulf and phagocytose fAβ readily; the question of
whether fAβ can be degraded intracellularly remains controversial. Early studies showed
that primary mouse microglia release fAβ after they have internalizated it (Chung et al.,
1999). Paresce et al found that microglia retain fAβ for a period of weeks without
degrading the peptides (Paresce et al., 1997). A subsequent study conducted by
Majumdar and collegues suggested that microglia have to be activated to enhance their
ability to degrade fAβ. Microglia in a nonactivated state were unable to degrade fAβ.
However, stimulating microglia with macrophage colony-stimulating factor (M-CSF)
enabled them to degrade fAβ efficiently through acidification of lysosomes (Majumdar et
al., 2007).
MICROGLIAL Aβ RECEPTOR COMPLEX
Microglia directly interact with and ingest fAβ via an ensemble of cell surface
receptors, including pattern recognition receptors (PRRs). PRRs, and most prominently
the Toll like receptors (TLRs), are commonly used by the innate immune system to
identify pathogen-associated molecular patterns (PAMPs) of bacteria and viruses. The
cell surface receptor complex of fAβ is composite of class A scavenger receptor (SR-A), the class B scavenger receptors CD36, α6β1 integrin, CD14, CD47 and TLR2, TLR4,
-35-
TLR6 and TLR9 (Paresce et al., 1996; Bamberger et al., 2003; Reed-Geaghan et al., 2009;
Stewart et al., 2010). Upon binding of fAβ, the receptor ensemble initiates the activation
of intracellular signaling cascades leading to the induction of phagocytic activity by
microglia. SRs were the first receptors reported to be involved in fAβ uptake. Paresce and colleagues demonstrated that microglial uptake of fAβ microaggregates was reduced by
competitive ligands for SRs, such as acetylated low-density lipoprotein (Ac-LDL), maleylated bovine serum albumin (M-BSA) or fucoidan (Paresce et al., 1996). Recently,
a study by Hickman et al. reported that the microglial mRNA levels of SR-A, CD36 and
RAGE were progressively and significantly reduced as mice aged (Hickman et al., 2008).
It suggests that the advance of AD pathogenesis may result from the decreased ability of
microglia to clear Aβ. Activating TLRs and their coactivator CD14 was shown to
stimulate the phagocytosis of fAβ (Liu et al., 2005; Tahara et al., 2006). Activation of
TLRs (TLR2, TLR4 or TLR9) with their specific ligands significantly enhanced the
uptake of fAβ by clonal BV-2 microglial and primary microglia. However, microglia
carrying defective TLR4 were less efficient than wildtype microglia in their ability to
take up fAβ after stimulation by lipopolysaccharide (LPS). The results suggest that TLR
signaling stimulates microglial phagocytosis of fAβ. Interestingly, fAβ itself activates
microglia and induces their phagocytic activity through TLR signaling (Bamberger et al.
2003; Koenigsknecht-Talboo and Landreth 2005; Koenigsknecht and Landreth 2004;
Reed-Geaghan et al. 2009). Blocking TLRs signaling by interfering with receptor-ligand
interactions or their downstream effectors also reduced fAβ-induced phagocytosis and
signaling (Bamberger et al., 2003; Koenigsknecht and Landreth, 2004). Microglia
deficient in TLR2, TLR4 or their coreceptor CD14 failed to induce phagocytosis in
-36-
response to fAβ stimulation (Reed-Geaghan et al., 2009). These results highlight the key
function of these PRRs in fAβ uptake and their stimulation of phagocytosis. The roles of
TLRs in AD pathogenesis have been evaluated in various animal models of AD, and the
effect of genetically inactivating the TLRs is confusing. APPswe/PSEN1dE9 mice with
inactive TLR4 exhibited increased cortical and hippocampal Aβ burden when compared
with mice with an intact TLR4 gene at 14-16 months of age. These authors argued that
the change in Aβ load was due to a change in microglial-mediated Aβ clearance that was
reliant upon TLR4 function (Tahara et al., 2006). In contrast, TLR2-null mice with
APPswe/PSEN1dE9 transgene showed delayed Aβ deposition through 6 months of age.
Interestingly, these animals had comparable deposition to their wildtype littermates at 9
month of age (Richard et al., 2008). It is noteworthy that although TLR2-null transgenic
mice had decreased plaque load, their cognition was dramatically impaired compared to
the TLR2-bearing littermate controls. Total Aβ42 levels were significantly increased in
TLR2-knockout mice. Thus, the role of TLRs in AD pathogenesis remains unclear. The
controversy over the outcomes may reflect the different ages of analysis.
ROLE OF INFLAMMATION IN FIBRILLAR Aβ CLEARANCE
The consequences of activating of TLR signaling on Aβ plaque burden in animal models of AD has also been controversial. Acute single intrahippocampal injection of the
CD14/TLR4 ligand LPS into Tg2576, an animal model of AD, resulted in clearance of diffuse, but not compact, Aβ plaques that required microglial activation (DiCarlo et al.,
2001; Herber et al., 2007). On the other hand, chronic administration of LPS by daily
-37-
infusion of LPS into the lateral ventricles for 2 weeks in APPV717F mice or weekly
injection of LPS intraperitoneally in APPSwe mice resulted in enhanced Aβ deposition
(Qiao et al., 2001; Sheng et al., 2003). The effects of chronic LPS exposure was
ameliorated by blocking the TNF signaling cascade with a dominant negative TNF
inhibitor in vivo, and prevented the acceleration of AD pathology (McAlpine et al., 2009).
These data clearly demonstrate that TLR activation can have quite different effects depending on the length of exposure to LPS.
We have shown that proinflammatory cytokines, like LPS, inhibit microglial
phagocytosis induced by fAβ in vitro (Koenigsknecht-Talboo and Landreth, 2005).
Exposure of microglia to fAβ activates microglia and induces their phagocytic activity
through TLR signaling (Bamberger et al., 2003; Koenigsknecht and Landreth, 2004;
Koenigsknecht-Talboo and Landreth, 2005; Reed-Geaghan et al., 2009). Blocking TLR
signaling or other elements of the fAβ receptor complex by interfering with receptor-
ligand interactions or their downstream effectors also reduced fAβ-induced phagocytosis
(Bamberger et al., 2003; Koenigsknecht and Landreth, 2004). Microglia lacking TLR2,
TLR4, or their coreceptor CD14, failed to induce phagocytosis in response to fAβ stimulation (Reed-Geaghan et al., 2009). These results combined highlight the key function of these PRRs in fAβ uptake and its stimulated phagocytosis. Interestingly, the fAβ-induced phagocytosis was inhibited when microglia were co-incubated with proinflammatory cytokines, including IL-1β, TNFα, IFNγ, MCP-1, and CD40L
(Koenigsknecht-Talboo and Landreth, 2005). The results were consistent with the effect observed in LPS-treated cells. Importantly, co-incubation of cells with anti-inflammatory
-38-
cytokines, including IL-4 and IL-10, or cyclooxygenase (COX) inhibitors, blocked the
ability of proinflammatory cytokines to suppress fAβ-elicited phagocytosis through the
inhibition of prostaglandin E2 (PGE2) production or its signaling pathways
(Koenigsknecht-Talboo and Landreth, 2005). Proinflammatory cytokines have also been
shown to inhibit microglial degradation of fAβ whereas anti-inflammatory cytokines
promote degradation in vivo. The inhibition of Aβ degradation was possibly a
consequence of the down-regulation of proteosomal enzymes (Yamamoto et al., 2008).
Consistent with their findings, knocking out the PGE2 EP2 receptor in a mouse model of
AD was reported to significantly reduce total Aβ levels and amyloid plaque burden
(Liang et al., 2005). Treating AD mice with anti-inflammatory agents such as LXR
agonists, PPARγ agonists and non-steroidal anti-inflammatory drugs (NSAIDs) resulted
in enhanced phagocytosis in response to fAβ and ameliorated AD pathology (Lim et al.,
2000; Heneka et al., 2005; Zelcer et al., 2007; Terwel et al., 2011). These findings support
the importance of inflammatory status in regulating microglial mediated Aβ clearance and suggesting the use of anti-inflammatory therapies in the treatment of AD.
ANTIBODY- AND COMPLEMENT-MEDIATED CLEARANCE OF FIBRILLAR Aβ
Schenk and colleagues reported that active immunization to Aβ in a mouse model of AD prevented plaque formation in younger animals and reduced the plaque burden and associated neuropathy in animals with established plaque pathology. Decoration of the plaques with IgG was observed in immunized animals, demonstrating that anti-Aβ antibodies could pass the BBB, albeit at low levels, and mediate the removal of plaques
-39-
from the brain (Schenk et al., 1999). Introduction of anti-Aβ antibodies directly into the
brain or peripherally resulted in a robust phagocytic response of microglia and
consequently the dissolution of Aβ deposition in the brain (Bard et al., 2000; Wilcock et
al., 2003). These data argue that the uptake and degradation of Aβ by microglia was a
result of the Aβ-antibody complex interacting with Fc receptors (FcR) which stimulated the phagocytic uptake of Aβ. These findings are consistent with previous reports of
enhanced phagocytosis of Aβ-IgG conjugates in vitro (Paresce et al., 1996; Brazil et al.,
2000). Interestingly, the presence of Aβ-specific antibodies in the blood and cerebrospinal
fluid (CSF) of healthy humans and AD patients have been reported (Du et al., 2001;
Hyman et al., 2001; Moir et al., 2005). The increase of auto-immune anti-Aβ antibodies has been viewed as a consequence of aging. However, the role of endogenous anti-Aβ antibodies in AD pathogenesis remains unclear. Indeed, the relative levels of anti-Aβ antibodies in AD patients and healthy individuals are highly variable. It is important to note that FcR-mediated phagocytosis was not inhibited by the presence of proinflammatory cytokines and this may underlie the efficacy of Aβ vaccination therapy in AD (Koenigsknecht-Talboo and Landreth, 2005).
The complement system has also been reported to be involved Aβ clearance
(Rogers et al., 1992; Rogers et al., 2002). Fibrillar Aβ is a strong stimulator of the
complement system and can activate the classical (antibody-dependent) pathway by
binding Clq and the alternative (antibody-independent) pathway by binding C3b (Rogers
et al., 1992; Jiang et al., 1994; Chen et al., 1996; Webster et al., 1997; Bradt et al., 1998).
Upon opsonization of Aβ by complement, microglia elicited more aggressive
-40-
phagocytosis via complement receptors (~1.5-fold increase over fAβ alone) (Brazil et al.,
2000; Webster et al., 2001; Rogers et al., 2002). Interestingly, the association of the
complement component C1q with Aβ inhibited microglial phagocytosis (Webster et al.,
2000). Thus, C1q may have opposing effects on ingestion of Aβ via the SR- and FcR-
mediated pathways, inhibiting naked Aβ uptake and enhancing the Aβ immune complex uptake. A recent study conducted by Maier et al. demonstrated that complement C3 deficiency in APP mice resulted in elevated cerebral Aβ levels and amyloid plaque burden. The authors also noticed that the activation status of microglia were switched from the classical activation M1 state to the alternative activation M2 state, suggesting the important role of complement system in Aβ clearance and microglia activation (Maier et al., 2008).
CHOLESTEROL AND ALHEIMER’S DISEASE
Epidemiological studies have shown that the high risk lipoprotein profiles for atherosclerosis and coronary artery disease (CAD), including increased levels of total plasma cholesterol and particularly high levels of LDL with low levels of HDL, are also commonly found in AD patients (Jarvik et al., 1995; Kuo et al., 1998; Martins et al.,
2009). Other CAD risk factors, including obesity, hypertension, and type II diabetes, are also linked to the increased risk of AD. Therefore, a possible link between cholesterol homeostasis and the development of AD has been suggested. In support of these epidemiological observations, intake of high-fat/high-cholesterol diets is reported to
-41-
associate with developing sporadic AD (Solfrizzi et al., 2003). Elevated dietary
cholesterol significantly exacerbated AD pathology in several mouse models of AD
(Refolo et al., 2000; Levin-Allerhand et al., 2002; Fitz et al., 2010). In contrast,
community-based case-control studies showed the association of the Mediterrian diet to
the reduced AD risk (Scarmeas et al., 2006). Since both BACE1 and γ-secretase are located on the lipid rafts, it has been postulated that cholesterol and lipid composition of the cellular membrane may influence APP processing and Aβ production. Several cholesterol loading and depletion studies found a correlation between cholesterol levels and the production of Aβ (Di Paolo and Kim, 2011). On the other hand, clearance of Aβ is mediated through the endolytic pathway, which has been suggested to be modulated by the cholesterol content in the cells (Jiang et al., 2008; Wahrle et al., 2008; Mandrekar et al., 2009). Yet, much less is known about this effect of cholesterol on the degradation of
Aβ, and will then be extensively explored in the thesis.
CHOLESTEROL IN THE CNS
In human, almost a quarter of unesterified cholesterol in the body is contained in
the CNS, which accounts for only 2% of the total body mass (Dietschy and Turley, 2001).
The vast majority of brain cholesterol resides in the specialized myelin sheaths of the
neurites. Mammalian cells continuously acquire cholesterol from two sources, cell-
autonomous de novo cholesterol synthesis and uptake of sterol carried in lipoproteins.
Owing to the blood-brain barrier (BBB), cholesterol metabolism in the CNS is isolated
-42-
from the rest of the body (Dietschy, 2009). BBB blocks the exchange of lipoproteins
between the CNS and plasma. And there is very little or no bulk-phase trans-endothelial transport of cholesterol. Many observations provide very compelling evidence that there is no net contribution of cholesterol from the periphery to the pool of sterol in the CNS, even before closing of the BBB in the fetus. In several species, the rate of de novo synthesis was found to be fully accounted for the rate of accumulation of sterol in the developing CNS while massive myelination occurs (Xie et al., 2000; Quan et al., 2003).
This rate of synthesis declines with maturation of the animals but maintains, albeit at a low rate, in the adult. Thus, almost all brain cholesterol is synthesized in situ.
CHOLESTEROL SYNTHESIS
Virtually all cells express the machinery to produce cholesterol from acetyl-CoA, while the rate of production varies among cell types. In the brain, cholesterol and apolipoproteins are synthesized mainly by glial cells and to a much lesser extent by neurons (Dietschy, 2009; Martins et al., 2009). The production of cholesterol is regulated by a family of membrane-bound transcription factors, termed sterol regulatory element- binding proteins (SREBPs) (Goldstein et al., 2002; Horton et al., 2002). Depletion of cholesterol induces the release of the SREBP cleavage-activating protein (SCAP) from
ER and escorts SREBP to Golgi apparatus (Goldstein et al., 2006). In the Golgi apparatus,
SREBP is sequentially cleaved by S1P, a serine protease, and S2P, a zinc metalloproteinase, to release its N-terminal domain, designated nuclear SREBP
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(nSREBP). Translocation of nSREBP to the nucleus enables its binding to the
nonpalindromic sterol response elements (SREs) and activates the transcription of several
target genes, including HMG-CoA reductase, the rate limiting enzyme for cholesterol
synthesis. Cells synthesize cholesterol in the endoplasmic reticulum (ER) and transfer it
to the plasma membrane. The transport of cholesterol between these two compartments is rapid, with a t1/2 of approximately 15 min. It is suggested that the transfer of cholesterol
from ER to plasma membrane is principally mediated through a non-vesicular lipid/sterol
transport mechanism and is independent of passage through the Golgi apparatus
(reviewed in Lev, 2010).
CHOLESTEROL UPTAKE AND INTRACELLULAR TRAFFICKING
In addition to de novo synthesis, local demand for cholesterol could be fulfilled, at
least partially, through uptake of sterol carried by lipoproteins from the adjacent area.
This pathway to acquire cholesterol is extremely important especially in the neurons,
since de novo synthesis of cholesterol occurs in ER, which is located in the cell body, but
not in their distal processes (Vance et al., 1994). The transport of cholesterol from the cell
body may not be fast enough to supply the urgent need in the distal axons. It is supported
by the finding that synapse formation between neurons is shown to require the presence
of astrocytes. Importantly, this requirement was found to be the cholesterol carried in
lipoproteins produced by astrocytes rather than other secreted factors, since it could be
obviated by supplying apoE-HDL to the neurons (Mauch et al., 2001). Cells take up
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lipoproteins from the interstitial fluid through the well-studied LDL receptor-mediated endocytosis (Simons and Ikonen, 2000). The LDL receptors (LDLRs) constitute a large family of structurally related proteins, which are expressed in a cell-type specific manner.
Neurons express several different LDL receptors, including LDLR and LRP1, in a region-
specific manner; astrocytes and microglia mainly express scavenger receptor B1 (SR-B1),
which is absent in neurons (Pfrieger, 2003). In the CNS, apoE- and apoA-I-HDLs are
recognized by these LDLRs, endocytosed via clathrin-coated pits and transported to
sorting endosomes, where HDLs dissociate with their receptor and sorted to the
lysosomal pathway (Ioannou, 2001; Dietschy, 2009). HDL particles are dismantled in the
late endosomes/lysosomes, releasing cholesterol and cholesterol ester. Apolipoproteins
are either transported to the recycling endosomes for resecretion or to the lysosomes for
degradation. The cholesterol esters are hydrolyzed by an acid-optimum cholesteryl ester
hydrolase as the endosome matures. Efflux of unesterified cholesterol from endocytic
vesicles to ER and cytosolic compartments is mediated through NPC proteins, which are
named because their disruption causes Niemann-Pick type C (NPC) disease. From there,
cholesterol is actively redistributed throughout the cell.
CELLULAR CHOLESTEROL HOMEOSTASIS
Because free unesterified amphipathic cholesterol is potentially toxic, excess free
cytosolic cholesterol triggers multiple feedback pathways to negatively regulate the
cellular cholesterol levels (Dietschy, 2009). First, cholesterol in the cytosol is esterified
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by acyl-CoA A:cholesterol acyltransferase (ACAT), resulting the more biological inert
hydrophobic cholesterol ester, which is then stored in lipid droplets. Second, cholesterol can be hydroxylated to 24S-hydroxycholeserol (24S-OHC) by the 24-hydroxylase,
CYP46. 24S-OHC activates the liver X receptor (LXR), which subsequently upregulates
the expression of reverse cholesterol transport (RCT)-related genes, including ABCA1,
ABCG1 and apoE, yet trans-inhibits the expression of HMG-CoA reductase (Repa and
Mangelsdorf, 2000). Elevation of cholesterol transporters enhances the efflux of cellular
cholesterol. The binding of cholesterol to the cholesterol-sensor domain of the SREBP-
cleavage-activating protein (SCAP) also confines SCAP and SREBP to the ER, thus
inhibits the translocation of nSREBP to the nucleus and subsequently, reduces the transcription of HMG-CoA reductase. Finally, expansion of the cytosolic free cholesterol pool also induces the binding of HMG-CoA reductase to INSIG-1 and -2, by which it
initiates its ubiquitination and subsequent degradation (Goldstein et al., 2006). In
combination with reduced expression of HMG-CoA reductase, these steps lead to very
rapid suppression of cholesterol synthesis in the cell. It should be emphasized that this
negative feedback on cholesterol synthesis occurs only when the sterols (cholesterol and
oxysterols) reach the metabolically active pool in the cytosol and nucleus (Dietschy,
2009). If cholesterol is trapped in the late endosomes/lysosomes and cannot reach the
active pool, as seen in NPC1-deficient cells, and the synthesis of cholesterol will not be
inhibited. Thus, even though the overall cellular content of cholesterol is expanded, the
metabolic active pool of cholesterol eventually shrinks, resulting in the rate of cholesterol synthesis is actually increased and no activation of the LXR occurs (Vance and Peake,
2011).
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CHOLESTEROL EXCRETION FROM THE CNS
Although cellular cholesterol levels are tightly regulated, in the adult animal the
mount of overall cholesterol synthesis still exceeds the need for sterol accretion in the
CNS. Mammalian cells lack the ability to directly degrade the four-membered ring
structure of cholesterol; therefore, there is a need for constant excretion of the extra
cholesterol cross the BBB into the plasma to maintain the steady state in the brain. The excessive cholesterol in the plasma is then transported to the liver, converted into bile acids and excreted into the lumen of intestine. It is estimated that the rate of cholesterol efflux from the CNS to the periphery equals 1.4 mg/kg/day in a 7-8 week old mouse. One mechanism is the passive transport of 24S-hydroxycholesterol (24S-OHC), a hydroxylated form of cholesterol. A cytochrome P450 species, termed CYP46, converts cholesterol into 24S-OHC (Lund et al., 1999; Bjorkhem and Meaney, 2004). This cholesterol hydroxylase is expressed exclusively in many, but not all, neurons. It is expressed particularly in a subset of large, metabolically active neurons including
Purkinje cells and pyramidal cells. Unlike cholesterol, 24S-OHC passes freely between lipophilic membranes and thus passively travels across the BBB (Bjorkhem and Meaney,
2004). Over 90% of plasma 24S-OHC originates from the brain. Deficiency in Cyp46
revealed essentially no change in cholesterol turnover in any organ of the body except the
brain where accumulation of cholesterol was observed and synthesis of cholesterol was
suppressed by 40% (Lund et al., 2003). Interestingly, progressively decreased 24S-
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OHC/cholesterol ratios have been observed in AD patients, indicating a defect of 24S-
OHC-mediated cholesterol excretion (Kolsch et al., 2003). Polymorphisms in CYP46 have been reported to be associated with the risk of LOAD, suggesting that these genetic variations might act via influencing brain cholesterol homeostasis (Garcia et al., 2009). It is also noteworthy that 24S-OHC is the canonical ligand of LXR. Reducing activation of
LXR may lead to direct impact on Aβ metabolism as described above and will be discussed in more detail later. It is reported that the efflux of 24S-OHC is 0.9 mg/kg/day, which accounts for roughly 65% of total sterol excretion from the mouse CNS (Xie et al.,
2003). Therefore, a second pathway must be responsible for the remaining 35% of sterol efflux and is possibly reflected in the cholesterol turnover within small neurons, glial cells and myelin. Although much less understood and yet controversial, it is proposed that this pathway may involve apoE-associated cholesterol transport to cross the BBB
(Dietschy, 2009). The observations of basolateral expression of LRP1 as well as apical expression of ABCA1 on the endothelial cells support the possible LRP1-mediated transcytosis of apoE-associated cholesterol.
CHOLESTEROL AND Aβ PRODUCTION
Several lines of evidence support the idea that most, if not all, classes of lipids are implicated in AD pathogenesis. Many studies have demonstrated that Aβ production is sensitive to cellular cholesterol levels. In APP transfected HEK cells or hippocampal neurons, depletion of cellular cholesterol strongly reduced Aβ production neuronal cell
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lines; while increasing cellular cholesterol levels induced the production (Simons et al.,
1998; Frears et al., 1999). Extensive research has been undertaken on the the trafficking
and/or proteolytic activity of APP and its processing enzymes including BACE1 and
presenilins. APP, BACE1 and presenilins are present in both cholesterol-rich raft and
non-raft regions of the membranes. However, APP processing occurring within lipid rafts
appears to incline to the amyloidogenic BACE1 cleavage, while outside lipid rafts, APP processed is predominantly through the non-amyloidogenic α-secretase pathway (Di
Paolo and Kim, 2011). Reducing membrane cholesterol levels by methyl-β-cyclodextrin
(MβCD), a cholesterol-extracting compound, inhibited activity of both BACE1 and γ- secretase, leading to reduction in Aβ generation. Cholesterol depletion decreases the association of BACE1 with lipid rafts, which correlates with decreased amyloidogenic processing of APP. In contrast, introducing a raft-targeting GPI anchor into BACE1 sequence or acute exposure of cholesterol induced the raft localization of BACE1, which in turn promoted generation of Aβ (Vetrivel and Thinakaran, 2010). In addition, Grimm and colleagues transfected hippocampal neurons with a C99 APP construct to observe the effect of cholesterol depletion on γ-secretase activity. With C99, the Aβ generation is only dependent on γ-secretase, yet independent of β-secretase activity. They reported that both
MβCD and lovastatin were able to decrease the production of Aβ. Moreover, raft association of γ-secretase subunits is sensitive to acute cholesterol depletion. Increased accumulation of APP CTFs in lipid raft microdomains was observed after the inhibition of γ-secretase activity, supporting that γ-secretase might preferentially cleave APP in lipid rafts (Di Paolo and Kim, 2011). These data support the correlation between cellular cholesterol content and APP processing, and suggest that reducing cholesterol levels may
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be beneficial to AD by stalling Aβ production.
Epidemiological studies report that there is up to a 70% lower prevalence and
incidence of AD in subjects chronically taking statins, which inhibit HMG-CoA reductase
and results in lower cholesterol levels (Jick et al., 2000; Wolozin et al., 2000). This strong
effect is observed even after correcting for confounding issues such as physician or
patient bias (Wolozin, 2004). Statins have also been shown to have beneficial effects in ameliorating pathology and behavior deficits of AD in several mouse models (Refolo et al., 2001; Petanceska et al., 2002). Cell culture studies further support that the usage of statins can lower Aβ generation (Fassbender et al., 2001; Paris et al., 2002). However, inconsistent results have been reported from several retrospective and prospective clinical studies examining the relationship between the use of statins and the incidence of AD
(Wolozin, 2004). The simvastatin treatment showed a beneficial effect of reducing cognitive deficit in a small cohort prospective study of 26 mild AD subjects; while pravastatin failed to reduce the incidence of dementia in a much larger prospective study, the PROSPER study, of 6000 subjects with high plasma cholesterol levels. Although the reasons for the failure of pravastatin to prevent AD in this study are unclear, it may in large part due to differences in study design and data analysis. For example, the selection of patient as well as the methods used to detect cognitive function. Further investigation will be needed to properly translate the preclinical findings to the clinical usage.
CHOLESTEROL HOMEOSTASIS-RELATED GENES AND AD
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ATP-CASSETTE TRANSPORTER A1
ABCA1 transfers phospholipid and cholesterol from the plasma membrane to
apoE and apoA-I and this process is known as the reverse cholesterol transport (RCT)
pathway. In Tangier disease, mutations in the Abca1 gene lead to defective cholesterol
efflux, thus resulting in excessive cholesterol ester deposition in cells (Orso et al., 2000).
It has been reported that ABCA1 is critical for apoE secretion in macrophages. Similar
observations reported that Abca1 deficiency significantly reduced apoE secretion in vitro
from astrocytes and microglia and in vivo. Consistent with a role for ABCA1 in apoE lipidation and metabolism, the level of ABCA1 protein was positively associated with the size and levels of apoE in the mouse brain (Hirsch-Reinshagen et al., 2004; Riddell et al.,
2008). The disease-related role of ABCA1 was first studied in APP processing. Fukumoto and colleagues demonstrated that blocking Abca1 expression in neuroblastoma Neuro2a cells by siRNA reduced Aβ secretion, while upregulating ABCA1 levels by the means of
endogeneous or synthetic LXR ligands caused significant increases in Aβ secretion
(Fukumoto et al., 2002). However, later in vitro studies reports opposite observations
(Koldamova et al., 2003; Sun et al., 2003). Although the effect of ABCA1 on Aβ
production is inconclusive, knocking out Abca1 in the mouse models of AD confers very
compelling evidence for its role in AD pathogenesis. Three independent studies
demonstrated that Abca1-deficiency in four distinct mouse models of AD resulted in
increased Aβ levels, parenchymal amloid plaques and cerebral amyloid angeopathy
(CAA) (Wahrle et al., 2004; Hirsch-Reinshagen et al., 2005; Koldamova et al., 2005a).
Moreover, a recent work from Holtzman’s group overexpressing Abca1 in PDAPP mice
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correlates the reduced Aβ levels and plaque burden to the increased dose of the Abca1
transgene (Wahrle et al., 2008). These results are strikingly contrast to studies in apoE deficient animals in which in amyloid plaques was significantly reduced. Although the cholesterol homeostasis was altered in these animals, it is important to note that unlike lowering cellular cholesterol levels by statins, APP processing and Aβ production were not affected by these genetic manipulations, indicating the influence of ABCA1 on AD pathology was a result of alternations in Aβ clearance. Our recent work further demonstrated that microglia deficient in Abca1, which have poorly lipidated apoE-HDLs, were unable to efficiently degrade Aβ (Jiang et al., 2008). Inducing production and lipidation of apoE by the LXR agonist GW3965 by contrast enhanced the degradation of
Aβ. These studies highlight the importance of apoE lipidation status in Aβ clearance and suggest that ABCA1 is essential for normal apoE lipidation and Aβ clearance.
LIVER X RECEPTORS
The liver X receptor (LXR) family consist of two isoforms α and β that are oxysterol-activated nuclear receptors and play an important role in regulating cellular as well as whole-body cholesterol homeostasis (Tontonoz and Mangelsdorf, 2003). Both
LXRs are expressed in the brain with LXRβ being the predominant isoform. Upon the binding of oxysterols, including 24S-OHC, LXR dimerizes with the retinoid X receptor
(RXR) and binds to LXR-response elements in the regulatory regions of its target genes, which leads to the recruitment of the transactivation complex and induces transcription.
LXR counteracts SREBPs, acting to lower cholesterol levels. A myriad of genes,
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including apolipoproteins (apoA-I, apoC, apoD and apoE), ABC transporters (ABCA1
and ABCG1) and HDL modifying enzymes (CETP and PLTP) have been identified as direct LXR targets. Overall, activation of LXR results in enhanced cholesterol absorption and efflux in the extraheptic tissues, including CNS, and cholesterol exchange among
lipoproteins for the elimination from the liver and intestine.
Because of its function on regulating apoE and ABCA1 levels, LXR agonists are
common reagents used in many studies to induce ABCA1 expression. Although as
mentioned above, activation of LXR in the cell-based studies led to inconclusive effects
on Aβ production, genetic manipulation and in vivo administration in the mouse models
of AD conveyed convincing results (Koldamova et al., 2005b; Riddell et al., 2007; Jiang
et al., 2008). Zelcer et al. reported that loss of either LXRα or LXRβ expression
exacerbated AD-related pathology in APP/PS1 mice (Zelcer et al., 2007). In contrast, we
have recently demonstrated that administration of GW3965, a LXR agonist, reduced the
plaque load and Aβ levels in Tg2576 mice and ameliorate the behavior deficit (Jiang et al.,
2008). Consistent with previous findings, APP expression and processing were not
changed in both studies, suggesting that changes in Aβ levels and AD pathology after
manipulating LXR levels or activation were the result of alternations in Aβ clearance.
Our in vitro experiments showed that degradation of Aβ both intracellularly by microglia
and extracellularly in the conditioned media were enhanced following GW3965
administration, supporting the effect of LXR on the clearance of Aβ. Importantly, the
enhanced Aβ degradation by LXR activation was largely impaired in the absent of apoE
or ABCA1, indicating that this effect was mediated through regulating the expression of
-53- apoE and ABCA1, as well as the lipidation status of apoE (Jiang et al., 2008).
NIEMANN-PICK TYPE C PROTEINS
NPC disease is characterized by the accumulation of unesterified cholesterol in the endolytic system is an autosomal recessive disease. It is caused by mutations in the two NPC proteins, NPC1 and NPC2, among which NPC1 mutations account for 95% of patients (reviewed in Rosenbaum and Maxfield, 2011). NPC1 is a membrane-bound glycoprotein located on the membranes of late endosomes/lysosomes. NPC2 is a soluble glycoprotein found in the lysosomal lumen. Both NPC proteins contain cholesterol- sensing domains; however, they bind cholesterol in opposite orientations. Crystal structure studies demonstrated that NPC1 likely binds the 3β-hydroxyl group of cholesterol with its N-terminal domain facing the lumen of the late endosomes/lysosomes, while NPC2 binds to cholesterol with the isooctyl chain buried in the binding pocket.
However, the highly dynamic nature of the endocytic recycling system makes the question of colocalization and functional linkage of the membrane-integrated NPC1 and soluble NPC2 very difficult to answer definitively. A recent finding showed that NPC1 accepted or donated cholesterol from liposomes >100-fold faster when NPC2 was present, yet a mutant NPC2 (Pro120Ser) deficient in binding cholesterol failed to accelerate this activity, suggesting that NPC1 and NPC2 work in tandem to export lipoprotein-derived cholesterol from late endosomes/lysosomes (Infante et al., 2008; reviewed in Miller and
Bose, 2011).
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Importantly, the lipidosis caused by NPC dysfunction leads to progressive AD- like neuropathology including the formation of neurofibrillar tangles, malfunction of neurons, massive neuronal loss and eventually death of the individuals. Some neuronal populations in NPC patients, especially in adult onset cases (5% of total NPC cases), also develop deposition of Aβ. It has been demonstrated that cholesterol may directly modulated the activity of BACE1 and γ-secretase (discussed below), suggesting that NPC deficiency induced abnormal accumulation of cholesterol may promote the production of
Aβ. Indeed, intracellular accumulation of Aβ was observed in the U18666A-, an NPC inhibitor, treated or NPC-deficient neurons (Runz et al., 2002; Jin et al., 2004) and in mouse and human brains (Saito et al., 2002; Burns et al., 2003; Jin et al., 2004). Although less studied, it is possible that deficiency of NPC also influences the cellular clearance of
Aβ, which will be explored in the thesis.
CONCLUSION AND RESEARCH OBJECTIVES
The amyloid hypothesis predicts that a decrease of Aβ levels in the brain will lead to reduction of plaque formation and ameliorate AD pathology. Thus, modulation of either production or clearance of Aβ could be a potential target for AD therapy. The effect of apoE in AD pathogenesis is very strong, although its exact role in AD pathogenesis remains controversial. Our recent findings in line with several other studies demonstrated that apoE plays important role in promoting Aβ degradation in vivo and in vitro. However, the underlying mechanisms remain unclear. Understanding the mechanisms will be
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challenging, but will provide a potential therapeutic angle to treat AD. In the thesis, I will
explore the mechanism of which apoE facilitate Aβ degradation in both intracellular and extracellular conditions.
In chapter 2, we investigate the effect of apoE in the intracellular Aβ degradation by microglia, concluding that the cholesterol efflux and lowering property of apoE on the microglia is important for its influence on Aβ degradation. We took advantage of a small mimetic peptide, which elicits cholesterol efflux as apoA-I, as well as using cholesterol modulating reagents to demonstrate that the cholesterol lowering property of apoE alone is required and sufficient to facilitate intracellular degradation of Aβ. In contrast, direct interaction between apoE and Aβ plays no role in this activity. Furthermore, we observed that intracellular trafficking of Aβ through the endocytic systems was accelerated by apoE. This is possibly through the cholesterol lowering effect of apoE on the recycling of
Rab7, a small GTPase protein that recruits a rab7-conatinig motor complex to late endosomes. In this chapter, we will provide the first mechanistic evidence linking cholesterol modulation to apoE-induced Aβ degradation.
In chapter 3, we study the effect of apoE in the IDE-mediated Aβ degradation. By
means of enzyme kinetic analysis, we demonstrate that in the presence of apoE, IDE-
mediated Aβ degradation has reduced the Km with unchanged Vmax, supporting that the
possible chaperone effect of apoE in this activity. By using different lipidated species of apoE in the assays, we demonstrated that highly lipidated apoE induced Aβ degradation more efficiently than the poorly lipidated ones. These results support that the association
-56- of Aβ to apoE is important for the elevated proteolytic activity of Aβ. In this chapter, we also demonstrated that the facilitation of Aβ degradation is apoE isoform-dependent, possibly owing to the difference in their lipidation profiles, which influence the affinity to
Aβ.
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FIGURES
Figure 1.1
Reprinted with permission from Van Dam et al. (2006) Nat Rev Drug Discov 5: 956-970
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Figure 1.1: APP mutations associated with early-onset Alzheimer’s disease
Mutations of APP are named after the nationality or location of the first family in which that specific mutation was demonstrated. The majority of mutations found in the APP gene are clustered in the vicinity of secretase-cleavage sites, thereby influencing APP processing. These mutations either favor the cleavage of β-secretase over α-secretase to produce more Aβ overall, like Swedish mutation, or dispose γ-secretase for producing more Aβ1-42, like French, London and Florida mutations. Some mutations are located within a specific region of Aβ peptide sequence (12-28 a.a.) which has been shown to mediates the interaction between Aβ and apoE. The Aβ sequence is indicated in red.
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Figure 1.2
Figure 1.2: Proteolytic processing pathways of APP
Non-amyloidogenic cleavage of APP by α-secretase between Lys16 and Leu17 of the Aβ
domain generates the soluble ectodomain of APP (sAPPα) and the C-terminal fragment
(CTFα or C83). Subsequent cleavage of CTFα by γ-secretase yields the the APP
intracellular domain (AICD) and a short fragment termed p3. Alternatively, amyloidogenic cleavage of APP by β-secretase (BACE1) occurs at the N-terminus of the
Aβ domain and generates the secreted sAPPβ as well as the C-terminal fragment (CTFβ
or C99). C99 is further cleaved by γ-secretase, resulting the secretion of Aβ and the
generation of AICD.
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Figure 1.3
A B
C
Reprinted with permission from Hatters et al. (2006) Trends Biochem Sci 31: 445-54
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Figure 1.3: Model of the structure of lipid-free apoE and the formation of an α-
helical hairpin conformation in apoE-HDL-like particles.
(A) The N-terminal domain of apoE consists of a four-helix bundle (helix 1, red; helix 2,
blue; helix 3, green; helix 4, yellow). The short cyan-colored helix is present in the mouse
apoE structure. The LDL-receptor-binding region is located on helix 4 and inthe cyan-
colored region near the start of the C-terminal domain.. The two polymorphic positions,
112 and 158, that distinguish the three common isoforms, also reside in the N-terminal
domain. High-affinity binding to the LDL receptor also requires Arg172 in the hinge
region that connects the N- and C-terminal domains. The C-terminal domain (gray) of
apoE contains the principle lipid-binding domain. (B) ApoE undergoes a considerable
conformational change when it binds to phospholipids. An α-helical hairpin conformation
puts all of the known elements of the LDL-receptor-binding motif, including Arg172, into a structural apex, which potentially explains why only lipid-bound apoE binds to the LDL receptor with high affinity. (C) The molecular envelopes of the two protein molecules are colored red and blue. The inner sphere (yellow) represents the hydrophobic chains of the phospholipids; the outer layer of the sphere (violet) represents the more polar region occupied by the phospholipid polar head groups.
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CHAPTER 2
APOLIPOPROTEIN E PROMOTES β-AMYLOID TRAFFICKING AND
DEGRADATION BY MODULATING MICROGLIAL CHOLESTEROL LEVELS
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Apolipoprotein E Promotes β-amyloid Trafficking and Degradation by Modulating
Microglial Cholesterol Levels*
This research was originally published in The Journal of Biological Chemistry:
C. Y. Daniel Lee, Wayne Tse, Jonathan D. Smith, and Gary E. Landreth. Apolipoprotein E
promotes β-amyloid trafficking and degradation by modulating microglial cholesterol
levels. J. Biol. Chem. 2012; 287(3):2032-44. ©the American Society for Biochemistry and
Molecular Biology
ABSTRACT
Allelic variation in the apolipoprotein E (APOE) gene is the major risk factor of
sporadic Alzheimer’s disease (AD). ApoE is the primary cholesterol carrier in the brain.
Previously, we demonstrated that intracellular degradation of β-amyloid (Aβ) peptides by
microglia is dramatically enhanced in the presence of apoE. However, the molecular
mechanisms subserving this effect remain unknown. This study reports a mechanistic link
between apoE-regulated cholesterol homeostasis and Aβ degradation. We demonstrate
that promoting intracellular Aβ degradation by microglia is a common feature of HDL
apolipoproteins, including apoE and apoA-I. This effect was not dependent on the direct
interaction of apoE and Aβ. Regulation of Aβ degradation was achieved by solely
manipulating cellular cholesterol levels. The expression and the activity of Aβ degrading
enzymes, however, were not regulated by cholesterol. We observed that reducing cellular
cholesterol levels by apoE resulted in faster delivery of Aβ to lysosomes and enhanced
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degradation. Moreover, apoE facilitated the recycling of Rab7, a small GTPase
responsible for recruiting the motor complex to late endosomes/lysosomes. These data
indicate that faster endocytic trafficking of Aβ-containing vesicles in the presence of apoE resulted from efficient recycling of Rab7 from lysosomes to early endosomes. Thus, apoE-induced intracellular Aβ degradation is mediated by the cholesterol efflux function of apoE, which lowers cellular cholesterol levels and subsequently facilitates the intracellular trafficking of Aβ to lysosomes for degradation. These findings demonstrate a direct role of cholesterol in the intracellular Aβ degradation.
INTRODUCTION
The polymorphism of the apolipoprotein E (APOE) gene is the major genetic risk
factor for sporadic, late onset Alzheimer’s disease (AD) (Corder et al., 1993; Schmechel
et al., 1993). There are three common apoE isoforms in human, apoE2, E3 and E4, which
differ in only two amino acid residues (Mahley and Rall, 2000). Possession of APOE4
alleles confers increased AD risk and an earlier age of onset in a gene dose-dependent
manner, while inheriting the apoE2 allele is protective (Corder et al., 1993; Corder et al.,
1994). Recently, Bateman and colleagues demonstrated that Aβ clearance is impaired in
human late onset AD patients (Mawuenyega et al., 2010). Importantly, the APOE4 allele
is associated with decreased Aβ clearance (Bales et al., 2009; Castellano et al., 2011). We
have demonstrated that apoE plays a direct role in the normal, physiological clearance of
Aβ from the brain. ApoE was shown to facilitate both intracellular and extracellular
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proteolytic degradation of Aβ in an isoform- and lipidation status-dependent manner
(Jiang et al., 2008). However, the molecular mechanisms by which apoE influences
proteolysis of Aβ remain unclear. In this study, we focus on the mechanism of apoE-
facilitated intracellular Aβ degradation by microglia.
ApoE is the predominant apolipoprotein in the central nervous system (CNS) and
acts to scaffold the formation of high density lipoprotein (HDL) particles. ApoE regulates
the redistribution and homeostasis of cholesterol within the CNS, that is necessary for the
maintenance of the structural and functional integrity of synapses and membranes
(Mahley, 1988; Kim et al., 2009). In the brain, apoE is mainly produced by astrocytes and
to a much lesser extent by microglia. Cholesterol is transferred to apoE through the
activity of ATP-binding cassette transporter A1 (ABCA1) and related transporters,
including ABCG1 and ABCG4, to form HDL particles, resulting the reduction of cellular
cholesterol levels (Grehan et al., 2001; Wahrle et al., 2004; Lund-Katz and Phillips, 2010).
Conversely, cells can use HDL as an exogenous source of cholesterol through a receptor
mediated endocytosis pathway (Xu et al., 1997).
A growing body of evidence suggests that dysregulated cholesterol metabolism may be involved in the pathogenesis of AD. Epidemiological studies also suggested a positive correlation of hypercholesterolemia and high incidence of coronary artery disease to the increased risk of AD (Martins et al., 2009). Inhibition of cholesterol synthesis by statins was shown to decrease the Aβ levels and AD pathology in several animal models of AD (Refolo et al., 2001; Petanceska et al., 2002). Moreover, high fat diets dramatically exacerbate AD-related pathology in mouse models (Refolo et al., 2000;
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Levin-Allerhand et al., 2002; Fitz et al., 2010). Although cholesterol has been widely investigated in the production of Aβ, there is limited knowledge about its involvement in the clearance of Aβ. Deficiency of Abca1 results in the accumulation of cholesterol and exacerbates the formation of amyloid plaques, while overexpression of Abca1 reduces brain Aβ levels and amyloid deposition (Wahrle et al., 2004; Hirsch-Reinshagen et al.,
2005; Koldamova et al., 2005a; Wahrle et al., 2008). APP processing and Aβ production were not affected by these genetic manipulations, indicating their influence on AD pathology was a result of alternations in Aβ clearance. Increasing apoE expression through liver X receptor (LXR) activation promotes Aβ degradation both in vitro and in vivo (Jiang et al., 2008). Importantly, the ability of apoE to facilitate Aβ clearance is dependent upon the lipidation status of apoE as highly lipidated species were more effective in promoting Aβ degradation. Indeed, microglia deficient in Abca1, which have poorly lipidated apoE-HDLs, exhibited impaired cholesterol efflux function and were unable to efficiently degrade soluble Aβ (sAβ). These reports combined suggest the importance of the cholesterol efflux function of apoE in its effects on intracellular sAβ degradation.
In the current study, we report that apoE facilitates intracellular Aβ degradation by microglia through modulation of cellular cholesterol levels. This activity does not require direct interaction between apoE and Aβ. Depleting cellular cholesterol through its transfer to apoE-containing HDL particles alone was sufficient to promote Aβ degradation.
Furthermore, we observed that the intracellular transport of Aβ through the endocytic system was accelerated in the presence of apoE. The cholesterol lowering effect of apoE
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enhanced the recycling of Rab7, a small GTPase protein in the motor complex on late
endocytic vesicles, which in turn facilitated the trafficking of Aβ-containing vesicles to
lysosomes for degradation. These findings provide a mechanistic explanation of apoE-
facilitated intracellular Aβ degradation and demonstrate a direct role for cholesterol in
AD.
MATERIALS AND METHODS
Reagents
Human apoE and apoA-I were purchased from rPeptide (Bogart, GA) and Sigma
(St. Louis, MO), respectively. The 4F peptide (Ac-DWFKAFYDKVAEKFKEAF-NH2)
(Handattu et al., 2009) was custom made by American Peptide (Sunnyvale, CA).
Lovastatin, methyl-β-cyclodextrin (MβCD), water-soluble cholesterol (cholesterol-loaded
MβCD) and U18666A were purchased from Sigma.
Primary microglial culture
Primary microglia were derived from the brains of C57BL/6 or Apoe-/- mice at postnatal day 1 – 3 using a mild trypsinization protocol as previously described (Saura et al., 2003; Koenigsknecht and Landreth, 2004). Purified microglia were then maintained in DMEM/F12 (Invitrogen, Carlsbad, CA) containing 2% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin for 3 – 5 days before further experiments.
Preparation of Aβ Peptides
Lyophilized unlabeled (American Peptide) or Alexa 555-labeled Aβ1-42 (AnaSpec,
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Fremont, CA) and Aβ12-28 (American Peptide) were dissolved to a final concentration of
1 mg/ml in DMSO and stored at -80°C until use. For flow cytometry, the Aβ1-42 was then labeled with Alexa 488 fluorophores (Invitrogen) using the manufacturer’s protocol as previously described (Jiang et al., 2008; Mandrekar et al., 2009). In brief, the Aβ reaction mixture was allowed to fibrilize at 37°C overnight. Unincorporated fluorophores were then removed by ultracentrifugation at 100,000 x g at 4°C. The pellet was re-suspended in DMSO, sonicated, and ultracentrifuged. The steps were subsequently repeated until most of the pellet fraction was solubilized in DMSO. The supernatant contains the operationally defined “soluble Aβ” which consists of primarily monomeric Aβ, with only low levels of small oligomeric species (Shen and Murphy, 1995).
Intracellular Aβ degradation assay
Primary microglia were plated overnight in 12-well plates at a density of 2.2 x 105 cells/well in DMEM/F-12 (Invitrogen) containing 2% FBS (Invitrogen). On the day before pretreatment, cells were switched to serum-free DMEM/F-12 overnight. Microglia were then treated with apoE, apoA-I, 4F peptide, U18666A or cholesterol-loaded MβCD at indicated concentrations for 24 h. For experiments in which MβCD or Aβ12-28 were used, microglia were incubated with these reagents for 30 min prior to the addition of Aβ.
For lovastatin treated groups, cells were incubated with lovastatin supplemented with 250
μM mevalonate and 2% lipoprotein-deficient fetal bovine serum (Sigma, St. Louis, MO) in DMEM/F-12 for 48 h (Chen et al., 2008). Following pretreatments, cells were then incubated with 2 μg/ml of Aβ1-42 for 18 h. After washing extensively with PBS to remove
residual extracellular Aβ, cells were lysed in 1% SDS containing protease inhibitor
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cocktail, including 2 μg/ml leupeptin, 2 μg/ml aprotinin and 250 μM PMSF. Remaining
intracellular Aβ was quantified by ELISA using 6E10 as the capture antibody and 4G8-
HRP as the detection antibody (Covance, Princeton, NJ). Synthetic Aβ1-42 was used to
generate a standard curve. Plates were developed using a TMB substrate kit (Pierce,
Rockford, IL) and the reaction was stopped by the addition of an equal volume of 1 M
HCl. The results were read using a Spectramax colorimetric plate reader (Molecular
Devices, Sunnyvale, CA). The amount of remaining intracellular Aβ was normalized to
the total protein in the lysates.
Flow Cytometry
Primary murine microglia were plated at a density of 7 x 105 cells/well in a six-
well plate in DMEM/F-12 containing 2% FBS. On the day before pretreatment, culture
media was replaced with serum-free DMEM/F-12 overnight. Cells were then pretreated
with the drugs of interest for 24 h, followed by administrating Alexa 488-labeled Aβ for 6
h. For experiments in which MβCD or Aβ12-28 were used, microglia were incubated with these reagents for 30 min prior to the addition of Alexa 488-labeled Aβ. Cells were then washed with PBS and fixed with 4% paraformaldehyde. Following fixation, cells were washed with PBS and collected for analysis by flow cytometry using the EPICS-XL
MCL (Beckman Coulter, Brea, CA).
Cellular cholesterol quantification
Cellular cholesterol levels were measured by an Amplex Red cholesterol assay kit
(Invitrogen) according to the manufacturer’s instruction and normalized to protein levels
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measured with a BCA assay kit (Pierce, Rockford, IL).
Competitive Inhibition Assay
Inhibition of Aβ1-42 binding to natural apoE3 particles in the presence of Aβ12-28
was performed as described (Sadowski et al., 2004). In brief, 100 μl of Aβ1-42 (2 μg/ml in
0.5 M sodium bicarbonate buffer, pH 9.6) was incubated on polystyrene 96-well plates at
4°C overnight to immobilize Aβ1-42. Plates were then washed with PBS plus 0.01%
Tween20 (PBST) and blocked with 1% skim milk in PBST for 1 h at 37°C. Immortalized
E3-DSE astrocytes (a gift from Dr. Holtzman, Washington University at St. Louis, MO) were cultured in serum-free DMEM/F-12 with 1 mM sodium pyruvate for 3 days to obtain condition media containing lipidated apoE3 particles. Phosphoramidon (10 μM)
was added to the conditioned media to block IDE and NEP activity (Jiang et al., 2008).
The conditioned media was preincubated with an increasing concentration of Aβ12-28 (0
to 10 μg/ml) for 1 h at 37°C and added to the 96-well plate containing immobilized Aβ
for additional 3 h. In parallel, a series of apoE dilutions was incubated on the wells
coated with an anti-apoE antibody (WUE-4, a gift from Dr. Holtzman) to generate a
standard curve for calibration. The plate was then washed with PBST and apoE bound to
Aβ1-42 was detected using a HRP-conjugated anti-apoE antibody (Abcam, Cambridge,
MA). The chromogenic reaction was developed with a TMB substrate kit (Pierce),
stopped by the addition of 1M HCL and measured using a Spectramax colorimetric plate
reader (Molecular Devices). OD values were first calibrated to the standard curve and
then converted to percentages, considering the binding of apoE3 to Aβ1-42 in the absence of Aβ12-28 as 100%. Nonspecific binding was determined by replacing coated Aβ1-42
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with bovine serum albumin and/or omitting the conditioned media in the assay.
Colocalization assay and Immunohisto-chemistry
Primary microglia were plated on coverslips in 6-well plates at a density of 3 x
105 cells/well plate in DMEM/F-12 containing 2% FBS. Cells were serum-starved overnight and pretreated with the drugs of choice for 24 h. To label the late endosomes and lysosomes, Alexa 488-labeled microspheres (20 nm, Invitrogen) were diluted (1:10) and incubated in PBS with 1 mg/ml bovine serum albumin (BSA) for 10 min at room temperature, washed with serum-free DMEM/F-12, and applied to the cells at 10 μl/ml
for 30 min. After being washed extensively to remove non-internalized microspheres,
cells were incubated for 4 h in 37°C to allow microspheres to accumulate in late
endosomes/lysosomes. Colocalization of Aβ and late endosomes/lysosomes was
measured by a pulse-chase experiment. Cells were incubated with 2 μg/ml Alexa 555- labeled Aβ, washed thoroughly, and further incubated in 37°C for 15 min. Cells were then washed with PBS and fixed in 4% paraformaldehyde. The nuclei were visualized by
DAPI staining. For measuring the colocalization of Aβ and Rab7, the cells were treated as stated above but omitted the step of applying microspheres. Rab7 was detected using an anti-Rab7 antibody (Cell Signaling, Danvers, MA) followed by a corresponding Alexa
488-conjugated secondary antibody (Invitrogen). Coverslips were mounted on glass
slides and observed using a Zeiss LSM 510 confocal microscope. The microscopic
images were subjected to the colocalization assay using NIH ImageJ software (Bethesda,
MD) with both “Intensity Correlation Analysis” (Li et al., 2004) and “Colocalisation Test”
plugins (Costes et al., 2004). The algorithm of these two methods takes a specific
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approach to automatically estimate the threshold for both channels and to exclude the
overlaps from randomly distributed fluorescent signals. In our samples, colocalization
coefficients calculated using both methods were very close. Thus, only the Pearson’s
correlation coefficients obtained from Intensity Correlation Analysis plugin were plotted.
Time-lapse live cell imaging
Primary microglial cells were plated on Delta T tissue culture plates at a density
of 3 x 105 cells/plate. Cells were incubated overnight in serum-free DMEM/F-12 and
treated with 1 μg/ml ApoE or 10 μM U18666A for 24 h as indicated. Cells were stained
with DiD (Invitrogen) for 5 min to visualize membrane and vesicles. Alexa 488-labeled
Aβ1-42 (1 μg/ml) was applied to the cell for 5 min. After a brief wash, live cell imaging was performed using a Zeiss LSM 510 confocal microscope. Heated stage and objectives
were used to maintain the temperature of culture media at 37°C. Immediately after
washing off extracellular Aβ, time-lapse images were taken every 10 s for 10 min. Images
of 512 x 512 pixels were acquired at 7.5 s/frame (Chen et al., 2008). The paths of
movement were tracked using MetaMorph software (Molecular Devices, Sunnyvale, CA) to calculate the velocity of these vesicles. 12 to 20 vesicles per cell were analyzed. It normally took 15 – 20 min for Aβ-containing vesicles to traffic from the plasma membrane to the perinuclear compartments. The fluorescently labeled vesicles then stayed in that region for a long period of time with minimal movement, indicating the fluorophores reached lysosomes and stayed. Since we were focusing on the trafficking of endolytic vesicles, these immobile vesicles were excluded in the statistic analysis. To avoid bias exclusion of certain vesicles, only four most motile Aβ-containing vesicles per
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cell were selected to represent the directed movement of endolytic vesicles. To eliminate
the perturbations from photo or mechanical drifting while recording, four most stationary
vesicles were set as the baseline motility (VS) and subtract that from the speed of motile
vesicles (VM):
V = VM – VS
The average trafficking speed in the first 6 min of the recording of the 4 most motile
vesicles was plotted as one data point in the figure.
Subcellular fractionation
Primary microglia plated in 1 x 106 cells/well in 6-well plates were serum-starved
overnight and treated with drug of interest for 24 h. Cells were washed with PBS and
lysed by incubation in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and 10 mM PIPES, pH 7.3) on ice for 15 min, followed by 10 s of sonication.
Cell debris was cleared by centrifugation at 500 x g for 5 min at 4°C. The supernatant was then centrifuged for 1 h at 110,000 x g at 4°C in a SW50.1 rotor (Beckman-Coulter,
Fullerton, CA). The resulting supernatant was collected and saved as the “cytosolic” fractions, and the pellet was resuspended in relaxation buffer as the “membrane” fractions.
These subcellular fractions were subjected to Western blot analysis to determine the relative amount of Rab7 in each fraction. The membrane marker flotillin was used to assess the efficacy of the fractionation procedure.
Rab-Guanine Nucleotide Dissociation Inhibitor (GDI) mediated Rab7 extraction
The in vitro extraction of Rab7 by GDI was evaluated as described (Hutt et al.,
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2000; Lebrand et al., 2002; Ganley and Pfeffer, 2006). Cells were harvested by scraping
in homogenization buffer (20 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1
mM dithiothreitol plus protease inhibitor cocktail), and were homogenized with 5 passes
through a 22-gauge needle. The homogenates were centrifuged at 3,000 x g at 4°C for 5
min. The resulting supernatants were further centrifuged at 110,000 x g for 1 h at 4°C.
The pellet was resuspended in GDI extraction buffer (20 mM HEPES, 100 mM KCl, 1
mM MgCl2, pH 7.4 plus protease inhibitor cocktail) and saved as “crude membrane
fractions”. Protein concentration of the fractions was determined by the BCA assay using
BSA as the standard. Crude membranes containing 20 – 25 μg of proteins were diluted in
GDI extraction buffer plus 1 mM GDP and 0.5 mg/ml BSA. Recombinant GDI (GenWay,
San Diego, CA) in the indicated amount was added to each sample for 15 min at 37°C.
Samples were then immediately centrifuged at 110,000 x g for 30 min. The supernatant
containing extracted Rab7 was collected and subjected to Western blot analysis. Separate
non-GDI extracted aliquots from the same crude membrane fractions were used as the
loading control of Rab7.
Western blot
Lysates or subcellular fractions were resolved in 12% Tris-glycine SDS-
polyacrylamide gels and transferred to PVDF membranes. Blots were probed with anti-
Rab7 (Cell Signaling), anti-flotillin (BD Biosciences, Franklin Lakes, NJ) or anti-β-actin
(Santa Cruz Biotechnology, Santa Cruz, CA) antibodies overnight at 4°C.
Chemiluminescent signals (Millipore, Billerica, MA) were visualized by exposure to
Kodak Biomax X-ray film. Band intensities were quantified using NIH ImageJ software.
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Statistics
All statistical analyses were performed using Prism software (GraphPad, San
Diego, CA). Graphs are shown as means ± SEM. Values statistically different from controls were calculated using a two-tailed t-test or one-way ANOVA with the Tukey-
Kramer multiple comparisons test to determine p values. In the Aβ12-28 competitive inhibition assay, data were analyzed by a one-site competition non-linear regression fit algorithm.
RESULTS
Facilitation of Aβ degradation is a common feature of HDL apolipoproteins
Microglia are able to internalize sAβ from extracellular milieu and degrade it intracellularly without resecretion (Jiang et al., 2008). The clearance of Aβ was assessed by measuring remaining Aβ in the cells after an 18 hr period that allows for extensive degradation of internalized sAβ. Consistent with our previous findings, preincubation of microglia with apoE for 24 h resulted in the enhanced intracellular degradation of Aβ in a dose-dependent manner (Fig. 2.1A) (Jiang et al., 2008). Similarly, another apolipoprotein, apoA-I, was also capable of promoting Aβ degradation in a dose-dependent manner, demonstrating that facilitation of Aβ degradation is a common feature of HDL apolipoproteins (Fig. 2.1D). To avoid the possibility of treatment-induced variation in Aβ uptake, the cumulative internalization of Aβ was monitored using flow cytometry with fluorescently labeled Aβ. Although the internalized Aβ was degraded over time, the
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fluorophore was retained in the cells, thus, accumulated fluorescence reflected the total
Aβ internalized (Mandrekar et al., 2009). No significant change in Aβ uptake after the treatment of apoE and apoA-I indicated that the difference in remaining intracellular Aβ resulted from the change in efficiency of Aβ degradation (Fig. 2.1B and 2.1E).
The direct association of apoE with Aβ, has been extensively investigated
(Strittmatter et al., 1993b; LaDu et al., 1994; Tokuda et al., 2000). ApoA-I is also shown to bind to Aβ (Koldamova et al., 2001). However, it is unknown if the facilitation of degradation requires direct interaction between apolipoproteins and Aβ or rather that this effect is due to the common lipid transport function of the apolipoproteins. The residues
12-28 of Aβ were identified as the binding site of apoE (Strittmatter et al., 1993b). It has been shown that using this small fragment of Aβ (Aβ12-28) was able to competitively block the binding of full-length Aβ to apoE (Sadowski et al., 2004), suppressing fibril formation and enhancing the survival of cultured neurons (Ma et al., 1996). However, we found that Aβ12-28 was not able to inhibit apoE-induced Aβ degradation by microglia
(Fig. 2.2B-C), although at the same concentration (5 μg/ml), Aβ12-28 was able to largely block the binding of Aβ1-42 and apoE (Fig. 2.2A). These data indicate that the direct
apoE-Aβ interaction is not required for apoE to promote intracellular Aβ degradation by
microglia.
Facilitation of Aβ degradation relies on the cholesterol efflux function of apolipoproteins
To ascertain whether the cholesterol efflux property of apolipoproteins is sufficient to enhance Aβ degradation, we employed an apoA-I mimetic peptide, 4F, that
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possesses the structure of the essential lipid binding domain but lacks the receptor
binding domain. Importantly, it has no sequence homology with apoA-I (Datta et al.,
2001; Navab et al., 2005b). The mimetic peptide has been shown to form HDL-like
particles and is capable of mediating cholesterol efflux similar to apoA-I-HDL (Navab et
al., 2004; Navab et al., 2005a). The 4F peptide reduced cellular cholesterol levels when
supplied to the cultured primary microglia, similar to apoE and apoA-I (Fig. 2.1C, 2.1F,
2.2F and 2.2I); while no Aβ binding activity to the 4F peptide was observed (data not
shown). The 4F peptide, therefore, provides an excellent tool to parse out which of the
two highly connected properties of apoE promotes Aβ degradation. Application of the 4F
peptide facilitated Aβ degradation in a dose-dependent manner (Fig. 2.2D). Control
studies demonstrated that Aβ uptake was not influenced by the treatment with the 4F
peptide (Fig. 2.2E). However, it remains possible that the mimetic peptides interact with
the endogenous apoE-HDL particles to prevent their turnover and eventually increase
their overall amounts (Navab et al., 2005a; Ou et al., 2005), and in this way promote Aβ
degradation rather than by the direct cholesterol lowering effect of the 4F peptide. To
evaluate this possibility, we performed the same assay in apoE-deficient microglia. The
ability to enhance Aβ degradation by the 4F peptide was not changed in the Apoe-/- background (Fig. 2.2G-H). To further confirm that the cholesterol efflux function mediates apoE-induced Aβ degradation, we blocked the ABCA1 activity with glyburide
(Fielding et al., 2000; Wang et al., 2001). ABCA1 mediates cholesterol efflux onto apolipoproteins to form HDL particles, and blocking the activity of ABCA1 by treatment of glyburide was able to inhibit apoE-induced Aβ degradation without influencing the uptake of Aβ (Fig 3). These results demonstrated that lowering cellular cholesterol levels
-78- by cholesterol efflux was sufficient to promote microglial Aβ degradation. Importantly, the data demonstrated that the direct interaction with Aβ is not necessarily required for the enhancement of intracellular Aβ degradation and that apolipoproteins act through their ability to promote cholesterol efflux from cells to enhance Aβ degradation.
Degradation of Aβ is influenced by the cellular cholesterol levels
If cellular cholesterol levels have critical regulatory roles of Aβ degradation, decreasing cholesterol should enhance Aβ degradation. To understand whether cellular cholesterol levels could directly regulate Aβ degradation, we used a variety of pharmacological reagents to directly manipulate cellular cholesterol levels. We first utilized methyl-β-cyclodextrin (MβCD), a cholesterol chelator, to decrease cellular cholesterol by direct extraction of cholesterol from the plasma membrane (Yancey et al.,
1996). A low dosage of MβCD was able to promote Aβ degradation without interfering with its uptake (Fig. 2.4A-B). Decreasing endogenous cholesterol synthesis by inhibiting
HMG-CoA reductase activity with lovastatin also resulted in the facilitation of Aβ degradation (Fig. 4D-E). Conversely, increasing cellular cholesterol levels by incubating microglia with cholesterol-loaded MβCD resulted in impaired Aβ degradation (Fig. 2.5A-
B). Since Aβ is degraded mainly by neprilysin in the late endosomes/lysosomes (Iwata et al., 2000), we tested if cholesterol accumulation preferentially in the endocytic system would also impair Aβ degradation. Cells take up HDL or LDL particles through LDL receptor-mediated endocytosis. Lipoproteins are then transported to late endosomes where cholesterol ester carried is hydrolyzed to free cholesterol (Maxfield and Wustner,
2002; Maxfield and Mondal, 2006). The transport of cholesterol from late endosomes and
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lysosomes to other cellular compartments is regulated by Niemann Pick C-1 protein
(NPC1), which is located on the membrane of late endosomes and lysosomes (Mukherjee
and Maxfield, 2004). Cells carrying defective NPC1 exhibit cholesterol accumulation in
late endocytic vesicles. U18666A is a widely used NPC1 inhibitor that acts to stimulate
the accumulation of cholesterol within the endocytic system (Feng et al., 2003).
Treatment of primary microglia with U18666A resulted in the increase of cellular
cholesterol levels and an inhibition of Aβ degradation (Fig. 2.5D-E). Control incubations
monitored total cellular cholesterol levels and verified that all drugs elicited their
expected actions (Fig. 2.4C, 2.4F, 2.5C and 2.5F). Since the cholesterol homeostasis is
tightly regulated in the cells, it is possible that manipulating cellular cholesterol levels
feeds back to regulate apoE expression, which in turn, affects Aβ degradation. To verify
this possibility, we performed the same treatments in Apoe-/- microglia to rule out the
possibility of the involvement of apoE-HDL particles in previous experiments. We found that Aβ degradation regulated by directly manipulating cellular cholesterol levels did not require the presence of apoE (Fig. 2.6). These data strongly demonstrate that modulating
cellular cholesterol levels alone is sufficient to regulate intracellular Aβ degradation.
Thus, intracellular Aβ degradation is likely to be facilitated by apoE’s ability to reduce cellular cholesterol levels via stimulating cholesterol efflux.
The transcription of Aβ degrading enzymes is not regulated by cholesterol levels
We further investigated whether the regulation of Aβ degradation by modulating cellular cholesterol levels was a result of the changes in the levels of Aβ degrading enzymes. Proteases that degrade Aβ include neprilysin (NEP), insulin degrading enzyme
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(IDE), matrix metalloproteinase 9 (MMP-9), angiotensin-converting enzyme (ACE),
endothelin converting enzyme 1 and 2 (ECE-1, -2) and plasminogen (Soto and Castano,
1996; Mukherjee and Hersh, 2002; Falkevall et al., 2006). Quantitative real-time PCR was performed to quantify their transcripts levels. We found no correlation linking cholesterol modulation and the levels of the transcripts analyzed (Fig. 2.7). Although the
expression of these enzymes was not changed, their functionality could be affected by
their distribution or microenvironments, such as pH and lipid rafts. Thus, we utilized
fluorogenic substrates which are specific to these enzymes and act as independent
indicators to analyze their enzyme activity. The results showed that the enzyme activities
were not influenced by apoE treatment (Fig. 2.8). Thus, the apoE- and cholesterol-
mediated regulation of Aβ degradation is not due to altered levels or activity of Aβ
degrading enzymes.
Cholesterol levels regulate Aβ transport
Since uptake of Aβ and the levels and activity of Aβ degrading enzymes were not
influenced by apoE, the step that apoE induces to facilitate Aβ degradation must take
place between the plasma membrane and lysosome. Previous studies demonstrated that
the motility of endosomes depends on the membrane cholesterol composition. Lowering
cholesterol was reported to enhance the motility of folate receptor containing endosomes
while increasing cholesterol paralyzes them (Chen et al., 2008). We thus hypothesized
that the trafficking of Aβ to lysosomes might be the rate limiting step for Aβ degradation
and regulated by the levels of cellular cholesterol. The cholesterol lowering effect of
apoE might facilitate the trafficking of Aβ to lysosomes and ultimately results in
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enhanced Aβ degradation. We first evaluated the delivery of Aβ to late
endosomes/lysosomes by a pulse-chase experiment. Fluorescently labeled Aβ was
incubated with microglia for 7 min, followed by a 15 min chase. The localization of Aβ to late endosomes/lysosomes was assessed by labeling this compartment with fluorescence- labeled microspheres, which are internalized via pinocytosis, trafficked through the endocytic pathway and accumulated in late endosomes and lysosomes (Falcone et al.,
2006; Mandrekar et al., 2009). The fluorescent microspheres faithfully labeled the
LAMP1-positive endosomes/lysosomes (Fig. 2.9D). With this method, we demonstrated that in the presence of apoE, Aβ was delivered faster to the late endosomes/lysosomes, reflected by its higher colocalization index compared with vehicle-treated controls (Fig.
2.9A-B, E). Conversely, when microglia were pretreated with U18666A to increase endosomal cholesterol levels, Aβ transport was slower, indicated by the lower colocalization index (Fig. 2.9C, E). To further confirm the idea of Aβ trafficking under
the influence of cellular cholesterol levels, we performed live cell imaging to directly
track the motility of Aβ. Fluorescently labeled Aβ was applied to primary microglia and
time-lapse confocal images were taken to calculate the velocity of the vesicular
movement. The Aβ containing vesicles moved significantly faster when cells were
pretreated with apoE compared to the non-treated controls, while the vesicles staggered after U18666A pretreatment (Fig. 2.9F). The results combined suggest that the cholesterol-lowering effect of apoE accelerates the trafficking of Aβ toward late endosomes/lysosomes. Consequently, enhanced Aβ degradation is observed.
Cholesterol regulates the trafficking of Aβ-containing endosomes via the recruitment of
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Rab7
It has been well established that endocytic trafficking is regulated by Rab proteins
and their effectors (Novick and Zerial, 1997; Seachrist and Ferguson, 2003). Among
these Rab proteins, Rab7 is recruited to endosomes, which are sorted and directed into a
lysosomal degradation pathway. Rab7 regulates the trafficking, maturation and fusion of
endosomes with lysosomes (Rupper et al., 2001; Poteryaev et al., 2010). The activity of
Rab7 was shown to be affected by endosomal cholesterol levels (Lebrand et al., 2002;
Chen et al., 2008). To characterize the possible role of Rab7 in the apoE-enhanced endocytic trafficking, we first performed the pulse-chase experiment as described above to analyze the colocalization of Aβ and Rab7. The amount of Aβ colocalized with Rab7
was increased in the cells pretreated with apoE yet decreased in the cells pretreated with
U18666A, suggesting the involvement of Rab7 in the trafficking of Aβ (Fig. 2.10A).
However, no significant changes in Rab7 protein levels were observed in the total cell
lysates (Fig. 2.10B). Detailed analysis also showed no shift in its subcellular distribution
between cytosolic and membrane fractions (Fig. 2.10C). Like other Rab proteins, Rab7 is
tightly associated with the membranes by virtue of the geranylgeranylation of cysteine
residues at its C-terminus (Novick and Zerial, 1997; Pfeffer and Aivazian, 2004). Doubly
prenylated Rab proteins are also present in cytosol where their prenylated cysteines are
tightly bound to the guanine nucleotide dissociation inhibitor (GDI). Rab proteins
normally cycle between the membrane and cytosol. Generally, after a vesicle fusion event,
Rab proteins are retrieved from the membrane by GDI, and then re-delivered to its
membrane of origin for reutilization (Cavalli et al., 2001; Pfeffer, 2001; Seabra et al.,
2002). Previous studies suggested that extraction of Rab7 from lysosomes for its
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recycling is affected by the cellular cholesterol levels (Lebrand et al., 2002). Reducing
cellular cholesterol levels enhance the recycling of Rab7 while accumulation of cellular
cholesterol paralyzed the endocytic system. We thus hypothesized that apoE might enhance the recycling of Rab7, since apoE is able to reduce cellular cholesterol levels
(Fig. 2.1C). The recycling of Rab7 was assessed by its extractability by GDI. We found that Rab7 from membrane fractions was more extractable in microglia pretreated with apolipoproteins or the 4F mimetic peptide, while it was less extractable in U18666A treated cells (Fig. 2.10D). The data is consistent with the finding on other cell types
(Lebrand et al., 2002) and suggests that lowering cellular cholesterol levels by apoE facilitates the recycling of Rab7 and subsequently results in accelerated endocytic trafficking of Aβ for lysosomal degradation.
DISCUSSION
In the current study we have demonstrated a link between cholesterol homeostasis and Aβ metabolism and provided a mechanistic explanation of how apoE facilitates intracellular Aβ degradation by microglia. We report that promoting microglial Aβ degradation is a common feature of apolipoproteins, including apoE and apoA-I.
Interfering with ABCA1 function by glyburide impaired the capacity of apoE to promote
Aβ degradation, consistent with the hypothesis that Aβ degradation promoted by apolipoproteins is mediated through reduction of cellular cholesterol levels rather than their affinity for Aβ. The 4F mimetic peptide provided us with another discriminatory
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ABCA1 (Remaley et al., 2003). Although the 4F peptide demonstrated no binding to Aβ, it was capable of promoting intracellular Aβ degradation, similar to the actions of apoE and apoA-I. This result strongly supports the hypothesis that apoE facilitates Aβ degradation via reducing cellular cholesterol levels. Indeed, oral administration of 4F peptide to a mouse model of AD for 4 months has been shown to reduce Aβ levels and plaque load and improve memory retention (Handattu et al., 2009). Although the authors concluded that this was the anti-inflammatory effect of the mimetic peptide, they could not exclude the involvement of cholesterol efflux with Aβ degradation. Since direct modulation of cellular cholesterol was capable of influencing the efficiency of intracellular Aβ degradation, our data suggest apoE facilitates intracellular Aβ degradation by means of regulating cellular cholesterol levels.
It has been documented that apoE co-localizes with amyloid deposits in AD patients and can also bind soluble Aβ found in the brain, CSF and plasma (Namba et al.,
1991; Wisniewski and Frangione, 1992; Wisniewski et al., 1993; Naslund et al., 1995;
Kuo et al., 1996). ApoE directly interacts with Aβ and this binding is both isoform and lipidation status-dependent (Wisniewski and Frangione, 1992; Strittmatter et al., 1993a;
Strittmatter et al., 1993b; LaDu et al., 1994; LaDu et al., 1995; Aleshkov et al., 1997).
ApoE2 binds Aβ more efficiently than apoE3 and apoE4. Lipidated native apoE has much higher affinity for Aβ than unlipidated forms of this apolipoprotein (Tokuda et al., 2000).
The isoform- and lipidation status-dependent binding affinity correspond with the genetic
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risk for AD imparted by the individual human isoforms, suggesting that apoE may act as
a molecular chaperone participating in the metabolism and amyloidogenesis of Aβ. Thus, it is reasonable to hypothesize that apoE may facilitate Aβ degradation through its chaperone activity. To our surprise, however, this interaction is not required for the apoE- stimulated intracellular Aβ degradation. Blocking the apoE-Aβ interaction by Aβ12-28 was not able to inhibit apoE-promoted Aβ degradation. Nevertheless, we could not
exclude the possible involvement of apoE-Aβ binding in the degradation of Aβ in vivo.
Our previous study demonstrated that apoE containing HDL particles stimulated the IDE- dependent proteolysis of sAβ in an ex vivo assay (Jiang et al., 2008). These data provide direct evidence that apoE promotes Aβ degradation possibly through at least two distinct mechanisms in vivo, suggesting that in the extracellular milieu apoE-HDL might act as a chaperone to enhance sAβ clearance and we are currently exploring this hypothesis.
As the major genetic risk factor for sporadic AD, apoE has been suggested to play an important role in the clearance of Aβ in an isoform-specific manner (Holtzman, 2004).
We previously reported that supplying primary microglia with apoE resulted in isoform- dependent enhancement of Aβ degradation, which apoE2 had the greatest effect while apoE4 had minimum (Jiang et al., 2008). In the current study, we demonstrated that cholesterol efflux is the key property of apoE to promote intracellular Aβ degradation.
Since the cholesterol efflux activity is apoE isoform-dependent (E2 > E3 > E4)
(Michikawa et al., 2000; Hara et al., 2003), the higher risk of AD observed in the apoE4 carriers may be due to the poorer efficiency of cholesterol efflux by apoE4. Cellular cholesterol levels governed by the activity of apoE isoforms may directly regulate Aβ
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degradation (Jiang et al., 2008). This hypothesis is further supported by the observation that the colocalization between Aβ and late endosomes/lysosomes was significantly reduced when microglia were pretreated with apoE4 versus apoE2 (unpublished data).
Increasing apoE levels or lipidation status, which reflects cholesterol efflux activities, by using liver X receptor agonists or overexpressing ABCA1 has been shown to ameliorate
AD pathology in several mouse models of AD (Koldamova et al., 2005b; Lefterov et al.,
2007; Riddell et al., 2007; Jiang et al., 2008; Wahrle et al., 2008). Intracellular cholesterol distribution and levels are crucial for cell functions, thus are highly regulated in the cells.
Cholesterol is highly enriched in the plasma membrane while it is low in the endoplasmic reticulum, where it is synthesized. The level of cholesterol decreases from trans- to cis-
Golgi apparatus (Pfrieger, 2003). Noteworthy, in the current study, changes in the levels of total cholesterol did not linearly correlate to the efficiency of intracellular Aβ degradation, especially when comparing the effectiveness of different cholesterol modulating reagents to regulate the degradation of Aβ. Apolipoproteins and the mimetic peptide were most effective to elicit heightened Aβ degradation activity with only moderate cholesterol reduction. Moreover, building up cholesterol primarily in the endocytic system by the NPC inhibitor led to impaired Aβ degradation. These results suggest that regulating cholesterol levels in certain subcellular compartments or the composition of cholesterol derivatives might be more important than just manipulating the overall levels of cellular total cholesterol in regulating Aβ degradation. Thus, using apoE to remove excessive cholesterol may be the most effective way to enhance Aβ clearance. However, it remains to be determined whether the beneficial effects would be similar under the genetic background of different Apoe alleles.
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A striking observation of this study is that the motility of Aβ-containing vesicles was increased upon apoE-dependent reduction of cellular cholesterol levels. Cholesterol has been shown to play an important role in the intracellular transport. It is not a passive component of endosomal membranes, but rather is directly involved in the sorting and transport of endocytic vesicles (Gruenberg et al., 2001; Helms and Zurzolo, 2004). Chen et al. reported that the motility of folate receptor (FR)-containing endosomes is regulated by the cholesterol (Chen et al., 2008). Cholesterol depletion by MβCD increased motility of FR endosomes, while increasing cellular cholesterol using U18666A retarded it. Our results are entirely consistent with their findings. We extended these observations and documented that the enhanced vesicle motility was not random, but was directed toward lysosomes as evidenced by enhanced colocalization of Aβ and lysosomes. The motility analysis of Aβ-containing vesicles showed the direct influence of cholesterol on vesicular trafficking speed, yet it is still possible that cholesterol may affect the sorting of Aβ in the endolytic system (Umebayashi, 2003). Active vesicle transport relies on a variety of motor proteins to transport cargo along cytoskeletal network. The recruitment of specific dynein or kinesin motor complexes onto vesicles is regulated by Rab GTPases. Among these Rab GTPases, Rab7 is shown to play a critical role in the endocytic processes, regulating the sorting of endosomes and biogenesis of lysosomes (Rupper et al., 2001;
Poteryaev et al., 2010). In Niemann-Pick C disease, dysfunction of NPC1 results in missorting and accumulation of cholesterol in the late endosomes. The cholesterol loading decreases the capacity of GDI to extract membrane-associated Rab7, thus inhibiting these vesicles from switching from minus- to plus-end-directed motility,
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Lebrand et al., 2002). Failure to recycle Rab7 to the membrane interferes with the switch in early-to-late endosome maturation and ultimately results in the paralysis of the endocytic system (Lebrand et al., 2002; Chen et al., 2008; Poteryaev et al., 2010).
Conversely, cholesterol depletion has been reported to enhance the motility of FR- containing endosomes. Reducing cellular cholesterol levels significantly decreased the association of Rab7 with FR endosomal membrane, suggesting the increased recycling of
Rab7 (Chen et al., 2008). In our study, the capacity of GDI to extract membrane- associated Rab7 was also enhanced in apoE-treated microglia, suggesting that facilitated
Rab7 recycling led to accelerated transport of Aβ-containing vesicles. It is notable that this property is related to the cholesterol efflux function of apoE since using the 4F mimetic peptide elicited the same results. Although the underlying reason why GDI extraction of Rab7 is modulated by cholesterol is unknown, results from previous studies suggested that this regulation perhaps involves oxysterol-binding protein (OSBP)-related protein 1L (ORP1L) through the Rab7 effector Rab7-interacting lysosomal protein (RILP)
(Lebrand et al., 2002; Rocha et al., 2009; Vihervaara et al., 2011). Whether this activity subsequently regulates the extraction of Rab7 by GDI requires further investigation. Our data indicate that the cholesterol content of endocytic vesicles is critical for Rab7’s action which impacts the efficiency of Aβ delivery to the lysosome for subsequent degradation.
In summary, we report that apoE promote intracellular Aβ degradation through reducing cellular cholesterol levels. This is the first evidence that provides a mechanistic explanation of the role of apoE in facilitating intracellular Aβ degradation. Reducing
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cellular cholesterol level by apoE does not change the expression and the activity of Aβ degrading enzymes, while promoting the trafficking of Aβ to lysosome for degradation by modulating Rab7 recycling. These findings demonstrated a direct role of cholesterol in
Aβ proteolytic degradation and provide a new insight into this aspect of AD pathogenesis.
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FIGURES
Figure 2.1
Figure 2.1: Facilitation of Aβ degradation is a common feature of apolipoproteins
(A, D) Wildtype primary microglia mice were preincubated with increasing
concentrations of purified ApoE (A) or ApoA-I (BD) for 24 h, followed by the
application of 2 mg/ml Aβ1-42. After 18 h incubation, intracellular Aβ levels were
quantified by ELISA, and normalized to total protein (n = 6). (B, E) Uptake of Aβ was assessed by applying Alexa 488-labeled Aβ1-42 to microglia after pretreatment with ApoE
(B) or ApoA-I (E). The accumulation of the fluorophore was analyzed by flow cytometry.
(C, F) Total cellular cholesterol levels were quantified and normalized to total protein (n
= 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle treated control.
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Figure 2.2
Figure 2.2: Facilitation of Aβ degradation relies on the cholesterol efflux function of apolipoproteins
(A) In a competitive inhibition assay, conditioned media containing native ApoE3 particles from E3-DSE astrocyte was incubated with Aβ12-28 at indicated concentrations for 30 min and transferred to Aβ1-42 coated plates for 3 h. ApoE bound to the immobilized
Aβ was detected using an ApoE-specific antibody. Results are expressed as a percentage of remaining ApoE bound, considering binding of ApoE to Aβ in the absence of Aβ12-28
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as 100%. The data represent the pooled outcome of 5 independent experiments and were
fitted into a one-site competition curve. (B, D, G) Wildtype (B, D) and Apoe-/- (G) primary microglia were preincubated with ApoE 1 μg/ml (B) or the 4F peptide at various concentrations, as indicated (D, G) for 24 h, followed by applying 2 μg/ml Aβ1-42. 5
μg/ml of Aβ12-28 was administered simultaneously with Aβ1-42 as indicated in (B). After
18 h incubation, cells were lysed and the intracellular Aβ levels were analyzed by ELISA
(n = 6 for (B, D); n = 3 for (G)). (C, E, H) Uptake of Aβ was monitored by applying
-/- Alexa 488-labeled Aβ1-42 to the wildtype (C, E) and Apoe (H) primary microglia after
pretreated with indicated reagents for 24 h. The accumulation of fluorophore was then
analyzed by flow cytometry. The histogram is representative of the outcome from three
independent experiments. (F, I) Total cellular cholesterol levels of wildtype (F) or Apoe-/-
(I) primary microglia treated with the 4F peptide were analyzed and normalized to total protein (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001 compared to the vehicle treated control.
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Figure 2.3
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Figure 2.3: Blocking cholesterol efflux impairs the ability of ApoE to enhance Aβ
degradation
(A) Wildtype primary microglia were preincubated with glyburide at indicated
concentrations in the presence or absence of 1 μg/ml ApoE, as indicated, for 24 h,
followed by the administration of 2 μg/ml Aβ42 for 18 h. The remaining intracellular Aβ levels were quantified by ELISA, and data were normalized to total protein (n = 3). (B)
Wildtype primary microglia were pretreated for 24 h. Alexa 488-labeled Aβ1-42 were then
applied for 6 h. Aβ uptake was analyzed by flow cytometry. The histogram plots are the
representative graphs of the outcome from three independent experiments. (C) Total cellular cholesterol levels were analyzed and normalized to total protein (n = 3). *p <
0.05, **p < 0.01 compared to the vehicle treated control; #p < 0.05, ###p < 0.001
compared to the 1 μg/ml apoE treated group.
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Figure 2.4
Figure 2.4: Reducing cellular cholesterol levels promotes Aβ degradation
(A, D) To lower cellular cholesterol levels, wildtype primary microglia were preincubated with MβCD (A) or lovastatin (D) at indicated concentrations for 30 min or 48 h, followed by administration of 2 μg/ml Aβ42 for 6 h or 18 h, respectively. Remaining intracellular
Aβ was analyzed using ELISA (n = 3). (B, E) Uptake of fluorescently labeled Aβ was monitored after pretreated with MβCD (B) or Lovastatin (E) for 30 min or 48 h, respectively, using flow cytometry. The accumulation of the fluorophore was then analyzed by flow cytometry. The histogram is representative of the outcome from three independent experiments. (C, F) Total cellular cholesterol levels were quantified and normalized to total protein (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001 compared to the vehicle treated control.
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Figure 2.5
Figure 2.5: Increasing cellular cholesterol levels impairs Aβ degradation
(A, D) To increase cellular cholesterol levels, wildtype primary microglia were preincubated with cholesterol-loaded MβCD (A) or U18666A (D) at indicated concentrations for 24 h, followed by administration of 2 μg/ml Aβ1-42 for 18 h. The remaining intracellular Aβ was analyzed using ELISA (n = 3). (B, E) Uptake of fluorescently labeled Aβ was monitored after pretreated with cholesterol-loaded MβCD
(B) or U18666A (E) for 24 h using flow cytometry. The histogram is representative of the outcome from three independent experiments. (C, F) Total cellular cholesterol levels were quantified and normalized to total protein (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001 compared to the vehicle treated control.
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Figure 2.6
Figure 2.6: Direct manipulation of cellular cholesterol levels regulates Aβ
degradation in the absence of apoE
(A) Primary Apoe-/- microglia were preincubated with 1 mM MβCD or 10 μM U18666A
as indicated for 30 min or 24 h, respectively, followed by administration of 2 μg/ml Aβ1-
42 for 6 h. The remaining intracellular Aβ were analyzed using ELISA (n = 3). (B-C)
Uptake of fluorescently labeled Aβ was monitored after preincubation with indicated
reagents for 24 h using flow cytometry. The histogram is representative of the outcome
from three independent experiments. (D) Total cellular cholesterol levels were quantified
and normalized to total protein (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001 compared to
the vehicle treated control.
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Figure 2.7
Figure 2.7: Transcription of Aβ degradation enzymes is not modulated by cholesterol
Wildtype primary microglia treated with apoE (1 μg/ml), apoA-I (5 μg/ml), 4F (10μg/ml) or U18666A (10 μg/ml) for 24 h and harvested for RNA extraction. Expression of transcripts was analyzed by real-time PCR with specific primers-probe sets as indicated.
The graphs represent pooled data from four independent experiments.
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Figure 2.8
Figure 2.8: ApoE has minimal effects on the activity of Aβ degradation enzymes
Wildtype primary microglia were pretreated with 1 μg/ml apoE for 24 h and lysed in PBS
with brief sonication. Fluorogenic substrates ES001 (A) for MMP-9, ES002 (B) for plasminogen and ES005 (C) for NEP, IDE, ECE-1 and ACE were utilized to measure the enzyme activity. The graphs represent pooled data from four independent experiments.
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Figure 2.9
Figure 2.9: Cholesterol levels regulate the trafficking of Aβ-containing endosomes
(A-C) A pulse-chase experiment was used to monitor the trafficking of Aβ after manipulating cellular cholesterol levels. Wildtype primary microglia were preincubated with vehicle (A), 1 μg/ml ApoE (B) or 10 μM U18666A (C) for 24 h. Prior to the application of Aβ, cells were pulsed with fluorescent microspheres (green) for 30 min and followed by incubation for 4 h to label the late endosomes/lysosomes. Alexa 555-labeled
Aβ1-42 (1 μg/ml) was applied to cells for 7 min. After extensive wash with DMEM/F-12, cells were incubated in fresh culture media for another 10 min. Representative images demonstrate the colocalization of Aβ (red) and late endosomes/lysosomes (green). Nuclei -101-
were stained with DAPI (blue). (D) To verify that fluorescent microspheres efficiently
label late endosomes/lysosomes, wildtype primary microglia were pulsed with fluorescent microspheres for 30 min and followed by incubation for 4 h. Cells were fixed and immunostained with an anti-LAMP1 antibody. The representative image demonstrates the colocalization of microspheres (green) and the late endosomal/lysosomal marker LAMP1 (red). Nuclei were stained with DAPI (blue). (E)
Images taken from (A-C) were analyzed with ImageJ for the colocalization of Aβ and late
endosomes/lysosomes (n = 5; *p < 0.05, *** p < 0.001 compared to the vehicle treated
control). (F) Wildtype primary microglia were preincubated with vehicle, 1 μg/ml ApoE
or 10 μM U18666A for 18 h. Alexa 488-labeled Aβ1-42 (2 μg/ml) were applied to cells for
7 min. After extensive washing with PBS, time-lapse images were taken to record the movement of Aβ in the cells. Images were acquired at every 10 s for 10 min and analyzed using Metamorph software. Each data point plotted is the average of the traveled distance of the 4 most motile fluorescent Aβ-containing endosomes in a cell (n = 4, *p < 0.05, **p
< 0.001).
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Figure 2.10
Figure 2.10: Cholesterol regulates the recycling of Rab7
(A) Wildtype primary microglia were preincubated with vehicle, 1 μg/ml ApoE or 10 μM
U18666A for 18 h. Alexa 555-labeled Aβ1-42 (1 μg/ml) was applied to cells for 7 min.
After extensive wash with DMEM/F-12, cells were incubated in fresh culture media for another 30 min. Rab7 was visualized by immunostaining. The colocalization of Aβ and
Rab7 were analyzed with ImageJ (n = 9; * p < 0.05). (B-D) Wildtype primary microglia
were treated with vehicle, ApoE (1 μg/ml), ApoA-I (5 μg/ml), 4F peptide (10 μg/ml) or
U18666A (10 μM) for 24 h. (B) Cell lysates were subjected to Western blot (WB)
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analysis using appropriate antibodies. Actin serves as a protein-loading control. (C)
Membrane (M) and cytosolic (C) fractions were isolated from cell lysates and analyzed by WB analysis. Flotillin-1 serves as a control for both fraction efficiency and protein- loading. (D) Crude membrane fractions (25 μg of total membrane protein) from cell lysates were incubated with GDI 0.2 μM for 15 min and pelleted with ultracentrifugation.
GDI-extracted Rab7 in the supernatants was analyzed by WB analysis. Total membrane fractions without the treatment of GDI serve as the protein-loading controls. The band intensity of extracted Rab7 was normalized to total Rab7 input from the membrane fractions and expressed as relative extractablilty compared to vehicle-treated control (n =
5). *p < 0.05, **p < 0.01 compared to the vehicle treated control.
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CHAPTER 3
APOE ACTS AS A CHAPERONE TO PROMOTE
IDE-MEDIATED Aβ DEGRADATION
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ABSTRACT
Apolipoprotein E (apoE) is the major risk factor for late onset sporadic
Alzheimer’s disease (AD). It is the predominant apolipoprotein in the central nervous system and a critical component of high density lipoproteins (HDL). Humans have three common apoE alleles (E2, E3, and E4), which differ in their amino acid composition at positions 112 and 158. Inheritance of apoE4 confers higher risk, earlier onset, and poorer clinical outcomes for AD. ApoE is known to avidly bind β-amyloid (Aβ) and is suggested to play a critical role in Aβ homeostasis. We previously reported that both intracellular and extracellular degradation of Aβ peptides was facilitated by apoE. The principal protease mediating the extracellular Aβ degradation is insulin degrading enzyme (IDE).
However, the molecular mechanism through which apoE facilitates the IDE-mediated extracellular degradation of Aβ remains unclear. In this study, we first evaluated IDE- mediated Aβ degradation by in vitro enzyme kinetic analysis. In the presence of apoE, the
Km value of Aβ degradation by IDE was reduced; however, no significant change in its
Vmax was observed. The results favored the chaperone hypothesis, in which the interaction of apoE with Aβ confers a conformation that makes it a favorable substrate of IDE and thus enhancing degradation. The affinity of apoE and Aβ was shown to be governed by the lipidation status of apoE. Native apoE particles secreted from Abca1-deficient astrocytes were poorly lipidated. These poorly lipidated apoE particles not only had less affinity for Aβ but also promoted IDE-mediated Aβ degradation less efficiently compared to the fully lipidated apoE particles from wildtype astrocytes. Furthermore, we observed apoE isoform-dependent facilitation of IDE-mediated Aβ degradation (E2 > E3 > E4),
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which correlated with the lipidation status of apoE as well as their affinity for Aβ.
Together, these data support the chaperone function of apoE in facilitating Aβ degradation by IDE. These findings provide a mechanistic explanation of apoE’s function in Aβ degradation and may explain the increased risk of AD conferred by apoE4.
INTRODUCTION
The progressive cerebral accumulation of the β-amyloid peptide (Aβ) is an early, invariant and necessary step in the pathogenesis of Alzheimer’s disease (AD) (Hardy and
Selkoe, 2002). It has been estimated that approximately 8% of total Aβ peptides in the brain are generated every hour and are also cleared at an equivalent rate in both humans and mice (Bateman et al., 2006). Thus, even moderately increased production or decreased clearance of Aβ eventually leads to an overall elevation of its steady state levels and ultimately the enhanced deposition in the brain. In most cases of sporadic AD, the rate of Aβ peptide synthesis is unchanged. Therefore, the decreased clearance of Aβ in the brains of AD patients is likely to be a key factor in the etiology of AD. In support with this idea, a recent study monitored the production and clearance of Aβ in the brain of late- onset AD patients and age-matched controls, and found that AD is associated with a 30% reduction in Aβ clearance without change in Aβ production (Mawuenyega et al., 2010).
One of the major pathways for removing excessive Aβ from the extracellular milieu is the degradation by proteases. Aβ has been shown to be sensitive to many
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proteases (Mukherjee and Hersh, 2002). Among these proteases, Insulin degrading
enzyme (IDE) is reported to be the principle enzyme responsible for extracellular Aβ degradation and is expressed by various cell types in the brain (Kurochkin and Goto,
1994; Mukherjee and Hersh, 2002; Jiang et al., 2008). IDE is a zinc metalloprotease and is secreted or associated with the cell surface, depending on the cell types. Microglia are found to secrete IDE, yet hippocampal neurons only possess membrane associated IDE.
In addition to insulin, the canonical substrate of IDE, monomeric Aβ has been reported to be the substrate of IDE (Qiu et al., 1998). IDE cleaves Aβ at His13-Gln14, His14-Gln15, and
Phe19-Phe20 bonds as initial cleavage sites, followed by additional cleavage at other sites
at Val18-Phe19, Phe20-Ala21, and Lys28-Gly29 bonds that occur at a slower rate (Mukherjee
et al., 2000). Cleavage of Aβ1–42 by IDE prevented the neurotoxic effects of the peptide
toward primary rat hippocampal neurons, as well as the deposition of the peptide onto
synthetic amyloid plaques, suggesting that IDE may be able to prevent the pathogenesis
of AD (Mukherjee et al., 2000; Song and Hersh, 2005). Genetic deletion of the Ide gene
in mice resulted in increased Aβ levels in the brain (Farris et al., 2003; Miller et al., 2003).
Knocking out Ide in the gene-trap mouse model led to significant elevation of Aβ1-40 and
Aβ1-42 levels (1.6- and 1.4-fold, respectively). Over 50% reduction of Aβ degradation in
both brain membrane fractions and primary neuronal cultures of Ide-/- mice were reported.
And as an internal control, similar deficiency of insulin degradation in the liver were
observed in these mice (Farris et al., 2003). In contrast, overexpression of IDE
dramatically reduced brain Aβ levels and retarded the formation of amyloid plaques by
~50% (Leissring et al., 2003). Although it remains unknown whether deficiency or
genetic variation in the IDE gene is capable of initiating AD in human, genetic analysis of
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allelic association of the IDE gene has been reported linking to LOAD (Bertram et al.,
2007). Thus, enhancing the activity of IDE may be a potential therapeutic target to effectively reduce steady-state cerebral Aβ levels and retard AD pathogenesis.
Allelic variation of apolipoprotein E (apoE) is identified as the major risk factor of AD. ApoE2, E3 and E4 are the three most common alleles of apoE in human.
Inheritance of APOE4 confers increased risk, earlier onset, and poorer clinical outcomes
for AD in a dose-dependent manner, whereas the APOE2 allele is protective. ApoE is the
predominant apolipoprotein in the brain. It is a critical component of high density
lipoproteins (HDL) and plays a major role in the distribution and homeostasis of
cholesterol. In the brain, apoE is mainly secreted by astrocytes and to a much less extend
by microglia. Nascent apoE is lipidated through the initial action of the ATP-binding
cassette transporter-1 (ABCA1) followed by other related ABC transporters, including
ABCG1 and ABCG4, to form HDL particles (Grehan et al., 2001). ABCA1 activity is
necessary for the homeostasis and lipidation of apoE (Krimbou et al., 2004). Genetic
ablation of Abca1 leads to significantly decreased apoE levels and poorly lipidated apoE particles in the brain and cerebrospinal fluid (CSF) (Wahrle et al., 2004). The efficiency of cholesterol efflux also varies among apoE isoforms (E2 > E3 > E4) and is reflective of
their capacity to be lipidated. We previously demonstrated that extracellular degradation of Aβ, which was mainly mediated by IDE, was facilitated in the presence of apoE (Jiang
et al., 2008). However, the molecular mechanism of which apoE facilitates the IDE-
mediated extracellular degradation of Aβ remains unclear. ApoE is known to bind Aβ
avidly and this binding affinity is dependent on the isoforms and lipidation status of apoE
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(LaDu et al., 1995; Aleshkov et al., 1997; Tokuda et al., 2000). Expression of human
ApoE isoforms in hAPP expressing mice lacking the murine Apoe gene conferred
isoform-specific and dose-dependent plaque burden (E2 < E3 < E4), which is consistent
with the risk of AD observed in human. Thus, the direct interaction of apoE and Aβ may
be critical for apoE to promote the proteolysis of Aβ (Bales et al., 2009; Castellano et al.,
2011).
In this study, we evaluated IDE-mediated Aβ degradation by in vitro enzyme kinetic analysis. The reduced kinetic constant (Km) with an unchanged maximum reaction
rate (Vmax) of Aβ degradation by IDE were observed in the presence of apoE, suggesting
the possible chaperone effect of apoE in this reaction. The reduction of Km value suggested that apoE-Aβ binding might transform Aβ into a more favorable IDE substrate, and thus enhance Aβ degradation. Reducing apoE-Aβ interaction by using less lipidated apoE particles collected from Abca1 deficient astrocyte-conditioned media resulted in less efficient IDE-mediated Aβ degradation. We further demonstrated that apoE facilitated IDE-mediated Aβ degradation in an isoform-dependent manner, paralleling their lipidation profiles. The results suggest that the lipidation status of apoE not only governs it affinity for Aβ, but also determines its efficiency to promote Aβ degradation by
IDE. Our data supported the chaperon hypothesis and implied that the increased AD risk conferred by apoE4 from its reduced ability to facilitate the proteolysis of Aβ than other isoforms.
MATERIALS AND METHODS
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Reagents
Purified human apoE and recombinant apoE isoforms was purchased from
rPeptide (Bogart, GA) and Bioenza (Mountain View, CA), respectively. IDE inhibitors,
phosphoramidon and 1,10-phenanthroline, were purchased from Enzo (Farmingdale, NY).
Preparation of Aβ Peptides
Lyophilized unlabeled Aβ1-42 and Aβ12-28 (American Peptide) were dissolved to
a final concentration of 1 mg/ml in DMSO and stored at -80°C until use.
Primary astrocyte culture
Primary astrocytes were derived from the brains of Abca1-/- mice or Abca1+/+
(denoted as wildtype) littermates at postnatal day 1 – 3 using a mild trypsinization protocol as previously described (Jiang et al., 2008). In brief, cortices were minced and dissociated by incubating in 0.025% trypsin/PBS for 30 min at 37°C. Dissociated cortical cells from one brain were seeded on two 150-mm culture dishes and incubated with DMEM/F-12 containing 10% FBS at 37°C in humidified 5% CO2/95% air. Medium was replaced every 5-7 days. After 2-3 weeks in vitro, adherent astrocytes and microglia
were dislodged by incubating with 0.015% trypsin/HBSS for 30-45 min at 37°C. Mixed
glia suspension were then seeded to culture dishes and incubated with DMEM/F-12
containing 10% FBS at 37°C. 15 min later, non-adherent cells were aspirated and media
were replenished. Cells were then incubated until confluent which was usually achieved
after 3-5 days. With this method, the purity of primary astrocytes was >90%. To obtain conditioned media, cells were washed extensively with PBS and cultured in serum free
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DMEM/F-12 for 3 days. Conditioned media were filtered through 0.22 μm filters to eliminate floating cells or debris (Millipore, Billerica, MA). The concentration of apoE in the conditioned media was analyzed by ELISA.
Chromatographic fractionation of HDL particles
Immortalized DSE astrocytes, carrying human apoE2, E3 or E4 (a gift from Dr.
Holtzman, Washington University at St. Louis, MO), were cultured in serum-free
DMEM/F-12 with 1 mM sodium pyruvate for 3 days to obtain conditioned media containing lipidated apoE particles. Conditioned media were filtered through 0.22 μm filters and concentrated 40X by ultrafiltration using Amicon with 10-kDa cut-off filters
(Millipore). The concentrates (500 μl) were then fractionated by size exclusion chromatography using a BioLogic FPLC chromatography system (Biorad Laboratory,
Hercules, CA) with the Superose 6 column (GE healthcare, Piscataway, NJ). 20 mM phosphate buffer (PBS, pH 7.4, containing 50 mM NaCl and 0.03% EDTA) was used as the eluent at a flow rate of 0.5 ml/min. Eluates were collected at 700 μl per fraction. The concentration of apoE in each fraction was analyzed by ELISA.
ApoE ELISA
ApoE ELISA was performed as described (Jiang et al., 2008). In brief, WUE4 monoclonal anti-ApoE antibody was used as the capture antibody, and a goat polyclonal
HRP-conjugated anti-ApoE antibody (Abcam, Cambridge, MA) was used as the detection antibody. Purified human plasma apoE (rPeptide) was used to generate a standard curve for each experiment. The plates were developed using a TMB substrate kit
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(Pierce, Rockford, IL) and the reaction was stopped by addition of an equal volume of 1
M HCl. The results were read using a Spectramax colorimetric plate reader (Molecular
Devices, Sunnyvale, CA).
ApoE immunoprecipitation
Monoclonal mouse anti-apoE antibody (WUE4, 2 μg) and 30 ul Protein G beads
(Santa Cruz) were added to astrocyte-conditioned media (500 μl). The mixture was rotated at 4°C for 2 h and centrifuged at 12,000 x g for 1 min. The pellet was washed with PBS and the immunoprecipitated apoE-containing HDL particles were used immediately in later assays.
Aβ degradation assay and Aβ ELISA
Recombinant IDE (500 ng/ml; R&D Systems, Minneapolis, MN) was incubated with 2 μg/ml Aβ1-42 in a Tris-based buffer (50 mM Tris, 1 M NaCl, 1 ug/ml BSA, pH 7.5).
Purified human plasma apoE (rPeptide) or immunoprecipitated apoE-containing HDL particles were added to the mixture as indicated. Remaining Aβ was quantified by ELISA using 6E10 as the capture antibody and 4G8-HRP as the detection antibody (Covance,
Princeton, NJ). Synthetic Aβ1-42 was used to generate a standard curve. Plates were developed using a TMB substrate kit (Pierce) and the reaction was stopped by the addition of an equal volume of 1 M HCl. The results were read using a Spectramax colorimetric plate reader (Molecular Devices). The levels of IDE and apoE were monitored using western analysis.
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Michaelis-Menten kinetic analysis of IDE activity
Aβ1-42 at indicated concentration was incubated with 500 ng/ml recombinant IDE
in the presence of 100 ng/ml apoE (rPeptide) for 15 min. The remaining Aβ was
quantified by ELISA. Enzyme activity was graphed as the double reciprocal Lineweaver-
Burk plot and accurate values of Vmax and Km were calculated using VisualEnzymic
software (Softzymics, NJ).
Nondenaturing gradient gel electrophoresis for ApoE lipidation status
Conditioned media or fractions collected after FPLC were subjected to native gel electrophoresis to determine ApoE lipidation status. Samples were resolved by 4-12%
Tris-glycine nondenaturing gel (Invitrogen, Carlsbad, CA). After transfer to PVDF membrane, apoE was visualized using Western analysis. The size of apoE-containing particles was determined by comparison to a native size marker (Amersham Biosciences,
Piscataway, NJ).
IDE activity in the conditioned media
IDE activity in the conditioned media was measured by monitoring the hydrolysis
of the fluorogenic substrate V (Mca-RPPGFSAFK(Dnp)-OH) (ES-005; R&D Systems,
Minneapolis, MN). The substrates are non-fluorescent but become fluorescent after the proteolytic cleavage by their specific enzymes. The enzyme activity was measured as the generation of fluorescent signal, which was recorded every minute for 1 h in real-time using a Spectramax fluorometric plate reader (Molecular Devices, Sunnyvale, CA).The concentration of IDE in the conditioned media was assessed by immunoprecipitation
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followed by Western analysis.
ApoE-Aβ affinity assay
Affinity analysis of the binding between apoE and Aβ was performed as described
(Jiang et al., 2008). In brief, 100 μl of Aβ1-42 (2 μg/ml in 0.5 M sodium bicarbonate buffer,
pH 9.6) was incubated on polystyrene 96-well plates at 4°C overnight to immobilize Aβ1-
42. Plates were then washed with PBS plus 0.01% Tween20 (PBST) and blocked with 1%
skim milk in PBST for 1 h at 37°C. Recombinant human apoE or astrocyte-conditioned
media was added to the 96-well plate containing immobilized Aβ for 3 h. In parallel, a
series of apoE dilutions at known concentrations was incubated on the wells coated with
an anti-apoE antibody (WUE-4, a gift from Dr. Holtzman) to generate a standard curve
for calibration. The plate was then washed with PBST and apoE bound to Aβ1-42 was detected using a HRP-conjugated anti-apoE antibody (Abcam, Cambridge, MA). The chromogenic reaction was developed with a TMB substrate kit (Pierce), stopped by the addition of 1M HCL and measured using a Spectramax colorimetric plate reader
(Molecular Devices). O.D. values were calibrated to the standard curve. Nonspecific binding was determined by replacing coated Aβ1-42 with bovine serum albumin and/or
omitting the conditioned media in the assay.
RESULTS
IDE-mediated Aβ degradation is facilitated in the presence of apoE
Our previous study demonstrated that conditioned media from both astrocytes and
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microglia was able to degrade Aβ. Application of insulin or IDE inhibitors,
phosphoramidon and phenanthroline, was capable of inhibiting the degradation of Aβ,
suggesting that the extracellular Aβ degradation is primarily mediated by IDE (Jiang et al.,
2008). We then performed the Aβ degradation assay with purified human recombinant
IDE, Aβ and apoE in the phosphate buffered solutions. With this minimal reconstitution,
apoE remained to facilitate IDE-mediated Aβ degradation in a dose-dependent manner,
indicating that this activity required only interactions among IDE, Aβ and apoE (Fig. 3.1).
ApoE acts as a chaperone to facilitate Aβ degradation by IDE
Two possible mechanisms may explain this phenomenon: apoE interacts with IDE
as a co-enzyme or activator to enhance its activity; or apoE acts as a molecular chaperone
that binds and transforms Aβ to a more favorable substrate for IDE-mediated degradation.
We first tested if apoE interacts with IDE. Co-immunoprecipitation demonstrated that no
constitutive binding of apoE to IDE was detectable (data not shown). However, we could
not exclude the possibility of weak binding or transient kiss-and-run interaction between apoE and IDE, which is not detectable by co-immunoprecipitation. To evaluate this possibility, we performed an enzyme activity assay by using a fluorogenic substrate to
monitor the enzyme reaction of IDE in real-time. If apoE directly enhanced the enzyme
activity of IDE, this heightened degrading activity should not be restricted to Aβ but
would apply to all substrates of IDE. However, no increased enzyme activity of IDE in the presence of apoE was observed (Fig. 3.2). These data suggest that it is unlikely that
apoE interact with IDE and enhance its enzyme activity.
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We then tested whether apoE acts as a molecular chaperone, which transforms Aβ to a favorable substrate of IDE for degradation. ApoE is known to interact with Aβ
(Tokuda et al., 2000) and we are able to measure that interaction by an immuno pull- down assay using immobilized Aβ to capture apoE (see Fig. 2.2A of Chapter 2). In the single substrate degradation, enzyme-catalyzed reactions are saturable and their rate of catalysis does not show a linear response to increasing concentration of substrate as best described in Michaelis-Menten kinetics model (Di Cera, 2009). Initially, the rate of reaction increases as the concentration of substrates (denoted as [S]) increases. However, as [S] gets higher, the enzyme becomes saturated and the rate of reaction reaches Vmax, the enzyme's maximum rate. Another measurement in the Michaelis-Menten model is the
Michaelis constant Km, which is a measure of the affinity of a substrate for the enzyme.
Vmax and Km depict the enzymatic property of a protease. In the presence of activators or
inhibitors, changes in the Vmax and Km values reflect underlying mechanisms (Di Cera,
2009). For example, altered Vmax with constant Km suggests that the reagent confers
competitive activation/inhibition toward the enzyme, while changed Km with unaffected
Vmax inclines to the probability of non-competitive activation/inhibition. Therefore, we
performed kinetic analysis to estimate the Vmax and Km of IDE in degrading Aβ to verify
the possible chaperone effect of apoE. Aβ was incubated with IDE in the presence of
apoE and the reaction was stopped in various time points to obtain a kinetic plot of the
Aβ degradation by IDE (Fig. 3.3A). In the presence of apoE, the decrease of intact Aβ
was significantly faster compared to controls in the first 10 min, indicating that apoE
enhanced the initial rate (Vint) of Aβ degradation by IDE. However, for prolonged incubation, the accumulated ratio of degraded Aβ reached similar levels in both groups,
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suggesting that apoE does not alter the capacity of IDE to degrade Aβ. Thus, it is less likely that apoE acts as a modifier of IDE to enhance its activity which is consistent with the conclusion from enzyme activity assays using fluorogenic substrates (Fig. 3.2). We performed Michaelis-Menten kinetic analysis by measuring the IDE activity in response to the increasing Aβ concentration in the presence of absence of apoE. The IDE activity is
calculated from the amount of Aβ degraded within the 15-min duration. The results were
then graphed as a Lineweaver-Burk plot to visualize the Vmax and Km value of IDE (Fig.
3B). In the presence of apoE, the Km of IDE activity in degrading Aβ was significantly
decreased from 1.34 μM to 0.86 μM, while the change in Vmax was minimal (5.52 to 5.67
μM/min). The results suggest that apoE acts as a non-competitive activator of Aβ
degradation by IDE. Since apoE does not interact with IDE to promote its activity, these
data favor the hypothesis that apoE acts as a chaperone which binds Aβ and alters its
conformation so that the affinity of Aβ for IDE increased. Thus, the interaction between
apoE and Aβ might directly influence the efficacy of promoting Aβ degradation.
Lipidation status of apoE regulates its ability to promote IDE-mediated Aβ degradation
A number of studies have shown that the affinity of apoE to Aβ is relied on its lipidation status. Delipidation of apoE dramatically decrease its affinity for Aβ (Tokuda et al., 2000). Therefore, we hypothesized that the difference in apoE’s affinity to Aβ may govern its efficacy of promoting Aβ degradation by IDE. We first performed the affinity assay by using immobilized Aβ to capture native apoE particles in the conditioned media of primary wildtype or Abca1-/- astrocytes. ABCA1 is necessary for maintaining the
normal level and lipidation of ApoE. Abca1 deficiency leads to a significant (>75%)
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reduction in brain ApoE levels and the remaining ApoE in the brain and CSF is poorly
lapidated (Wahrle et al., 2004). Significantly smaller apoE-HDL particles were produced
by Abca1-/- astrocytes, indicating that they were less lipidated compared to wildtype
HDLs (Fig. 3.4A). Consistent with previous studies, apoE bound to Aβ in a lipidation
status-dependent manner. Higher lipidation of apoE conferred higher affinity to Aβ (Fig.
3.4B). Degradation of Aβ was then tested in these conditioned media. IDE has been
reported to be the major Aβ degrading enzyme in the astrocyte-conditioned media (Qiu et
al., 1998; Jiang et al., 2008). We found that conditioned medium, which contained IDE, from wild-type astrocytes efficiently degraded Aβ, while conditioned medium obtained
from Abca1-/- astrocytes that contain poorly lipidated forms of apoE left significantly
higher (4-fold) levels of remaining Aβ (Fig. 3.4C). The reduced Aβ degradation was not
due to the changes in the levels (Fig. 3.4D) or overall enzymatic activity (Fig. 3.4E) of
IDE in the conditioned medium derived from Abca1-/- compared to wild-type astrocytes.
These data indicated that the lipidation status of apoE regulates its effectiveness to
promote the proteolysis of Aβ in the conditioned media, which is mainly performed by
IDE. To further verify that the lipidation status of apoE affects its ability to enhance IDE-
mediated Aβ degradation, we performed ex vivo Aβ degradation assay using recombinant
IDE in the presence of lipidated apoE. ApoE particles were immunoprecipitated from the
conditioned media of primary wildtype or Abca1-/- astrocytes. We found that apoE
produced from Abca1-/- astrocytes, which was poorly lipidated, was less capable of
enhancing IDE-mediated Aβ degradation compared to the highly lipidated forms of apoE
from wildtype cells (Fig 4F). The results suggested that the capacity of apoE to promote
IDE-mediated Aβ degradation is governed by the affinity between apoE and Aβ,
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supporting the hypothesis of apoE’s chaperone effect.
ApoE isoforms differentially facilitate Aβ degradation by IDE
The variation in the two amino acid residues among apoE isoforms has been
demonstrated to confer different lipidation propensity of apoE, which apoE2 is the
highest lipidated while apoE4 is the lowest (Hopkins et al., 2002). As we demonstrated
above, the lipidation status of apoE affects its affinity for Aβ and subsequently alters its
capacity to promote Aβ degradation by IDE. We hypothesized that the ability to promote
IDE-mediated Aβ degradation is dependent on the apoE isoforms. The apoE lipidation
profile of the conditioned media from astrocyte cell lines carrying one of human APOE
alleles, which is knocked in the endogenous Apoe locus, was first analyzed. The conditioned media was fractionated using Fast Protein Liquid Chromatography (FPLC) and the recovery of apoE in the fractions was quantified by ELISA. The first peak of recovered apoE2 fraction was at fractions 18 to 19, while apoE3 peaked between 21 and
22, and apoE4 was slightly trailed at 22 (Fig. 3.5A). Thus, apoE2 was lipidated the most as apoE3 and apoE4 followed subsequently, supporting the dependency of the lipidation of apoE on its genotypes in previous findings (Hopkins et al., 2002). The analysis of the interaction between native apoE particles and Aβ demonstrated apoE2-HDL has higher
affinity to Aβ than apoE3, while apoE4 has the lowest affinity for Aβ (Fig. 3.5B).
Addition of the immunoprecipitated apoE particles from the conditioned media of these
human APOE knock-in astrocytes to the IDE-mediated Aβ degradation assay showed that
the capacity of apoE to promote Aβ degradation by IDE is related to the apoE isoform.
IDE degraded Aβ more efficiently when supplied native apoE2 than apoE3 or apoE4
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particles (Fig. 3.5C). However, the difference of apoE isoforms in promoting Aβ degradation was diminished when using delipidated apoE since the delipidated apoE isoforms possess similar affinity to Aβ (Fig. 3.5D-E). The data suggest that the apoE isoform-specific efficiency in promoting Aβ degradation is dependent upon the lipidation profiles, which determine the affinity to Aβ.
Aβ12-28 promotes Aβ degradation by IDE through enhancing allosteric reaction
To further confirm that apoE-Aβ interaction is required to promote Aβ degradation, we utilized a small peptide derived from Aβ, Aβ12-28, to competitively interfere with the binding of apoE and Aβ. The 12 to 28 amino acid residues of Aβ were identified as the binding site of apoE (Strittmatter et al., 1993b). Using this small fragment of Aβ (Aβ12-28) was shown to competitively block the binding of full-length
Aβ to apoE (please refer to Fig. 2.2 in Chapter 2) (Sadowski et al., 2004). However, when we applied this small peptide to the Aβ degradation assay, it surprisingly enhanced the enzyme activity of IDE toward Aβ rather than blocked the effect of apoE in promoting
Aβ degradation by IDE (Fig. 3.6A). The heightened activity of Aβ degradation was independent of the presence or the isoforms of apoE (Fig. 3.6B), yet it was dependent on the dose of Aβ12-28 (Fig. 3.6C). It has been reported that IDE is an allosteric enzyme, of which enzyme activity is enhanced as the concentration of substrates increasing and displaying sigmoidal substrate versus activity kinetic profiles (Song and Hersh, 2005).
Structural analysis demonstrated that the IDE recognition and cutting sites resided between the 16 to 23 amino acid residues of Aβ (Shen et al., 2006), indicating that the
Aβ12-28 may serve as a substrate of IDE. Thus, the attempt of using Aβ12-28 to block
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apoE-Aβ binding eventually evoked the allosteric enzyme reaction of IDE which in turn,
enhance the degradation of Aβ.
DISCUSSION
In this study, we demonstrated that apoE promotes Aβ degradation by IDE, which is consistent with our previous finding (Jiang et al., 2008). Further examination showed that this effect is dependent on the isoform as well as the lipidation status of apoE. These results are similar to the effect of apoE on intracellular Aβ degradation as described in chapter 2 (Lee et al., 2011). However, the underlying mechanisms are apparently distinct since there is no trafficking issue of Aβ in the extracellular condition. Here we provided several lines of evidence supporting the chaperone effect of apoE on promoting Aβ degradation by IDE. No direct association between apoE and IDE excluded the possibility of apoE being a coenzyme of IDE. In contrast, the efficiency of apoE to induce Aβ degradation was strongly correlated to its affinity for Aβ. Poorly lipidated apoE was less capable to enhance Aβ degradation. These results indicate that apoE may act as a molecular chaperone for Aβ. The Michaelis constant Km and the maximum
reaction rate Vmax are important parameters to evaluate the influence of an activator or an
inhibitor to the enzyme activity. We observed a reduced Km with an unchanged Vmax of
IDE activity in the presence of apoE, indicating that comparing to Aβ alone, apoE-Aβ
complex has higher affinity for IDE and thus the proteolytic reaction is accelerated. This
agrees with the prediction of apoE as a chaperone.
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Enzymes are biological catalysts, which speed up the chemical reaction by decreasing the amount of free energy required to form an activated complex and consequently transit to the end stage of the reaction. In many cases, a second component
(cofactor, coenzyme or metal-ion activator) needs to be present in order to activate the enzyme before catalysis can actually occur. In the absence of this second component, even if the substrate is present at the active region of the enzyme, the catalysis will not happen. IDE is a metalloprotease. The presence of zinc ion is necessary for its enzyme activity. Crystal structure studies have shown that IDE consists of two ~56 kDa N- and C- terminal domains (IDE-N and IDE-C, repectively), joined by a 26-amino acid loop (Shen et al., 2006). The structure of IDE resembles a clam shell, with two hemisphere-shaped
IDE-N and IDE-C forming an enclosed chamber to encapsulate the substrate. The catalytic site is located within IDE-N, while the substrate recognition relies on the assist from IDE-C. The truncated enzyme with IDE-N alone has less than 2% activity of the full-size wildtype enzyme (Li et al., 2006b). Tang and colleagues suggested that IDE adapts at least two conformations, designated as “open” and “close” states (Shen et al.,
2006). In the open state, substrates move in and out of the catalytic chamber freely. Once the chamber closes, previously bound substrates are entrapped to allow hydrolysis, but new substrates are blocked for entering the catalytic chamber. After the enzyme reopens, degraded substrates are released and the chamber is free to enter. In this model, the conformation switching between the open and close states is the rate limiting step of the catalytic reaction. In addition, crystal structure of IDE indicated that the extended hydrogen bonding between the two halves of IDE creates a latch that tends to keep the
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enzyme closed. Notably, introducing mutations disrupting these hydrogen bonds
increases the proteolytic activity by 40-fold. The data suggest that factors that destabilize the hydrogen-bonding will increase the proteolytic efficiency, whereas stabilizing the closed state will slow the activity of the protease. ATP is also shown to increase the IDE activity by >20-fold (Song et al., 2004). The triphosphate moiety of ATP is demonstrated to be responsible for the activation. Further verification by Im et al. indicated that association with ATP induced intramolecular conformational changes that favor the open state of IDE and as a consequence the enzyme activity is increased. In our study, the degradation of Aβ by IDE was facilitated in the presence of apoE. Absence of the evidence supporting the association between apoE and IDE excludes the possibility that apoE may act similarly to ATP to change the conformation of IDE by direct binding.
However, it is still possible that apoE directly modifies or indirectly induces the
modification of IDE. A recent work by Tang’s group showed that the oxidative burst of
BV-2 microglial cells leads to oxidation or nitrosylation of IDE, leading to the reduced
activity (Ralat et al., 2009). ApoE has been shown to express antioxidative property,
suggesting that apoE may protect IDE from the modification by reactive oxygen or
nitrogen species and thus retain its full enzyme activity. Although the assay using
fluorogenic substrates showing the overall activity of IDE was unchanged regardless the
presence of apoE, we cannot completely exclude the possibility that the effect of apoE on
IDE activity is substrate-dependent.
The interaction between apoE with Aβ has been extensively investigated
(Strittmatter et al., 1993b; LaDu et al., 1994; Tokuda et al., 2000), suggesting the possible
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molecular chaperone effect of apoE in the IDE-mediated Aβ degradation. Several chaperones have been connected with protein degradation. The most studied chaperones are the family of 70 kDa heat shock proteins (Hsp70s) and their constitutive counterparts
Hsc70s participate actively in chaperone-assisted degradation pathways (Arndt et al.,
2007; Kettern et al., 2010). Upon encountering a misfolded protein, Hsp70 either promotes the protein folding correctly or induces degradation. However, our understanding of what determines a chaperone-bound protein entering a “folding” or a
“degradation” pathway is rather limited. In the degradation pathway, Hsp70 binds to the nonnative proteins and present them to the E3 enzyme of the ubiquitin-proteasome system. After a cascade of enzyme activities, the non-native proteins are conjugated with multiple ubiquitins and transferred to the 26S proteasome for degradation. The binding of apoE to Aβ may resemble the Hsp70-nonnative protein binding and present Aβ as a favorable substrate to IDE, thus enhancing the recognition and consequent degradation.
Ladu and colleagues reported that the binding of Aβ to apoE is dramatically attenuated with delipidation and denaturation, which are the standard procedures for purification of apolipoproteins (LaDu et al., 1995). The results explained the inconsistent conclusions from earlier studies investigating the affinity of apoE isoforms and Aβ (Strittmatter et al.,
1993a; LaDu et al., 1994; Manelli et al., 2004). Later, by using native, lipidated apoE, researchers demonstrated that apoE bind Aβ in an isoform-dependent manner (E2 > E3 >
E4) (LaDu et al., 1994; Aleshkov et al., 1997; Tokuda et al., 2000). Tokuda et al. further demonstrated that the difference among isoforms is diminished after delipidation. This
result is consistent with Ladu’s finding and emphasizes the importance of
lipid/cholesterol in the interaction between apoE and Aβ (LaDu et al., 1995; Tokuda et al.,
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2000). In the current study, we observed that highly lipidated apoE from wildtype
astrocytes was more effective than poorly lipidated apoE from ABCA1-deficient astrocytes. ApoE2 promotes Aβ degradation more efficiently than apoE3 and apoE4.
These results indicate that the efficiency of apoE to promote IDE-mediated Aβ degradation relied upon its affinity for Aβ. This agrees with the prediction for the chaperone effect of apoE. And importantly, these results are also in concert with the risk of developing AD conferred by different isoforms (E2 > E3 > E4).
An unexpected result in this study is that Aβ12-28 promotes Aβ degradation by
IDE. The 12-28 residues of Aβ are its binding site of apoE (Strittmatter et al., 1993b).
Using a small peptide, Aβ12-28, derived from this sequence was capable of block the
binding of full-length Aβ to apoE (Sadowski et al., 2004). Thus, it is reasonable to predict
that the effect of apoE on facilitating Aβ degradation would be inhibited by Aβ12-28.
However, the result is exactly opposite. The activity of IDE is known to be allosterically
regulated by the ratio of the concentrations of substrates and enzymes. With a constant
concentration of IDE, the enzyme activity is increased sigmoidally as the concentration
of substrates elevating (Song and Hersh, 2005). Shen et al. suggested that dimerization of
IDE stabilizes the close state of IDE. High ratio of substrate/enzyme prohibits the
dimerization of IDE thus enhances the enzyme activity (Shen et al., 2006). Interestingly,
the crystal structure demonstrated that IDE recognizes and cleaves Aβ within the region
of 16-23 amino acid residues, indicating that Aβ12-28 could be a substrate of IDE.
Therefore, instead of competitive inhibition from Aβ12-28 to full length Aβ, the
increased concentration of substrates elicited the allosteric activity of IDE and eventually
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enhanced the degradation of Aβ as we observed. This result may also explain the
observation by Sadowski et al. that application of Aβ12-28 led to dramatically reduced
amyloid deposition in a mice model of AD (Sadowski et al., 2004). The reduced Aβ
deposition might result from the increased activity of IDE rather than inhibition of
fibrillization of Aβ. This possibility may be verified by examining the levels of sAβ in
those mice.
The technical difficulty of this study is to consistently purify active apoE-HDL
particles that promote IDE-mediated Aβ degradation. Although we were able to
demonstrate that immunoprecipitated apoE particles actively facilitated Aβ degradation,
this activity was greatly impaired or diminished when apoE particles were dissociated
from the antibodies and agarose beads. It is unlikely that binding with the antibody or the
agarose bead is required for apoE’s activity since directly using astrocyte-conditioned
media elicited similar effects of promoting Aβ degradation. HDLs are known to associate
with a variety of proteins in vivo, which are important to maintaining the functional and
structural integrity of HDLs (Davidsson et al., 2010). The high salt buffer used to dissociate apoE-HDL particles from the immunoprecipitants may strip off some of these
associated proteins or change the conformation of apoE particles, thus interfering with
their activity toward Aβ degradation. We also tried other crucial purification methods,
including heparin affinity column and KBr gradient ultracentrifugation, which are
traditionally used to purify lipoproteins from the plasma (Weisgraber and Mahley, 1980;
Aguilar et al., 1983). We also tested the reconstituted artificial apoE-DMPC particles in the Aβ degradation analysis (Wientzek et al., 1994; Zhu et al., 2007). However, none of
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these methods successfully gave us consistently effective apoE activity toward Aβ degradation. Since these procedures are usually time consuming, it is also possible that oxidation or aggregation of apoE may occur during the procedures and eventually affects the activity of apoE (Zhang et al., 2007; Asztalos et al., 2011). An important conclusion from these unsuccessful attempts is that the structure integrity or unidentified associated proteins may be essential for apoE to promote IDE-mediated Aβ degradation efficiently.
These factors may be regulated by the lipidation status or cholesterol content of the apoE-
HDL particles and thus is of important to be further investigated.
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FIGURES
Figure 3.1
125
100 (%) β 75 * ** ** 50
25 Remaining A Remaining
0 0 10 20 50 100 500 hApoE (ng/ml)
Figure 3.1: IDE-mediated extracellular degradation of Aβ is facilitated in the presence of ApoE
Aβ1-42 (2 μg/ml) was incubated with 500 ng/ml recombinant IDE in the presence of purified human plasma apoE at the indicated concentrations for 30 min. Remaining Aβ was analyzed by ELISA. The data represent pooled results from three independent experiments (*, p < 0.05; **, p < 0.01 compared to the no-apoE control).
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Figure 3.2
Figure 3.2: ApoE is not an activator of IDE
Recombinant IDE (500 ng/ml) was incubated with the fluorogenic substrate ES005 (10
μM) in the presence of purified human plasma apoE at indicated concentration. The enzyme activity was measured as the generation of fluorescent signal, which was recorded every minute for 1 h in real-time. The plot represents the results of three independent experiments.
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Figure 3.3
Figure 3.3: Kinetic analysis supported that apoE mighty act as a chaperone to facilitate Aβ degradation by IDE
(A) Aβ1-42 (1 μg/ml) was incubated with 500 ng/ml recombinant IDE for the indicated
time in the presence of 100 ng/ml purified human plasma apoE. Remaining Aβ was
analyzed by ELISA. The data represent pooled results from three independent
experiments. (B) Aβ1-42 at indicated concentration was incubated with 500 ng/ml
recombinant IDE in the presence of 100 ng/ml purified human plasma apoE for 15 min.
The remaining Aβ was analyzed by ELISA. Enzyme activity was demonstrated as the
double reciprocal Lineweaver-Burk plot and analyzed by VisualEnzymics 2008 software
(Softzymics, NJ). The Km of IDE degrading Aβ1-42 was significantly decreased from 1.34
μM to 0.86 μM in the presence of apoE, while the Vmax was only minimal changed (5.52
to 5.67 μM/min).
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Figure 3.4
Figure 3.4: Lipidation status of apoE regulates its ability to promote IDE-mediated
Aβ degradation
(A) Cell lysates from primary microglia from different Abca1 genotypes were resolved
with native gel electrophoresis and Western blotted for ApoE. The larger ApoE particles
indicate a greater degree of lipidation. (B) ApoE fractions purified by FPLC from ACM were incubated in Aβ1-42 (2 μg/ml) coated wells at indicated genotypes and
concentrations of apoE for 2 h. Bound apoE were detected by ELISA. (C) Conditioned
media from Abca1 wildtype, heterozygous or knock-out astrocytes was incubated with 1
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μg/ml Aβ1-42 for 24 h. The amount of Aβ1-42 remaining in the medium after 24 h was
quantified using ELISA. Data represent three independent experiments (**, p < 0.01 compared to Abca1+/+; ##, p < 0.05 compared to Abca1+/-). (D) IDE was
immunoprecipitated from conditioned medium obtained from astrocytes of the indicated
genotypes. The immunoprecipitates were then resolved by SDS PAGE and immunoblotted for IDE. (E) ApoE-containing HDL particles were collected from astrocyte conditioned media derived from wildtype, Abca1-/- and Apoe-/- mice by
immunoprecipitation. The group using immunoprecipitants from Apoe-/- astrocyte-
conditioned media provided a negative control for this experiment. (F) 2 μg/ml Aβ1-42 was incubated with 500 ng/ml recombinant IDE in the presence of immunoprecipitated apoE-HDL particles for 1 h. The reaction solution was then resolved by SDS-PAGE and
Western blotted for IDE, apoE or Aβ.
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Figure 3.5
Figure 3.5: ApoE isoforms differentially facilitate Aβ degradation by IDE
(A) Conditioned media of astrocyte cell lines carrying one of human APOE alleles which
knocked in the endogeneous Apoe locus, were collected and fractionated by FPLC on a
Superose 6 column eluted with PBS at a flow rate of 0.5 ml/min. ApoE was quantified by
ELISA and expressed as the proportion of total apoE. The presence of apoE in the eluted
fractions was analyzed using apoE ELISA. (B) Lipidated ApoE fractions purified with
FPLC from human Apoe knock-in astrocyte-conditioned media were incubated in Aβ1-42
(2 μg/ml) coated wells at indicated concentrations for 2 h. Bound ApoE were detected by
ELISA. (C) 1 μg/ml Aβ1-42 was incubated with 500 ng/ml recombinant IDE in the
presence of human Apoe knock-in (E2, E3 and E4) astrocyte-conditioned media for 15
min. ApoE levels in each reaction were adjusted to give a final concentration of 100
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ng/ml. The levels of remaining Aβ in the solution were quantified using ELISA. The data
represent pooled results from three independent experiments (**, p < 0.01; ***, p < 0.001
compared to the no-apoE control). (D) Delipidated recombinant human apoE isoforms at
indicated concentrations were incubated in Aβ1-42 (2 μg/ml) coated wells for 2 h. After
washed three times with PBS, remaining bound apoE was detected by ELISA. (E) 1
μg/ml Aβ1-42 was incubated with 500 ng/ml recombinant IDE in the presence of
delipidated recombinant human apoE (100 ng/ml) with the indicated genotypes for 15
min. The levels of remaining Aβ were quantified by ELISA (*, p < 0.05 compared to the
no-apoE control).
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Figure 3.6
Figure 3.6: Aβ12-28 promotes Aβ degradation by IDE through enhancing allosteric reaction
(A-B) Aβ1-42 (1 μg/ml) was incubated with 500 ng/ml recombinant IDE and Aβ12-28 (5
μg/ml) in the presence of 100 ng/ml of purified human plasma apoE (A) or delipidated recombinant human apoE with the indicated genotypes (B) as indicated for 15 min. The
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500 ng/ml recombinant IDE in the presence of various concentration of Aβ12-28 for 15 min. The levels of remaining Aβ were quantified by ELISA.
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CHAPTER 4
DISCUSSION
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The ε4 allele of APOE is the major genetic risk factor for AD. Although numerous
studies have been conducted to elucidate the underlying mechanisms of the increased risk
in APOE4 carriers, our understanding of the role of apoE in AD pathogenesis remains
limited. We have recently demonstrated that apoE facilitates Aβ degradation both intracellularly and extracellularly in an isoform- and lipidation status-dependent manner
(Jiang et al., 2008). However, the mechanisms underlying this effect remain to be clarified and are the primary focus of this thesis.
In the chapter 2, we studied the mechanism of apoE in promoting microglial degradation of Aβ. Our study highlights the correlation between cellular cholesterol levels and the efficiency of intracellular degradation (Fig. 4.1). This is the first evidence that links these two important biological functions of microglia. We demonstrated that
apoE promotes microglial degradation of Aβ through its ability to elicit cholesterol efflux
which lowers cellular cholesterol levels. This step is necessary since blocking cholesterol
efflux by inhibiting ABCA1 activity completely antagonizes the effect of apoE on Aβ degradation. We also discovered that direct manipulation of cellular cholesterol levels is sufficient to regulate intracellular Aβ degradation by microglia. Lowering cellular cholesterol levels enhances Aβ degradation, while elevating cellular cholesterol levels impedes it. These findings support the importance of cholesterol homeostasis in the regulation of Aβ catabolism. It was to our surprise that cholesterol does not influence the expression or the activity of Aβ degrading enzymes. However, we observed that cellular cholesterol levels influences the trafficking of Aβ. Lowering cellular cholesterol levels by apoE accelerates the transport of Aβ to lysosomes for degradation, while inducing
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cholesterol accumulation in the cells by the NPC inhibitor retards it. These results argue
that the trafficking of Aβ is the rate-limiting step of intracellular Aβ degradation. Faster
delivery of Aβ to the lysosomes leads to more efficient degradation of it. Notably, we
found that the cholesterol regulates trafficking of Aβ through Rab7, a key component of
the dynein motor complex on endosomes. The recycling of Rab7, which is mediated by
GDI, is crucial for regulating endosomal movement. Lowering cellular cholesterol levels by apoE enhances the recycling of Rab7 and subsequently accelerates the endocytic trafficking of Aβ. As a result, the efficiency of Aβ degradation is elevated. Our data support the importance of the cholesterol efflux property of apoE and highlight the underappreciated biological function of cholesterol in Aβ transport and degradation.
In the chapter 3, we investigated the role of apoE in the IDE-mediated Aβ degradation. Interesting, in contrast to intracellular degradation, apoE facilitates IDE- mediated Aβ degradation through its direct interaction with Aβ. In the presence of apoE, the Km of IDE for Aβ was reduced while the Vmax remained unchanged, suggesting apoE
promotes the association/recognition of IDE and Aβ. Lack of a direct apoE-IDE
interaction, as well as the fact that apoE does not enhance IDE’s activity toward other
substrates indicate that apoE may not be a direct activator of IDE. Conversely, manipulating apoE’s affinity for Aβ regulates the effectiveness of apoE in promoting the
degradation. These results support that apoE acts as a molecular chaperone in
transforming Aβ into a favorable substrate for IDE. Importantly, we demonstrated that the
lipidation status of apoE affects its efficiency in promoting IDE-mediated Aβ degradation.
ApoE2, which is the most highly lipidated apoE isoforms, shows the highest affinity for
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Aβ and greatest efficiency in facilitating Aβ degradation. In contrast, apoE4 is poorly
lipidated and therefore has low Aβ binding affinity and induces Aβ degradation
inefficiently. These results are consistent with the risk for AD associated with individual
apoE isoforms and support the role of apoE in the homeostasis of Aβ and AD
pathogenesis.
In this thesis we provide in-depth understanding of the mechanism of apoE in
facilitating the degradation of Aβ. One of the most intriguing findings is the involvement
of cholesterol in the intracellular degradation of Aβ by microglia. The use of cholesterol
lowering drugs, especially statins, has been shown to ameliorate AD pathology in mice and linked to reduced AD risk in observational epidemiology studies. Other cholesterol metabolism modulating reagents, including ACAT inhibitors, are shown to be beneficial in mouse models of AD. However, the mechanistic understanding of these phenomena remains unclear and the results from prospective case-controlled clinical trials are controversial. In this chapter, we will discuss these preclinical and clinical studies with the implication of our findings. We will also discuss the involvement of the cholesterol derivatives in regulating Aβ degradation. Importantly, the heterogeneity of microglia resulted from their origins and activation status and their involvement in AD pathogenesis have been debated in the field of AD research. We will discuss these controversial studies in more detail and also cover the role of apoE in the immunomodulation, which may also influence the clearance of Aβ.
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EFFECT OF CHOLESTEROL MODULATION ON Aβ LEVELS IN VIVO
There is a large body of literature, including epidemiological studies and
preclinical research, showing a putative connection between elevated cholesterol level
and increased AD risk (Shepardson et al., 2011a). High total cholesterol levels at midlife
have been associated with a significant increased incidence of AD (Kivipelto et al., 2002;
Solomon et al., 2007). Furthermore, AD patients with high LDL-C or total cholesterol are
reported to have faster cognitive decline than those having relative normal cholesterol
levels (Helzner et al., 2009). These observations suggest that lowering cholesterol levels
may be a beneficial for AD treatment or even prevention. Currently, the statins are one of
the most effective agents available for lowering total serum and LDL cholesterol. In
support with the epidemiological observations, lowering cholesterol synthesis by
inhibiting HMG-CoA reductase activity has been verified in many animal models of AD
and yielded generally encouraging results. For example, treatment with lovastatin
strongly reduced Aβ1-40 and Aβ1-42 levels in the TgCRND8 mouse model of AD
(Chauhan et al., 2004). Simvastatin rescued the behavior deficit of Tg2576 mice in the
Morris water maze test (Li et al., 2006a). However, the outcomes of using statins in
human patients remain controversial.
The conclusions from human observational studies, which specifically measured the risk for developing AD or the prevalence of AD, generally correlate statins with reduced risk or incidence, while the effectiveness of individual statin derivatives remains variable among studies (Table 4-1). Wolozin and colleagues analyzed 57,104 individuals
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60 years or older and found that the prevalence of probable AD in the cohort taking lovastatin, pravastatin or both, is 60-73% lower than the total patient population, while simvastatin has no effect (Wolozin et al., 2000). A prospective population-based study conducted by Haag et al. reported that during 9 years of follow-up, statin use was associated with a decreased risk of AD compared with individuals who had never used cholesterol-lowering drugs (Haag et al., 2009). However, not all studies conclude that statins are beneficial. Rea and colleagues reported that use of statins does not alter the risk for AD (Rea et al., 2005).
In contrast to the observational studies, the results of randomized case-controlled trials of the use of statins for AD patients are contradictory (Shepardson et al., 2011b). A small case-controlled prospective study of 63 individuals with mild to moderate AD with randomly assigned treatment of atrovastatin (80 mg/day) or placebo showed that the atrovastatin treatment significantly improved cognitive function at 6 months and remained effective until the study ended at 12 months (Sparks et al., 2005). A similar study recruited 614 patients with mild to moderate AD, who had been taking donepezil for over 3 months. Patients were randomized to receive atrovastatin (80 mg/day) or placebo for 18 months. However, the cognitive evaluation of these patients showed no significant difference, regardless the treatment (Feldman et al., 2010). A recent review by
Shepardson et al. provided an insightful explanation for the variability among these studies, suggesting that the selection of patients, BBB permeability of statins and the methods for measuring cognitive function may be the major causes for the disparate outcomes of the various studies (Shepardson et al., 2011b).
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Notably, most of the studies attributing the effect of lowered cholesterol to the production of Aβ may only explain one part of its effect on Aβ homeostasis. Our findings of the function of cholesterol in regulating intracellular Aβ degradation by microglia represent another aspect of the function of cholesterol in the homeostasis of Aβ.
Furthermore, the results from our studies also suggest that the regulation of Aβ degradation may be dependent on the subcellular distribution of cholesterol. In chapter 2, it is noteworthy that although all the cholesterol modulating reagents revealed dose- dependent regulation in Aβ degradation, neither the absolute value nor the changes of the level of cellular total cholesterol linearly reflects the efficiency of intracellular Aβ degradation when comparing among different cholesterol modulating reagents. With only moderate cholesterol reduction, apolipoproteins and the mimetic peptide promote Aβ degradation activity more effectively than other reagents. Acute application of MβCD resulted in much greater reduction of cellular cholesterol levels, yet the induction of Aβ degradation was similar to the effect of other less effective. A similar situation was observed with the treatment of lovastatin. Increasing cellular cholesterol through inhibition of NPC1 activity by U18666A apparently inhibited Aβ degradation more efficiently than the use of cholesterol-loaded MβCD did despite similar total cholesterol levels. NPC1 inactivation sequesters cholesterol within the late endosomes/lysosomes
(Miller and Bose, 2011). Thus, the accumulation of cholesterol is mainly in the endocytic system. It has also been shown that the late endosomes/lysosomes cholesterol pool is the preferential source of cholesterol for ABCA1-mediated cholesterol efflux (Chen et al.,
2001). These results suggest that the activity of Aβ degradation may be regulated mainly
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CHOLESTEROL DERIVATIVES AND ACAT INHIBITORS IN Aβ
DEGRADATION
Homeostasis of cholesterol and its derivatives are tightly regulated and have been reported to influence Aβ production. In addition to the function of cholesterol and oxysterols mentioned in chapter 1, inhibition of acyl-CoA:cholesterol acyltransferase
(ACAT) has been shown to negatively affect amyloid pathology in a mouse model of AD
(Hutter-Paier et al., 2004). Kovacs and colleagues reported that ACAT inhibitors indirectly affect APP processing without altering BACE1 or γ-secretase activities.
Treatment of CP-113,818, an ACAT inhibitor, delays APP maturation and increases immature APP retention in the ER, suggesting that the inhibition of ACAT activity may disrupt the trafficking of APP in the early secretory pathway thus limiting its availability for the secretases (Hutter-Paier et al., 2004; Huttunen et al., 2009; Bryleva et al., 2010).
Similarly, this interruption in the vesicular trafficking may also apply to the degradation of Aβ. Therefore it was of interest to see if inhibition ACAT activity also affected Aβ degradation by microglia. Indeed, inhibition of ACAT activity by Sandoz 58-035 and CI-
976 was able to promote Aβ degradation (Fig. 4.2). Quantification of mRNA levels of Aβ degrading enzymes revealed no significant change upon inhibiting ACAT function (Fig.
4.3). It is similar to which was observed in microglia treated with apolipoproteins or other
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cholesterol modulating drugs in chapter 2 (Fig. 2.7). These results, in combination with previous findings, suggesting that inhibition of ACAT may enhance the endocytic trafficking of Aβ. Although it is seemingly contradicts Kovac’s conclusion that inhibition of ACAT delays vesicular trafficking (Bhattacharyya and Kovacs, 2010), it is possible that cholesterol differentially regulates the transport within the secretory and endocytic pathways.
ACAT converts cholesterol to cholesterol ester. Thus, inhibition of ACAT strongly reduces the cholesterol ester levels and leads to the expansion of free cholesterol pool, while the total cholesterol levels could be the same or decreased (Rodriguez et al., 1999;
Cignarella et al., 2005; Sankaranarayanan et al., 2010). Measuring the levels of cholesterol metabolites in the Aβ degradation assays showed no change in total cholesterol levels in the presence of ACAT inhibitors. We observed significantly
decreased cholesterol ester levels that were accompanied by slightly increased free
cholesterol, indicating the effectiveness of ACAT inhibition. Efficient Aβ degradation
was consistent with lowered cholesterol ester levels. However, this result is not consistent
with the conclusion reported in chapter 2, which showed that facilitated Aβ trafficking
and degradation by the treatment of apolipoprotein or cholesterol lowering reagents is a
result of reduced total cholesterol levels. When comparing different treatments of
cholesterol modulation free cholesterol levels followed the same trend as total cholesterol,
while changes in cholesterol ester levels were rather inconsistent across all treatments.
Thus, elevated Aβ degrading activity following inhibition of ACAT may be mediated
through a separate pathway or due to indirect regulation of Aβ trafficking. A recent study
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showed that knocking out ACAT1 in mice increases 24S-OHC levels in the brain and
ameliorates AD pathology (Bryleva et al., 2010). With all these results, we may conclude that regulation of the composition of cholesterol derivatives might be more important (or
sufficient) than just manipulating the overall levels of cellular total cholesterol in
regulating Aβ degradation.
RESIDENT AND INFILTRATING MICROGLIA IN THE CLEARANCE OF Aβ
Historically, microglia were thought to enter the brain only during embryogenesis
and then undergo modest self renewal, and in response to injury or pathogens they were
stimulated to proliferate (Streit and Xue, 2009). A number of studies have demonstrated
that in the AD brain microglia are unable to eliminate β-amyloid deposits by
phagocytosis (Wegiel et al., 2001; Wegiel et al., 2003; Wegiel et al., 2004). These
findings support the view that plaque accumulation in the brain is due to the inability of
microglia to effectively clear either soluble or fibrillar forms of Aβ. Specifically, it has
been argued that phagocytic functions of microglia are impaired and this has been
postulated to be a result of the proinflammatory environment of the AD brain (Bamberger
et al., 2003; Koenigsknecht-Talboo and Landreth, 2005). In 1989, Wisniewski reported that following stroke in individuals with existing plaque pathology, the plaques were
cleared owing to the action of peripheral macrophages that invaded the ischemic brain,
suggesting that these plaques could be removed by phagocytosis and that endogenous
microglia were distinct from macrophages in their ability to perform this function
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(Wisniewski et al., 1989; Wisniewski et al., 1991a). However, the hematopoietic origin of
microglia makes it difficult to discriminate between the infiltrated and resident microglia.
Despite tremendous efforts in the past years, monocyte-derived and resident CNS
parenchymal microglia remain virtually indistinguishable on the basis of known
immunophenotypic markers, although they might be functionally heterogeneous
(Guillemin and Brew, 2004). The advent of new techniques to selectively express
fluorescent reporters in circulating myeloid cells allowed the demonstration that these
cells could infiltrate the brain in a number of CNS disease models in which lethally
irradiated hosts received adoptively transplanted fluorescent bone marrow cells (Kennedy
and Abkowitz, 1997; Simard and Rivest, 2004; Malm et al., 2005; Simard et al., 2006).
These studies showed that a considerable percentage (up to 30%, one year after
transplantation) of microglia is derived from donor bone marrow under homeostatic
conditions (Kennedy and Abkowitz, 1997; Simard and Rivest, 2004). Recently, several
studies took advantage of the genetic models with GFP expressed in myeloid progenitors
engrafted into irradiated AD mice. In these bone marrow chimeric animals, fluorescent
donor bone marrow derived monocytes were found to invade brain parenchyma, acquire
microglial morphology and associate with Aβ deposits. Specifically, the infiltrated
microglia were found to associate with ~20% of the amyloid plaques and were able to
internalize Aβ deposits (Bolmont et al. 2008; Malm et al. 2005; Simard et al. 2006).
Simard et al. further demonstrated that bone marrow-derived monocytes/macrophages could infiltrate the AD brain and this was associated with plaque clearance. Jucker and colleagues then went on to show that microglia normally clear plaques by eliminating host microglia which carried CD11b-driven thymidine kinase (TK) by chronic
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intracerebroventricular ganciclovir delivery and showing these animals exhibited greater
plaque burdens (Simard et al., 2006). However, the idea that monocytes can traffic into
the brain has been challenged by experiments using parabiosis or limiting the irradiation
to periphery without the exposure of CNS. The authors found no evidence of microglia
progenitor recruitment from the circulation in both experiment models of denervation and
neurodegenerative disease, facial nerve axotomy and ALS, respectively (Ajami et al.,
2007). Mildner and colleagues further demonstrated that demyelinating and neurodegenerative CNS disease models without BBB disruption were not sufficient to induce substantial microglia engraftment during adulthood. The authors concluded that additional endogenous host factors, such as irradiation damage-induced gene expression, were required to condition the adult brain for microglia engraftment (Mildner et al.,
2007). Thus, their data suggested that the influx of peripheral monocytes into the CNS observed in the previous studies using bone marrow transplants may be an artefact.
Whole-body irradiation at the dosages commonly used for myeloablation might lead to the temporary disruption of the BBB, allowing the entry of circulating monocytes into the
CNS that would normally not cross an intact BBB. In addition, donor cells are routinely harvested by mechanically flushing whole bone marrow from bones followed by intravascular injection into recipients. Thus, it is possible that these progenitor cells would not enter the bloodstream and consequently infiltrate CNS under physiological conditions (Ajami et al., 2007). However, it is noteworthy that in the later stage of AD,
BBB break down has been reported in human patients and mouse models (Kalaria, 1997;
Wisniewski et al., 1997; Skoog et al., 1998). Thus as disease progress, it is possible that bone marrow derived monocytes can cross the BBB and migrate to the plaques (Lebson
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et al., 2010).
The idea that endogenous microglia might not be the principle cells to remove
amyloid plaques has been further reinforced by a recent study from Grathwohl et al.
(2009), in which they selectively ablate endogenous microglia using a CD11b-driven TK
coupled with intracerebroventricular ganciclovir administration to the brain of mouse
models of AD. The loss of microglia had no effect on amyloid plaque number or size over a 2-4 week period. The authors concluded that the CNS resident microglia play a
very limited role in restricting the formation and growth of amyloid plaques. However, one major confound in these experiments is that the infiltrated monocyte-derived
microglia, which are also CD11b+, might also be eliminated by ganciclovir ever since
they infiltrated the CNS. Another rather important observation was that there was a 3 to
4-fold increase in soluble Aβ40 and Aβ42 fractions after ablation of microglia, which
strongly suggested the role of microglia in clearance of soluble Aβ, although there is still
a poor understanding of the dynamics linking soluble Aβ levels and plaque formation.
The morbidity and mortality that accompany ganciclovir treatment, the loss of other cell
populations besides microglia, most notably pericytes, the loss of the integrity of the
vasculature and perturbation of normal homeostatic mechanisms in the brain also need to
be considered in the interpretation of the results (Grathwohl et al., 2009). However, a
primary conclusion from this study is that it has reinforced the view that endogenous
microglia in the AD brain are inefficient in remodeling or removing amyloid plaque from
the brain.
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Recently, several studies have demonstrated that CCL2, also known as monocyte chemotactic protein-1 (MCP-1), expression is induced following various CNS insults and its receptor, CCR2, is involved in the attraction and infiltration of mononuclear phagocytes into the brain (Calvo et al., 1996; Glabinski et al., 1996; Babcock et al., 2003).
Deletion of Ccr2 in an AD mouse model resulted in a substantial reduction of microglial accumulation around the plaque and an increase of Aβ deposition (El Khoury et al., 2007).
These data suggest that bone marrow-derived monocytes are capable of infiltrating the
CNS and may play an important role in AD pathogenesis. Thus, the role and to what extent infiltrating microglia play in the clearance of Aβ still need to be further investigated.
CLEARANCE OF Aβ BY OTHER GLIAL CELLS
Astrocytes have been reported to be able to internalize and degrade Aβ. In vivo, intracellular Aβ has been detected in the lysosomal granules of subpial astrocytes indicating phagocytic and lysosomal activity (Funato et al., 1998; Nagele et al., 2003).
Ultrastructural analysis showed that astrocytes separated fibrillar amyloid from neurons by extending hypertrophic processes and internalizing the amyloid into endosomes/lysosomes, suggesting their role in degradation of Aβ (Funato et al., 1998;
Wegiel et al., 2000; Wegiel et al., 2001). Our laboratory demonstrated that astrocytes were able to take up fluorescently labeled sAβ, though were less efficient than microglia
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(Mandrekar et al., 2009). Furthermore, adult mouse astrocytes have also been shown to
effectively degrade Aβ deposits in brain sections obtained from a mouse model of AD
(Wyss-Coray et al., 2003; Koistinaho et al., 2004). It is noteworthy that astrocytes
prepared from adult Apoe−/− mice were unable to degrade Aβ deposits present in PDAPP
mouse brain sections. It suggests that ApoE is essential for astrocytes to bind, internalize
and degrade Aβ deposits in brain sections in vitro (Koistinaho et al., 2004).
ANTI-INFLAMMATORY PROPERTIES OF APOE
It has been shown that the function of apoE is closely linked to the modulation of
inflammatory responses. In addition to its well-established role in cholesterol and lipid
homeostasis, apoE also holds immunomodulatory properties including regulating the
activation status of macrophages, inhibiting the proliferation of T cells and regulating the
expression of cytokines (Zhang et al., 2011). Exogeneous apoE application suppresses
lipopolysaccharide (LPS)-induced secretion of IL-1β, IL-6 and TNFα by the RAW 264.7
macrophage cell line through repressing the TLR-4 activation-induced phosphorylation
of c-Jun and c-Jun N-terminal kinase (JNK) (Baitsch et al., 2011). Microglia and
astrocytes derived from the Apoe knock-out mice have higher expression of TNFα and
IL-6 after LPS stimulation (Lynch et al., 2001). Conversely, preincubating Apoe-/- microglia with exogeneous apoE dramatically suppresses the secretion of TNFα
(Laskowitz et al., 2001). In response to LPS administration in vivo, Apoe-deficient mice have greater mRNA levels of IL-6, IL-12, TNFα and INFγ than wildtype mice (Ali et al.,
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2005). In contrast to repressing the expression of proinflammatory cytokines, treatment of microglia and macrophages with apoE elevated the production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) in response to the stimulation of LPS and INFγ
(Vitek et al., 1997). Moreover, apoE modulates inflammatory and immune responses in an isoform-dependent manner. Treating microglia derived from the target-replacement mice carrying one of human APOE isoforms with LPS elicits isoform-dependent expression of IL-1β, IL-6 and TNFα (E2 < E3 < E4) (Maezawa et al., 2006). Target- replacement mice carrying the APOE4 gene have greater systemic and brain elevation of
TNFα and IL-6 after acute LPS injection compared with the APOE3 carriers (Lynch et al.,
2003). These immunomodulatory properties of apoE may relate to its function as a lipid/cholesterol binding protein since knocking out Abca1 eliminates apoA-I-induced anti-inflammatory activity in macrophages (Tang et al., 2009; Yvan-Charvet et al., 2010).
Although the inflammatory and activation status of microglia is reported to regulate their ability to clear fAβ (as reviewed in chapter 1), the immunomodulatory properties of apoE in regulating microglial degradation of sAβ remains to be elucidated.
CONCLUSION
In this dissertation, we have explored the mechanisms of apoE in facilitating intracellular and extracellular degradation of Aβ. The findings highlight the importance of the cholesterol efflux property of apoE and provide the first direct evidence linking cholesterol to the intracellular transport and degradation of Aβ. In addition, in the IDE-
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mediated Aβ degradation, our data indicate that apoE acts as a molecular chaperone to
promote this reaction. And the Aβ-binding affinity governed by the lipidation status of
apoE confers isoform-dependent efficiency in promoting Aβ degradation by IDE.
Altogether, these results advance our understanding of the mechanism of apoE in the
proteolytic clearance of Aβ. The amyloid hypothesis predicts that a decrease of Aβ levels in the brain will ultimately lead to reduction of AD pathology and ameliorate cognitive impairment. Therefore, understanding the mechanisms underlying Aβ clearance will facilitate the development of effective therapies for AD.
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FIGURES
Figure 4.1
Figure 4.1: The mechanism of apoE in facilitating intracellular Aβ degradation by microglia
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Figure 4.2
Figure 4.2: Inhibition of ACAT activity does not regulate the transcription of Aβ degradation enzymes
Wildtype primary microglia treated with apoE (1 μg/ml), an ACAT inhibitor (Sandoz 58-
035, 1 μg/ml) or U18666A (10 μg/ml) for 24 h and harvested for RNA extraction.
Expression of transcripts was analyzed by real-time PCR with specific primers-probe sets as indicated. The graphs represent pooled data from four independent experiments.
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Figure 4.3
Figure 4.3: Inhibition of ACAT activity facilitates Aβ degradation
(A) Wildtype primary microglia were preincubated with Sandoz 58-035, an ACAT
inhibitor at indicated concentrations for 24 h, followed by administration of 2 μg/ml Aβ1-
42 for 18 h. The remaining intracellular Aβ was analyzed using ELISA (n = 3). (B) Uptake
of fluorescently labeled Aβ was monitored after pretreated with Sandoz 58-035 for 24 h using flow cytometry. The histogram is representative of the outcome from three independent experiments. (C) Cellular total and free cholesterol levels were quantified
-157- and normalized to total protein (n = 3). The levels of cholesterol ester were calculated by subtracting the amount of free cholesterol from the total cholesterol. **p < 0.01 compared to the vehicle treated control.
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CHAPTER 5
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