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AXL receptor tyrosine kinase regulates Apolipoprotein E expression Zhao, Wenchen 2018

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AXL RECEPTOR TYROSINE KINASE REGULATES APOLIPOPROTEIN E EXPRESSION by  Wenchen Zhao  B.Sc., The University of British Columbia, 2016  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2018  © Wenchen Zhao, 2018  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  AXL Receptor Tyrosine Kinase Regulates Apolipoprotein E Expression  submitted by Wenchen Zhao in partial fulfillment of the requirements for the degree of Master of Science in Pathology and Laboratory Medicine  Examining Committee: Cheryl Wellington, Pathology and Laboratory Medicine Supervisor  Helene Cote, Pathology and Laboratory Medicine Supervisory Committee Member  Haakon Nygaard, Experimental Medicine Supervisory Committee Member   Neil Cashman, Pathology and Laboratory Medicine Additional Examiner Susanne Clee, Cellular and Physiological Sciences Additional Examiner  Additional Supervisory Committee Members: Aly Karsan, Pathology and Laboratory Medicine Supervisory Committee Member  Supervisory Committee Member   iii  Abstract Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. One of the hallmarks of AD is the accumulation of amyloid plaques in the brain. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. The APOE gene is the most validated genetic risk factor for late onset AD, and has well-established associations with amyloid deposition and clearance from the brain. We and others have shown that lipidation of apoE can assist amyloid clearance, raising interest in augmenting apoE function as a proposed therapeutic strategy for AD. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. A subset of AXL receptor tyrosine kinase inhibitors, which we term AXL modulators were identified as positive hits. The objective of this thesis is to dissect the mechanism of action (MoA) by which AXL modulators upregulate apoE expression. We initially understood their dependency on AXL by treating AXL-/- CCF-STTG1 cells generated using CRISPR-Cas9 method with the lead compound. We then determined if Liver X Receptor (LXR) activity was required utilizing LXR knock-out (KO) mouse embryonic fibroblasts (MEF) cells. Immunoblotting analysis of AXL protein indicated the ability of AXL modulators to promote AXL receptor cleavage and stabilize the intracellular domain (ICD). To investigate the role of AXL-ICD in apoE homeostasis, various Axl variants, including WT AXL, kinase-dead AXL mutant, Axl-ICD, and AXL N-terminal fragment were stably reconstituted in AXL-/- CCF-STTG1 cells. ApoE baseline expression was significantly upregulated only upon reconstitution of ICD-containing AXL variants. In summary, AXL protein plays a significant role in apoE homeostasis through its intracellular domain.      iv  Lay Summary In Canada, a new case of Alzheimer’s Disease (AD) is diagnosed every 7 minutes, making dementia one of the most pressing health challenges. Apolipoprotein E (apoE), the major component of “good” cholesterol - High-density lipoprotein (HDL), has been shown to help clear amyloid, a sticky protein that damages healthy nerves. Therefore, boosting the amount of apoE in the brain has become a proposed strategy to treat AD. Our lab has recently discovered a molecule that can strongly raise apoE production from human astrocytes, the star-shaped cells that produce apoE in brain. This molecule appears to modulate a cell surface protein called AXL. My works focused on finding out how exactly apoE production can be changed by modifying the AXL protein. Overall, this study helped us learn more about how apoE production is regulated in brain and could provide more insights into discovering drug candidates to treat AD. v  Preface For Chapter 3, the high-throughput phenotypic screen for apoE modulators, AXL-/- and reconstituted engineered CCF-STTG1 cell lines were conducted and generated by AstraZeneca. The experiments and analyses were mostly performed by Wenchen Zhao, under the consultation of Dr. Cheryl Wellington, with technical support especially from Dr. Jerome Robert, who conducted most steps of the cholesterol efflux assay. Dr. Jianjia Fan also provided technical supports and helped with troubleshooting.   vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Abbreviations ................................................................................................................... xi Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 Alzheimer’s Disease ....................................................................................................... 1 1.1.1 Definition .................................................................................................................... 1 1.1.2 AD neuropathology ..................................................................................................... 3 1.1.3 The amyloid cascade hypothesis ................................................................................. 4 1.1.4 APOE: the primary genetic risk factor of AD ............................................................. 5 1.2 CNS lipoprotein .............................................................................................................. 6 1.3 Influence of APOE on AD neuropathology .................................................................... 7 1.3.1 APOE and Aβ pathology ............................................................................................. 7 1.3.2 APOE and Aβ-independent pathways in AD neuropathology .................................... 8 1.4 ABCA1: the key protein that lipidates apoE ................................................................... 9 1.5 LXR: a master regulator of lipid homeostasis .............................................................. 10 vii  1.6 Targeting apoE for AD ................................................................................................. 11 1.6.1 Manipulations of apoE quantity ................................................................................ 11 1.6.1.1 Upregulation of apoE levels.............................................................................. 11 1.6.1.2 Downregulation of apoE levels ......................................................................... 12 1.6.2 Manipulations of apoE properties ............................................................................. 13 1.7 High-throughput phenotypic screen for apoE modulators ............................................ 14 1.8 AXL receptor tyrosine kinase in neurodegenration ...................................................... 15 1.8.1 Overview ................................................................................................................... 15 1.8.2 AXL in neurodegeneration ....................................................................................... 15 1.9 Rationale and hypothesis .............................................................................................. 16 Chapter 2: Materials and Methods ............................................................................................18 2.1 Cell Models and Reagents ............................................................................................ 18 2.2 Cell Culture and Treatment ........................................................................................... 18 2.3 Quantitative RT-PCR .................................................................................................... 19 2.4 ApoE ELISA ................................................................................................................. 20 2.5 Electrophoresis and Immunoblotting ............................................................................ 20 2.6 Cholesterol Efflux Assay .............................................................................................. 21 2.7 Cignal LXR Reporter Luciferase Assay ....................................................................... 22 2.8 Immunocytochemistry .................................................................................................. 22 2.9 Statistics ........................................................................................................................ 23 Chapter 3: Results........................................................................................................................24 3.1 Compound A1 upregulates apoE and ABCA1 expression ........................................... 24 3.2 Compound A1 does not activate the LXR pathway...................................................... 28 viii  3.3 Compound A1 has no or minimal SREBP-1c induction in liver cells .......................... 30 3.4 Compound A1 requires AXL to upregulate apoE expression ...................................... 31 3.5 AXL RTK plays a role in regulating apoE baseline expression in CCF-STTG1 cells . 32 3.5.1 Abolishment of Axl expression in mouse does not change brain apoE level............ 33 3.6 Compound A1 upregulates AXL intracellular domain expression ............................... 34 3.7 AXL-ICD regulates apoE baseline expression ............................................................. 36 3.8 AXL-ICD localizes in nucleus ...................................................................................... 37 Chapter 4: Discussion ..................................................................................................................38 Chapter 5: Conclusions ...............................................................................................................44 5.1 Future directions ........................................................................................................... 45 5.2 Limitations and caveats................................................................................................. 46 Bibliography .................................................................................................................................48  ix  List of Tables  Table 1.1. Summary of apoE allele and genotype distribution in healthy and AD population. ..... 6  x  List of Figures  Figure 1.1. APP processing pathways............................................................................................. 4 Figure 1.2. LXR/RXR binds to LXRE and regulates target gene expression. .............................. 11 Figure 3.1. Compound A1 upregulates apoE and ABCA1 expression in CCF-STTG1 cells. ..... 24 Figure 3.2. Compounds A1 enhances ABCA1 activity. ............................................................... 26 Figure 3.3. A1 upregulates apoE secretion and ABCA1 expression in HMC3 and primary human astrocyte. ....................................................................................................................................... 27 Figure 3.4. A1 upregulates apoE and ABCA1 expression in WT mouse primary mixed glia. .... 28 Figure 3.5. A1 does not require LXR pathway to upregulate apoE and ABCA1 expression. ...... 29 Figure 3.6. A1 does not activate LXR pathway. ........................................................................... 30 Figure 3.7. No or minimal SREBP-1c induction by compound A1 in liver cells. ........................ 31 Figure 3.8. A1 requires AXL to upregulate apoE expression. ...................................................... 32 Figure 3.9. AXL knockdown downregulates apoE expression..................................................... 33 Figure 3.10. Lack of Axl expression does not alter mouse brain apoE level. ............................... 34 Figure 3.11. A1 increases AXL-ICD expression. ......................................................................... 35 Figure 3.12. Compound A1 upregulates AXL-ICD expression in other cell types. ..................... 35 Figure 3.13. Reconstitution of AXL in AXL-/- CCF-STTG1 elevated apoE baseline expression.36 Figure 3.14. AXL-ICD predominantly localizes in nucleus. ........................................................ 37 Figure 5.1. Proposed mechanism of apoE regulation by AXL-ICD. ............................................ 44  xi  List of Abbreviations ABCA1 ATP-binding cassette transporter A1 Aβ  amyloid β AD   Alzheimer’s Disease  AICD  APP intracellular domain  ApoE  apolipoprotein E APP   amyloid precursor protein BBB  blood-brain-barrier CAA   cerebral amyloid angiopathy CNS   central nervous system CSF   cerebrospinal fluid CTF  C-terminal fragment DAM  disease-associated microglia DMSO  dimethyl sulfoxide ELISA  enzyme-linked immunosorbent assay FBS  fetal bovine serum Gas6  growth-arrest-specific gene 6  HDL  high-density lipoprotein HTS  high-throughput screen ICD   intracellular domain LDL  low-density lipoprotein LDLR   low-density lipoprotein receptor LRP1   low-density lipoprotein receptor-related protein 1 xii  LOAD  late-onset Alzheimer’s Disease LPS  lipopolysaccharide LXR  liver X receptor  LXRE  LXR response element MoA  mechanism of action NFT   neurofibrillary tangle PCR  polymerase chain reaction PS1  Presenilin 1 PS2  Presenilin 2 NTF   N-terminal fragment RIPA  radioimmunoprecipitation assay RTK  receptor tyrosine kinase RXR  retinoid X receptor  SDS  sodium dodecyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis SNP  single nucleotide polymorphism SREBP-1c Sterol regulatory element-binding transcription factor 1c TR  targeted replacement  WT  wild type   xiii  Acknowledgements First and foremost, I would like to express my sincere gratitude to my supervisor, Dr. Cheryl Wellington for taking me as a graduate student and giving me the opportunity to work on my project. Throughout my graduate study, I have been given generous freedom with proper guidance to come up with my own hypothesis and design my own experiments. I am always grateful for this environment where I am free to think. Next, I would like to thank all the past and present Wellington lab members for their continued support. I would like to thank Dr. Iva Kulic and Dr. Jianjia Fan for initially hiring me as a co-op student and directly working with me throughout my graduate education. As scientists they have taught me how to properly design rigorous experiments to test the hypothesis and how to critically analyze the data. As friends they are genuinely concerned about my well-being and future career. Additionally, my fellow lab members Dr. Jerome Robert, Dr. Tom Cheng, Dr. Sophie Stukas, Emily Button, Sonja Soo, Asma Bashier, and Guilaine Boyce have all been very supportive and help me troubleshoot in numerous occasions. I would also like to thank past and present co-op students who contributed to the project including Cameron Parro, Amber Chou, and Amanda Clark.   I would like to thank AstraZeneca for funding this project and providing important materials and tools including the compound and genetically engineered astrocytes. I would also like to extend my thanks to my committee members: Dr. Helene Cote, Dr. Haakon Nygaard, and Dr. Aly Karsan for providing important and helpful guidance. Last but not least, I would like to offer my gratitude to my parents and family who have supported me throughout my years of education, both morally and financially. xiv  Dedication I dedicate my thesis to my parents, Fumin Zhao and Ying Wu. They are always there for me no matter what happens. Without their continued support, I would not have been studying in such a great university and been as successful as today. I am always grateful for having them as my parents and will continue to be a better self. Thank you!1  Chapter 1: Introduction  1.1 Alzheimer’s Disease  Alzheimer’s Disease (AD), the most common form of dementia, currently affects 40% of North Americans over 85 years of age. In Canada, AD accounts for over 60% of dementia cases and more than half a million Canadians are living with this disease [1]. The estimated global burden of dementia is projected to be 100 million cases by 2050. In North America, AD leads to caregiving costs of approximately $50,000- $85,000/year/person and incalculable emotional costs to patients and their caregivers [1]. Clinical symptoms of AD primarily include cognitive impairments [2], particularly loss of short term memory in the early stages of AD. As the disease progresses, patients’ cognitive and executive functions such as speaking and understanding start to be gradually impaired until complete cognitive impairment, which severely impacts patients’ ability to perform routine tasks and renders them to be heavily relying on their caregivers [2].   Despite huge efforts in investigating the cause of the disease and working toward a cure, currently there is still no effective treatment nor prevention for AD. As the AD population continues to grow rapidly, the AD burden will become uncontrollably high if any effective therapies are not identified and implemented.   1.1.1 Definition Alzheimer’s disease was first described in 1906 by a German physician, Alois Alzheimer, who reported “ A peculiar severe disease process of the cerebral cortex” on the 37th Meeting of South-West German Psychiatrists in Tubingen [3]. Alzheimer first discovered and described the 2  histological features of AD including severe brain atrophy and neuron degeneration by examining the brain autopsy of one of his patients, Auguste D. He also described two distinct pathological histological features that were later identified as the two classical neuropathological AD hallmarks, namely neurofibrillary tangles and amyloid plaques. He also documented the clinical symptoms of this patient including reduced memory and comprehension, paranoia, hallucination, and severe psychosocial impairment [4].   In 2011, National Institute on Aging has published updated criteria for clinical diagnosis of AD. The new diagnostic guidelines describe three stages of AD: (a) Preclinical – “brain changes, including amyloid buildup and other nerve cell changes”, clinical cognitive symptoms are not yet obvious; (b) mild cognitive impairment – cognitive impairments such as memory or comprehension loss that are more serious than normal population but do not prevent patients routine tasks; (c) Alzheimer’s dementia – severe cognitive impairments such as memory loss, visual/spatial problems that are severe enough to impair a persons’ ability to carry on daily tasks [5]. Importantly, the revised criteria pointed out distinction between pathophysiological process of AD and the clinically observable syndromes because the observations made over the past 27 years suggest that pathological-clinical correlation is not always consistent. For example, one of AD hallmarks, amyloid plaques, can be profoundly present without any obvious clinical cognitive symptoms [5].   In 2018, National Institute on Aging and Alzheimer’s Association Research Framework, based on scientific progress from 2011-2018, further defined AD by its “underlying pathologic processes that can be documented by postmortem examination or in vivo by biomarkers” [6], 3  shifting the definition of AD from syndromal to biological-based. Biomarkers include three major groups: amyloid β deposition, pathologic tau, and neurodegeneration [AT(N)] and cognitive impairment is treated as the symptom rather than definition of the disease [6].   1.1.2 AD neuropathology The fundamental characteristics of AD pathology, despite of the revision of AD criteria in 2011, have remained the same: the presence of amyloid plaques in cortex and intracellular neurofibrillary tangles [5].   Amyloid plaques are composed of amyloid β (Aβ) peptides, which are generated from proteolytic cleavage of the amyloid precursor protein (APP) [7]. It is an integral transmembrane protein expressed in many tissues including brain. Its primary function is still unknown, though it has been implicated to play a role in synapse formation and neural plasticity [8]. APP can be cleaved via two pathways: non-amyloidogenic or amyloidogenic mediated by α-, β-, and γ- secretases [8]–[11]. The non-amyloidogenic APP processing requires sequential cleavage by α- and γ- secretases giving rise to secreted form of APPs-α, a p3 peptide and APP intracellular domain (AICD) [12] whereas the amyloidogenic process involves cleavages by β- and γ- secretases generating secreted APPs-β, AICD as well as Aβs [13]. The lengths of Aβs ranges from 37 to 43 amino acids, with Aβ40 and Aβ42 being the most frequent cleavage products. Aβ42 is more hydrophobic due to the extra two hydrophobic amino acids and aggregates more easily compared to Aβ40, which makes it more toxic [14].  4   Figure 1.1. APP processing pathways. APP can be cleaved by three secretase complexes: α-, β-, and γ- secretases. In the non-amyloidogenic pathway, α- secretase cleave the APP generating soluble APP (APPs-α) and an 83-aa C-terminal fragment (CTF), which can be further cleaved by γ-secretase to generate APP intracellular domain (AICD). In the amyloidogenic pathway, APP is first cleaved by β-secretase to generate a different soluble APPs-β and a 99-aa CTF, which is further processed by γ-secretase to produce Aβ peptides that are either 40- or 42-aa in length.   Unlike amyloid plaques, which are hypothesized to exert toxicity in the extracellular space, the other hallmark of AD, the neurofibrillary tangle (NFT), is found intracellularly within affected neurons. NFTs are formed by hyperphosphorylation of a microtubule-associated protein called tau [15], [16]. Tau is primarily located in the axons of neurons to assist cargo transportation as part of microtubule structure [17]. However, tau becomes pathologically hyperphosphorylated in AD brain, which prevents it from being attached to microtubule and thus increasing the free tau concentration. Hyperphosphorylated tau is prone to self-aggregation and eventually become NFTs via an unknown mechanism [18].    1.1.3 The amyloid cascade hypothesis The amyloid cascade hypothesis has dominated the AD research for almost thirty years. It states that the deposition of Aβ peptide in the brain initiates a cascade of events that ultimately give rise to AD pathology [19]. This hypothesis was raised by the discoveries of genetic causes of early-onset AD and Down’s syndrome. Early-onset AD refers to the cases where AD dementia 5  occurs before 65 years old. It has been found that mutations in Presenilin-1 and Prsesenilin-2, which are both components of the γ-secretases, as well as mutations in APP can lead to increased production of Aβ peptides, thus causing early-onset AD [20]–[22]. Down syndrome patients have an extra copy of chromosome 21 (trisomy 21) on which APP gene is located. They also have excessive amyloid deposition and develop AD pathology much younger [23]. Taken together, amyloid has been suggested to be the culprit of AD pathology, leading to decades of efforts to prevent its production. However, all therapeutic approaches targeting amyloid that reached Phase III clinical trials have failed, which has raised the controversy about the role of amyloid in AD pathology and stimulated interest in investigating other novel targets to treat AD.  1.1.4 APOE: the primary genetic risk factor of AD Apolipoprotein E (ApoE), which is secreted from astrocytes and microglia, serves as the major lipid carrier in the brain [24]. It is the most established genetic risk factor for late-onset AD (LOAD), which accounts for more than 99% of AD cases [25]–[27]. Human express three APOE alleles; APOE-ε2 (protective), APOE-ε3 (neutral) and APOE-ε4 (detrimental) due to two single nucleotide polymorphisms (SNPs) in its exon 4 (rs429358, rs7412) and resulting amino acid substitutions of arginine or cysteine at positions 112 and 158; ε2 corresponds to Cys112/Cys158, ε3 to Cys112/Arg158, and ε4 to Arg112/Arg158 [28]. The presence of one APOE-ε4 copy shifts the age of onset an average of 2 to 5 years earlier, whereas carrying two APOE-ε4 alleles shifts onset 5 to 10 years earlier.  At least one copy of APOE-ε4 is present in ~14% of the population and ~ 60% of AD patients [27]–[29]. In contrast, APOE-ε2 which has an allelic frequency of ~7% has been shown to have neuroprotective effect and can reduce AD risk by ~50% [30], [31].  6  Frequency APOE Allele APOE Genotype ε2 ε3 ε4 2/2 2/3 3/3 2/4 3/4 4/4 Control 0.07 0.79 0.14 0.007 0.11 0.623 0.019 0.222 0.019 AD 0.04 0.58 0.38 0.003 0.046 0.343 0.026 0.434 0.148 Table 1.1. Summary of APOE allele and genotype distribution in healthy and AD population.  Taken together, these studies suggest that APOE genetic status will likely play a major role in future personalized medicine. However, despite the pivotal role that apoE plays in AD pathogenesis, our understanding of its regulation and function in the brain is appallingly poor.   1.2 CNS lipoprotein The brain is the most cholesterol-rich organ in the body, containing ~25% of total body cholesterol with only 2% of total body weight [32]. Cholesterol is one of the major components of neuronal and glial membranes as well as myelin and is synthesized in situ due to the presence of blood-brain-barrier (BBB) that prevents almost all peripheral cholesterol from entering the brain [33].   Cholesterol is transported in the form of lipoproteins which are composed of lipids and proteins. The major function of lipoproteins is to deliver lipids to cells or tissues that use or store lipids. In central nervous system (CNS), apoE serves as a major protein component of lipoproteins, which resemble plasma high-density lipoprotein (HDL) in density, size and composition [34]. Brain and peripheral HDL particles are still different in that apoE is the major apolipoprotein in brain lipoproteins while apoA-I serves as the primary apolipoprotein species in circulating HDL [34]. 7  Thus, brain HDL is commonly referred to as HDL-like particles in order to distinguish from peripheral HDL. In brain, astrocytes and microglia cells are recognized major producers of apoE protein [35], [36] and pericytes are now also known to produce apoE [37]. After being produced by glial cells, HDL-like particles are composed of apoE, phospholipids and cholesterol. The particles are then transported and bind to apoE receptors expressed on target cells such as neurons and deliver lipids.   In brain, HDL-like particle metabolism, there are several other proteins and enzymes that also play critical roles. ATP-binding cassette transporter A1 (ABCA1), for example, has been found to load lipids onto apoE, forming the nascent particles. Loss of ABCA1 can significantly impair the lipidation status of apoE [38]. The low-density lipoprotein (LDL) receptor (LDLR) family members including LDLR and LDLR-related protein 1 (LRP1) serve as major apoE receptors [39], which are also important in apoE homeostasis.      1.3 Influence of APOE on AD neuropathology ApoE is the most validated genetic risk factor of LOAD, which plays important roles in AD neuropathology via both Aβ-dependent and independent pathways.  1.3.1 APOE and Aβ pathology As mentioned in section 1.1.4, APOE-ε4 significantly associates with AD occurrence, supported by increased amount of Aβ particularly the more toxic oligomeric form found in post-mortem APOE-ε4 carrier AD brains [40], [41]. Carrying APOE-ε4 increases plaque deposition in brain 8  [42]–[44] and exacerbates formation of cerebral amyloid angiopathy (CAA) in the brain vasculature [45], [46].   In mouse studies, lack of murine Apoe significantly reduces amyloid deposition without affecting Aβ production [47], indicating the potential role of apoE in conversion of soluble Aβ to its fibrillar form. In addition, Apoe-targeted replacement (TR) mice were generated by replacing coding exons 2-4 of murine Apoe with the corresponding region of human APOE. As a result, the amyloid load in TR mice were differentially affected with apoE4 > apoE3 > apoE2 [48], further supporting the critical role of apoE isoforms. Intriguingly, the presence of human apoE significantly delayed the onset of amyloid deposits in contrast to murine apoE [48]–[50]. Taken together, although apoE has been validated to be important in influencing Aβ metabolism, murine and human apoE species difference should not be neglected due to their differential effects.  1.3.2 APOE and Aβ-independent pathways in AD neuropathology NFT caused by hyperphosphorylated Tau is the other hallmark of AD. Not only does APOE-ε4 increases amyloid burden, it also associates with increased levels of NFTs in post-mortem AD brains [51]–[53]. Similar events have also been observed in apoE4 transgenic mice [54] and apoE4-TR mice [55]. In AD transgenic mice carrying early-onset familial AD mutations, apoE4 also exacerbates NFTs formation [56].   In addition, neuroinflammation is another feature of AD pathology [57], [58]. It has been found that APOE-ε4 carriers have elevated levels of inflammatory biomarkers present in plasma 9  compared to non-carriers [59]. In mouse studies where apoE-TR mice were treated with lipopolysaccharide (LPS), apoE4-TR mice showed an increased inflammatory response indicated by elevated brain pro-inflammatory cytokines compared to apoE3-TR mice [60], [61].   Furthermore, synaptic loss or dysfunctions strongly associates with cognitive impairments in AD patients and in AD mouse models [62]–[64]. ApoE has also been suggested to play a role in synaptic integrity, as APOE-ε4 AD brains show more synaptic loss compared to APOE-ε3 carriers [41], [62].   Last but not least, apoE is also involved in maintaining BBB integrity as APOE-ε4 AD brains showed more severe pericyte degeneration compared to APOE-ε3 brains [65]. Taken together, apoE has diverse roles within the brain, raising many questions about how to approach apoE as a therapeutic target for AD.   1.4 ABCA1: the key protein that lipidates apoE After being secreted, apoE assembles with other proteins and lipids to form HDL-like particles in the brain. During the process, ABCA1, which is a transmembrane protein, plays a paramount role in loading lipids onto the apoE protein. ABCA1 is a well-studied protein in peripheral HDL biogenesis, and its role in CNS has been known since 2004. Our lab and others have established the critical role of ABCA1 in lipidating apoE, regulating apoE secretion as well as determining apoE levels in brain due to the fact that in ABCA1-deficient mice, brain apoE levels as well as lipidation was significantly reduced [66], [67]. Taken together, ABCA1 is the major protein that regulates lipidation and turnover of apoE. 10   ABCA1 has also been shown to affect amyloid burden in AD mouse models. Our lab and others have generated ABCA1-deficient AD mouse models and all showed that although APP processing or steady state Aβ levels were not affected, amyloid deposition was significantly exacerbated, providing strong evidence that ABCA1 is important in amyloidogenesis via an Aβ-independent pathway [68]–[70].  On the other hand, overexpressing ABCA1 in AD mouse showed increased lipidation of apoE and drastically reduced amyloid deposition [71]. These results suggest that the lipidation status, rather than the absolute amount of apoE, may be more important in Aβ metabolism and it therefore has been hypothesized that lipid-rich apoE facilitates the Aβ clearance.   1.5 LXR: a master regulator of lipid homeostasis Both ABCA1 and apoE expression are under the control of a nuclear receptor liver X receptor (LXR) [72], [73]. In humans, LXR exists in two isoforms: LXRα, which is highly expressed in peripheral tissues such as liver, kidney and intestine [74], [75], and LXRβ which is ubiquitously expressed and is the main isoform present in the CNS [76]. LXR was initially targeted to treat atherosclerosis leading to the development of the synthetic LXR agonist TO901317 (Tularik) that activates both LXRα and LXRβ [77], [78].   In the nucleus, LXR functions as heterodimers with another nuclear receptor retinoid X receptor (RXR) [79]. LXR/RXR complex can be activated by LXR agonists or 9-cis-retinoic acid, which is the endogenous RXR agonist. The complex recognizes and binds to a unique DNA sequence called LXR response element (LXRE) leading to the activation or suppression of many 11  downstream target genes including APOE and ABCA1 depending on if the ligand exists (Figure 1.2).   Figure 1.2. LXR/RXR binds to LXRE and regulates target gene expression. Depending on if ligands are absent (A) or present (B), LXR/RXR heterodimer complexes recruit either co-repressor or co-activator to repress or activate downstream target gene expression.   1.6 Targeting apoE for AD As apoE is undeniably important in AD neuropathology, manipulations of the APOE gene and apoE functional properties have been raised as potential therapeutic approaches for AD. This section summaries some current investigations into apoE-centric approaches.  1.6.1 Manipulations of apoE quantity It is still controversial whether increasing or decreasing apoE levels in brain will be beneficial or detrimental. Conflicting results have been published in clinical trials evaluating apoE levels in cerebrospinal fluid (CSF) and plasma [80]–[82]. Thus, studies involving both upregulation or downregulation of apoE levels haven been conducted and summarized below.  1.6.1.1 Upregulation of apoE levels As mentioned in section 1.5, apoE expression is controlled by LXR and RXR receptors. In AD mouse models, oral administration of an RXR agonist, bexarotene, upregulated apoE levels, significantly reduced amyloid deposition, and improved cognitive functions [83]. Its beneficial 12  effects on rescuing cognitive functions were also reproduced in AD mice expressing human APOE-ε3 and APOE-ε4 [83] as well as APOE-ε4 mice without an amyloid background [84]. However, bexarotene’s effect on amyloid burden is still controversial due to conflicting results [85]. Bexarotene also has significant adverse effects including liver failure in some mouse studies [86], [87].   In addition to RXR agonists, LXR agonists such as TO901317 have also been investigated in AD mouse models and shown to reduce amyloid deposition and rescue cognitive functions [88]–[91]. However, it is still unknown whether the beneficial effects were mediated via upregulated apoE levels. It has also been suggested that increased ABCA1 activity by LXR agonists is more of importance which will be discussed below. Despite the promising beneficial effects, LXR agonists adversely activated sterol regulatory element-binding transcription factor 1c (SREBP-1c) pathway in liver, leading to hypertriglyceridemia and hepatic steatosis (fatty liver), which prevented them from passing the preclinical trails [92], [93]. It has not yet been possible to develop a drug that stimulates LXR target genes without triggering SREBP-1c lipogenesis pathway.  1.6.1.2 Downregulation of apoE levels Several mouse studies have shown strong evidence to support that reducing apoE levels is protective against amyloid. First of all, in AD mice lacking Apoe, no amyloid deposits were found, in sharp contrast to Apoe-expressing AD mice that displayed abundant amyloid deposition [47]. Also, APOE haploinsufficiency in apoE-TR mice attenuated amyloid deposition across different APOE genotypes [94], [95]. An intraperitoneal administration of an anti-apoE antibody 13  into AD mice has improved cognitive function and reduced brain Aβ load [96]. In terms of tauopathy, tau transgenic mice with human apoE knock-in displayed significant brain atrophy and neuroinflammation whereas tau mice with apoE knockout background was largely protected from these changes [97].  However, an overall reduction of apoE levels is not without its own risks, as apoE also plays important roles in the brain’s innate immune system and neuroinflammation, as well as for synaptic and BBB integrity as mentioned in section 1.3.2. A key question is whether reducing apoE may increase susceptibility for other brain insults.   Nonetheless, as APOE-ε4’s detrimental role has been well-established, selectively reducing apoE4 species may be a viable approach. In fact, one study using anti-apoE4 monoclonal antibody showed improved cognitive performance in apoE4-TR mice [98].  ApoE aggregates particularly driven by apoE4 have been found in amyloid plaque and predicted to be harmful [99]–[103]. Therefore, specifically reducing aggregated apoE but not functional free apoE may also be viable. A recent study targeting aggregated apoE was found to inhibit amyloid accumulation [104].  1.6.2 Manipulations of apoE properties As discussed in both sections 1.4 and 1.6.1.1, increasing the lipidation of apoE by promoting ABCA1 activity has demonstrated beneficial effects in AD mouse models. Likewise, induction of ABCA1 expression via inhibition of microRNA-33 increased lipidation of apoE and 14  significantly decreased amyloid deposition [105]. Of importance, apoE4 is significantly less lipidated than apoE2 and apoE3 both in humans [106] and in apoE-TR mice [107] potentially due to its poor ability to bind lipids.  The interaction between apoE and Aβ has also gained increasing attention due to their co-localization in amyloid plaques in human brains [108]. It is therefore hypothesized that apoE directly impacts Aβ aggregation, deposition and clearance [109]. Indeed, a synthetic peptide Aβ 12-28P that blocks apoE and Aβ binding led to reduced amyloid deposition and improved cognitive function [110], [111]. Targeting aggregated and unlipidated apoE in amyloid plaques by antibody also showed less amyloid burden, further supporting the benefits of blocking their interaction [104].   1.7 High-throughput phenotypic screen for apoE modulators As discussed in 1.6.1.1, although LXR agonists showed promising therapeutic benefits in multiple AD mouse models, they have severe hepatotoxicity effects which preclude them from being further developed and evaluated. Therefore, it is important to discover novel compounds that upregulate APOE and ABCA1 expression without activating SREBP-1c pathway in liver.  In collaboration with AstraZeneca, a high-throughput phenotypic screen (HTS) to identify potential small molecules that upregulate apoE secretion was conducted in human CCF-STG1 astrocytoma cells. Screening conditions were similar to those previously published by our laboratory [112] and will be reported separately. A class of compounds, known as AXL receptor tyrosine kinase (RTK) modulators, was identified to be potent apoE inducers and subsequently 15  validated in our laboratory. Thus, it is of our interest to investigate the mechanism of action (MoA) by which modulating AXL manipulates apoE levels.    1.8 AXL receptor tyrosine kinase in neurodegenration 1.8.1 Overview AXL is a transmembrane protein that belongs to the TAM (Tyro3, AXL, Mer) RTK family. It was first discovered as a oncogene in patients with chronic myelogenous leukemia [113] and later determined to play important roles in diverse cell functions such as cell survival, proliferation, migration and differentiation. Acting primarily as a phagocytosis receptor that responds to cell apoptosis, it transduces signals when binding to its extracellular ligand, growth-arrest-specific gene 6 (Gas6) [114], [115]. AXL has been actively investigated in the oncology field as it is upregulated in several cancers [116] and has been found to play a role in metastasis [117], [118], drug resistance [119], [120] and poor survival [121]–[123]. However, its role in neurodegeneration has yet to be fully established. To our knowledge, we are the first to report the crosstalk between AXL and apoE signaling.   1.8.2 AXL in neurodegeneration Although the role of AXL in the brain is not well-studied, several clinical studies have pointed out the potential importance of this protein. One study from the Alzheimer’s Disease Neuroimaging Initiative examined the correlation between brain amyloid burden and plasma analytes and found that along with apoE, soluble AXL significantly associates with fibrillar Aβ levels in AD patients [124]. In another similar studies trying to identify plasma biomarkers in AD patients, AXL plasma levels were found to be significantly different across different APOE 16  genotype groups (ε2/ε3 <ε3/ε3) [125]. However, the sample size in this study was relatively small (N=33). AXL was also identified as a potential biomarker for intracranial aneurysm (IA) rupture due to its significant increased expression in patients with IA [126].  AXL is gaining increasing attention for its role in microglia. In AXL-deficient mice, the microglial response to brain damage was significantly impaired due to delayed microglial recruitment to sites of injury [127]. In addition, in a mouse model of Parkinson’s disease, AXL expression in microglia was significantly increased in the neuroinflammatory region [127]. Another study isolated microglia from several CNS disease mouse models and examined its transcriptomic profile. Compared to homeostatic microglia, the disease-associated microglia (DAM) suppressed expression of homeostatic genes but significantly upregulated a panel of inflammation- and phagocytosis- related genes including Axl and Apoe [128]. Taken together, along with apoE, AXL seems to play an important role in microglia in response to brain damage, yet its detailed role as well as the relationship with apoE are still unknown.  1.9 Rationale and hypothesis As the most validated genetic risk factor for Alzheimer’s disease (AD), apolipoprotein E (apoE) is a priority therapeutic target. Human express three APOE alleles; APOE-ε2 (protective), APOE-ε3 (neutral) and APOE-ε4 (detrimental). APOE-ε4 increases AD risk and reduces age of onset. At least one copy of APOE-ε4 is present in ~17% of the population and ~ 60% of AD patients. ApoE has an undeniable role in regulating Aβ deposition, and also affects inflammation, blood brain barrier (BBB) integrity, synaptic plasticity, cell signaling and lipid transport. We and others have shown that lipidation of apoE can promote amyloid clearance, raising interest in 17  augmenting apoE function as a proposed therapeutic strategy for AD. Synthetic LXR agonists have been shown to enhance Aβ clearance possibly through ABCA1-mediated apoE lipidation. However, due to their severe adverse effects in liver, the field is interested in discovering apoE and ABCA1 modulators without targeting LXR directly. Thus, in collaboration with AstraZeneca, we have performed a high-throughput phenotypic screen in CCF-STTG1 astrocytoma cells looking for apoE modulators. A class of compounds, known as AXL modulators, was identified as positive apoE inducers.   The overall hypothesis of this thesis is that AXL plays a role in regulating apoE expression. The main goal of this thesis is to investigate the mechanism of action of AXL modulators by which they induce apoE expression. This thesis studies the hypothesis mainly through in vitro cell models with following aims: Aim 1: To validate the drug effect of AXL modulator A1 on apoE and ABCA1 in different apoE-secreting cell types. Aim 2: To determine if AXL modulator increases apoE and ABCA1 expression via a LXR-dependent pathway. Aim 3: To investigate the potential mechanism of action by which AXL modulators regulate apoE expression. 18  Chapter 2: Materials and Methods  2.1 Cell Models and Reagents Human CCF-STTG1 astrocytoma cells, AXL-/- and AXL-reconstituted CCF-STTG1 were obtained from AstraZeneca (Sweden). Wild-type HEK293, human HepG2 hepatoma and human microglia clone 3 cell line (HMC3) were purchased from ATCC (Virginia, USA). Immortalized LXR double knockout (LXRα-/LXRβ-) and LXR expressing (LXRα+/LXRβ-) mouse embryonic fibroblasts (MEFs) [129] were kindly provided by Dr. Peter Tontonoz (California, USA). Primary human astrocytes and hepatocytes were purchased from ScienCell (California, USA). Primary mouse mixed glia were cultured from postnatal day 0-2 wild type pups as described [130]. Compound A1 and the LXR antagonist GSK2033 were provided by AstraZeneca. The LXR agonists TO901317 was purchased from Sigma-Aldrich and was used as the positive control for all experiments. Stocks of all compounds were prepared in dimethyl sulfoxide (DMSO).  2.2 Cell Culture and Treatment Parental CCF-STTG1 cells were cultured in a mixed media consisting of 3 parts of High-Glucose Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine (Sigma, cat# D6429) and 1 part of Ham's F12 (Sigma, cat# N6658), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S, Gibco). AXL-/- and AXL-reconstituted CCF-STTG1 cells were cultured in Roswell Park Memorial Institute medium (RPMI, Gibco) 1640-GlutaMAXTM supplemented with 10% FBS and 1% P/S. Primary mouse mixed glia and mouse embryonic fibroblasts cells were cultured in DMEM (Gibco) supplemented with 10% FBS, 2 19  mM L-glutamine and 1% P/S; HepG2 cells were maintained in the same growth media further supplemented with 1 mM sodium pyruvate and 1x non-essential amino acids (Invitrogen). Primary human astrocytes and hepatocytes were cultured in their respective growth media (containing 2% FBS) provided by ScienCell. HMC3 cells were grown in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS.  For immunoblotting, mRNA, and apoE ELISA assays, cells were seeded in either 12-well (CCF-STTG1: 300,000 cells/well; MEF: 100,000 cells/well; HepG2: 400,000 cells/well; primary mouse mixed glia: 250,000 cells/well; primary human astrocyte: 300,000 cells/well; primary human hepatocyte: 150,000 cells/well; HMC3: 100,000 cells/well) or 24-well (CCF-STTG1: 150,000 cells/well) plates in their respective standard growth media. After 24 h, cells were treated with treatment media (parental CCF-STTG1: 3:1 DMEM:F12 with 1% P/S and 1% FBS; AXL-/- and AXL-reconstituted CCF-STTG1: RPMI 1640-GlutaMAXTM with 1% P/S and 1% FBS; MEF and primary mouse mixed glia: 1:1 DMEM:F12 (Gibco, cat# 11330) with 1% P/S and no serum; primary astrocyte and hepatocyte: their respective growth media; HepG2 and HMC3: 1:1 DMEM:F12 with 1% P/S and 1% FBS) containing DMSO, positive control TO901317, or test compounds for the indicated time intervals. For dose response experiments, compounds were serially diluted 1:3 to generate a range of concentrations. For all experiments, the final concentration of the vehicle DMSO was equalized for all treatment conditions.  2.3 Quantitative RT-PCR Cells were lysed in TRIzol (Invitrogen). RNA was extracted and treated with DNase I according to the manufacturer’s protocol (Invitrogen). Real-time quantitative PCR (qRT-PCR) was done 20  with SYBR Green reagents (Roche) on a LightCycler96 system (Roche). The qRT-PCR primer sequences used in this study were previously described [131]. Each sample was assayed at least in duplicate, normalized to GAPDH (human) or β-actin (mouse).  2.4 ApoE ELISA Secreted apoE levels in culture media were measured by a sandwich apoE ELISA protocol described previously [131]. Fluorescence was read at 325Ex/420Em on an Infinite M200 Pro plate reader (Tecan Life Science, Switzerland).  2.5 Electrophoresis and Immunoblotting For native PAGE, media samples were mixed with non-denaturing loading dye to a final concentration of 0.04% bromophenol blue, 4.0% glycerol, and 100 mM Tris (pH 6.8) and resolved on 6% non-denaturing Tris-HCl polyacrylamide gels. To visualize apoE, native gels were transferred as described below and probed with 1:1000 anti-apoE antibody (Cell Signaling Technology, cat# 13366S) overnight. For denaturing PAGE, cells were washed with 1x PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (20 mM Tris, 1% NP40 Alternative, 5 mM EDTA, 50 mM NaCl, 10 mM Na pyrophosphate, 50 mM NaF, and complete protease inhibitor (Roche), pH 7.4). Protein concentration was determined by BCA protein assay (Pierce). Cellular proteins (20–40 μg/well) were mixed with loading dye with a final concentration of 2% SDS and 1% β-mercaptoethanol, incubated for 5 min at 95 °C and resolved on 10% Tris-HCl polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride (PVDF, Millipore) membranes at 24 V overnight at 4 °C. After blocking with 5% non-fat milk in PBS for 1 h, membranes were probed overnight at 4 °C with 1:1000 monoclonal mouse-anti-ABCA1 21  (Neuromics, Minnesota, USA, cat# MO13101), 1:1000 rabbit-anti-AXL (Cell Signaling Technology, cat# 8661), or 1:10,000 anti-GAPDH or anti-β-actin (Millipore) loading controls for 30 min.  Membranes were washed with PBST (1x PBS with 0.05% Tween-20) and then incubated for 1 h with horseradish peroxidase (HRP)-labeled anti-mouse (1:1000 for ABCA1 detection, 1:5000 for GPADH or β-actin detection) or anti-rabbit (1:1000 for AXL and native apoE) secondary antibodies (Jackson Immuno-Research). Results were visualized using chemiluminescence (ECL, Amersham) and blot images were captured with a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad). Band density was quantified using ImageJ software (version 1.47q, National Institutes of Health).   2.6 Cholesterol Efflux Assay CCF-STTG1 cells were seeded at 150,000 cells/well in 24-well plates and cultured for 24 h before labeling for 24 h with 1 μCi/ml of 3H-Cholesterol (PerkinElmer Life Sciences) in growth media supplemented with DMSO, 1 μM TO901317, 1 μM AXL modulator A1. Labeled cells were then washed and equilibrated in serum-free media for 60 min. Serum-free media containing the same drug treatments were then added to the cells in the absence (NA, no acceptor) or presence of 10 μg/ml of exogenous lipid-free apoA-I (a kind gift from CSL Behring, Switzerland). After 24 h at 37 oC, culture media was collected and cells were lysed by addition of 0.1 M NaOH and 0.2% SDS, followed by incubation at room temperature for a minimum of 1 h. Radioactivity in media and cell lysate samples was quantified by scintillation counting (PerkinElmer). The percentage cholesterol efflux was calculated as the total counts per minute (CPM) in the media divided by the sum of the CPM in the media plus in the cell lysate.  22  2.7 Cignal LXR Reporter Luciferase Assay To determine whether the compound affects LXR pathway we used LXRα Cignal Reporter Assay kit (Qiagen) in CCF-STTG1 cells according to the manufacturer instructions. The LXRα reporter system is a mixture of a LXRα-responsive luciferase construct and a constitutively expressing Renilla luciferase construct (40:1) under the transcriptional control of a minimal CMV promoter and tandem repeats of LXRE. CCF-STTG1 cells were transfected with expression vectors containing WT or mutant LXRα without LXRE, followed by treatment with either 1 μM TO901317, 1 μM A1 or 3 μM A1 24 h post transfection. The LXRα activity was monitored by a dual luciferase assay (Promega) 24 h post treatment.  2.8 Immunocytochemistry Immunocytochemistry was conducted using Immunofluorescence application solutions kit (Cell Signaling Technology, cat# 12727). AXL-/- CCF-STTG1 cells were grown to approximately 80% confluency on glass cover slips (ThermoFisher Scientific) in 24-well tissue culture plates and transfected with pcDNA3.1 vectors expressing either WT AXL or AXL-ICD (aa 473-894) using lipofectamine 2000 according to manufacturer instructions (ThermoFisher Scientific) for 24 h. Cells were then washed by PBS and incubated in ice-cold 100% methanol for 10 min at -20 °C. After PBS rinsing for 5 min, cells were blocked in blocking buffer for 1h, followed by incubation with 1:200 rabbit-anti-AXL antibody diluted in antibody dilution buffer overnight at 4 °C. Cells were then washed in PBS 3x for 5 min each, followed by incubation with secondary goat anti-rabbit AF594 (ThermoFisher Scientific, 1:500). Cells were then washed again and mounted in ProLong Diamond antifade mountant with DAPI (ThermoFisher Scientific).  Fluorescent images were acquired with a SP8 confocal microscope (Leica, Canada). 23   2.9 Statistics Statistical analysis was done using randomized block ANOVA with experimental runs as blocks to minimize inter-experimental variation. [132]. For immunoblot analysis, raw densitometry data (target protein value over loading control protein value) were first log transformed and then analyzed by a blocked two-way (“Experiment” and “Drug” as the two factors) ANOVA model with “Experiment” being the blocking factor and with a Dunnett’s multiple comparison post-test (i.e. each drug condition compared to vehicle control). For qRT-PCR analysis, ΔCT values (target gene CT minus reference gene CT) were used in the same blocked two-way ANOVA model. For cholesterol efflux and MEF LXR-dependency experiments, a blocked three-way (“Drug”, “Experiment” and “ApoA-1 treatment/genotype” as the three factors) ANOVA with “Experiment” as the blocking factor and with Sidak's multiple-comparison tests was used to compare either the test compounds’ effect over vehicle control within each genotype/treatment condition or the effect of genotype/treatment themselves under each test compound condition.   For immunoblot and qRT-PCR results, data are plotted as mean fold-change over vehicle control ± 95% confidence interval (calculated from the aforementioned ANOVA analysis) of the indicated number of independent experiments. For ELISA, cholesterol efflux and LXR luciferase assay results, data are presented as mean measurement ± standard deviation from the indicated number of experiments. All statistical analyses were performed using SPSS (version 23) and P-values < 0.05 were considered significant. Prism 5 (GraphPad Software) was used to graph all data. 24  Chapter 3: Results  3.1 Compound A1 upregulates apoE and ABCA1 expression Compound A1 was identified through a high-throughput phenotypic screen performed at AstraZeneca using human CCF-STTG1 astrocytoma cells to identify modulators of apoE secretion and further validated in our laboratory. The compound was able to significantly induce apoE and ABCA1 expression at both mRNA and protein levels at 3 μM, the maximum effective dose after 72 h of treatment (Figure 3.1).   Figure 3.1. Compound A1 upregulates apoE and ABCA1 expression in CCF-STTG1 cells. (A) Half-log concentration response curve (0.03 – 30 μM) of secreted apoE by CCF-STTG1 cells treated with A1 for 72 h. Data are expressed as % apoE secretion relative to DMSO (0%) and 1 μM of the positive control LXR agonist TO901317 (100%). Error bars represent mean +/- range of duplicate wells in one representative assay. (B) ApoE and (C) ABCA1 mRNA levels were measured by qRT-PCR. (C) Cellular apoE and (D) ABCA1 protein levels were measured by immunoblot in CCF-STTG1 cells after 72 h treatment with vehicle control DMSO, 1 μM TO901317, 3 μM A1. Graphs represent fold-change over DMSO control (dashed line) and +/- 95% confidence intervals from N independent experiments indicated in brackets. ** p < 0.01, *** p < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests. (F) Representative immunoblot of apoE and ABCA1 expression upon drug treatment. Actin was used as housekeeping loading control. 25   To determine whether increased ABCA1 expression translates into increased overall ABCA1 activity, we assessed the effect of A1 on cholesterol efflux activity to apoA-I, the classical assay of ABCA1 activity. The positive control compound TO901317, which induces both apoE and ABCA1 expression, significantly increased cholesterol efflux both in the presence or absence of apoA-I. By contrast, A1 significantly enhanced cholesterol efflux compared to the DMSO vehicle control only in the presence of exogenous apoA-I relative to TO901317 at 1 μM suboptimal dose (Figure 3.2A). As lipidated apoE has been shown to be beneficial, we next assessed whether overall apoE lipidation was affected by drug treatment. Drug treated conditioned media were resolved on non-denaturing Tris gels to reveal the particle size distribution of the apoE-containing lipoproteins secreted by CCF-STTG1 cells. As expected and similar to TO901317, A1 increased the amount of the HDL-like sized apoE particles ranging from ~ 7 to 17 nm in diameter (Figure 3.2B). These results demonstrate that A1 increases ABCA1 expression and activity in CCF-STTG1 cells and elevate particles that resemble native lipidated apoE.    26   Figure 3.2. Compounds A1 enhances ABCA1 activity. (A) CCF-STTG1 cells were labeled with 3H-cholesterol followed by treatment of DMSO, 1 μM TO901317, 3 μM A1 for 24 h. Cholesterol efflux over 24 h in the absence (NA) or presence of 10 μg/ml of lipid-free apoA-I along with the above drug treatment was evaluated. Graphs represent mean % efflux and SD of three independent experiments. * p < 0.05, *** p < 0.001 comparing drug effect over respective DMSO control; ### p < 0.001 comparing between NA vs. apoA-I within each drug condition by blocked three-way ANOVA post-hoc tests. (B) Particle size distribution of apoE-containing lipoproteins in the unconcentrated 72 h-conditioned media from drug-treated CCF-STTG1 were assessed by 6% native PAGE followed by immunoblotting for apoE. Ladder on the left represents Stokes diameter.   As microglia cells are also major apoE secretors in human CNS, we tested A1 in HMC3 cells acquired from ATCC, which are immortalized human microglia. As expected, secreted apoE and ABCA1 expression were also increased. As HMC3 and CCF-STTG1 cells are immortalized human cell lines, we next confirmed the drug activity of A1 in more physiologically relevant primary human astrocytes acquired from ScienCell. As shown in Figure 3.3, 3 μM A1 was able to significantly upregulate secreted apoE and ABCA1 expression after 72 h treatment in all cell types. Note that in primary human astrocytes and HMC3 cells, the LXR agonist, TO901317, failed to upregulate apoE secretion whereas the A1 effect on apoE was significant, demonstrating potentially differential MoA of apoE regulation by A1. Also, it is worth noting that while TO901317 is more robust of inducing ABCA1 than apoE expression, A1 has opposite effect, i.e. stronger apoE induction than ABCA1.  27   Figure 3.3. A1 upregulates apoE secretion and ABCA1 expression in HMC3 and primary human astrocyte. (A-C) Secreted apoE levels were measured by apoE ELISA after 72 h treatment with vehicle control DMSO, 1 μM TO901317 or 3 μM A1 in indicated cell types. (D-F) ABCA1 protein levels were measured by (G-I) immunoblot in CCF-STTG1 cells after 72 h treatment with vehicle control DMSO, 1 μM TO901317 or 3 μM A1. Graphs represent fold-change over DMSO control (dashed line) and +/- 95% confidence intervals from N independent experiments indicated in brackets. ** p < 0.01, *** p < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests.  We also examined the compound effect on primary mouse mixed glia cells cultured from wild-type C57BL/6 mice. Again, A1 was able to significantly induce apoE and ABCA1 expression (Figure 3.4). Overall these data demonstrate that A1 has robust apoE and ABCA1 activities across different major CNS cell types that secrete apoE. 28   Figure 3.4. A1 upregulates apoE and ABCA1 expression in WT mouse primary mixed glia. (A) Secreted apoE levels, (B) cellular apoE and (C) ABCA1 levels were measured after 96 h treatment with vehicle control DMSO, 1 μM TO901317 or 3 μM A1. Graphs represent fold-change over DMSO control (dashed line) and +/- 95% confidence intervals from N independent experiments indicated in brackets. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests.  3.2 Compound A1 does not activate the LXR pathway As both apoE and ABCA1 are LXR target genes, we next tested whether A1 requires activation of LXR pathway by two methods. First, immortalized mouse embryonic fibroblasts (MEFs) that lack both LXRα and LXRβ expression and isogenic MEFs that were reconstituted with LXRα were used to determine whether A1-induced upregulation of apoE and ABCA1 expression requires LXR activity. As expected, TO901317 failed to induce apoE and ABCA1 expression in LXRα and LXRβ double knockout MEFs. Intriguingly, A1-induced apoE and ABCA1 activity still remained when LXR is absent, demonstrating LXR-independent drug activity (Figure 3.5). 29   Figure 3.5. A1 does not require LXR pathway to upregulate apoE and ABCA1 expression. LXR-knockout (LXRα-/β-) and LXRα-expressing (LXRα+/β-) MEF cells were treated with DMSO, 1 μM TO901317 or 3 μM A1 for 48 h. ApoE and ABCA1 mRNA levels were measured by qRT-PCR. Graph represents fold-change over respective DMSO control (dash line) +/- 95% CI from 5 experiments. ** p < 0.01, *** p < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests.  Second, we confirmed the lack of involvement of the LXR pathway using a LXR luciferase reporter assay. Briefly, vehicle control, TO901317 or A1 was added to CCF-STTG1 cells transfected with a mixture of LXR-responsive Firefly luciferase construct and constitutively expressing Renilla luciferase construct as internal control. Luciferase activities were measured after 24 h of treatment. While TO901317 exhibited strong normalized Firefly activity at 1 μM, A1 showed no activity at 3 μM (Figure 3.6), clearly demonstrating again that this compound does not alter LXR pathway.  30   Figure 3.6. A1 does not activate LXR pathway. (A) Demonstration of dual-luciferase constructs. Briefly, Firefly luciferase is under the control of tandem repeats of LXRE, which is not present in negative control. Renilla luciferase is under the control of CMV enhancer/promoter and constitutively expressed. (B) DMSO vehicle control, 1 μM TO901317 or 3 μM A1 was added to CCF-STTG1 cells transfected with dual luciferase constructs. Luciferase activities were measured after 24h of treatment. Error bars represent standard deviation from technical triplicates. **** p < 0.0001 by ANOVA post-hoc analysis.   Taken together, A1 does not directly bind and agonize LXR receptor, nor does it require LXR activity to modulate apoE and ABCA1, suggesting the involvement of other novel signaling pathways.  3.3 Compound A1 has no or minimal SREBP-1c induction in liver cells The major liability of LXR agonists is lipogenesis and hepatic steatosis caused by induction of the LXR target SREBP-1c. Importantly, A1 did not upregulate SREBP-1c in HepG2 hepatoma cells (Figure 3.7A) and led to minimal SREBP-1c induction in primary human hepatocytes (Figure 3.7B), suggesting that A1 may avoid the hepatotoxicity of direct LXR agonists. 31   Figure 3.7. No or minimal SREBP-1c induction by compound A1 in liver cells. (A) HepG2 cells and (B) primary human hepatocytes (hHepatocyte) cells were treated with DMSO, 1μM TO901317 or 3μM A1 for 48 h. SREBP-1c mRNA levels were measured by real-time qPCR. Data are expressed as fold change (mean +/- 95% confidence intervals from 3 independent experiments) relative to DMSO control treatment (dashed line). *** p < 0.001 compared to DMSO by blocked two-way ANOVA post-hoc tests.  3.4 Compound A1 requires AXL to upregulate apoE expression The novel compound A1 is from a family of pyrazine derivatives recognized as inhibitors of AXL kinase discovered by AstraZeneca. To determine whether apoE and ABCA1 activity of A1 requires either AXL, we assessed AXL knockout CCF-STTG1 cells generated by the CRISPR/Cas9 method that were confirmed to have no detectable AXL protein (Figure 3.8A) and compared with parental WT CCF-STTG1 cells. While TO901317 still significantly upregulated apoE expression across cell types, A1’s activity was completely abolished in AXL-/- CCF-STTG1 cells, supporting AXL as the drug target (Figure 3.8B). 32   Figure 3.8. A1 requires AXL to upregulate apoE expression. (A) Representative immunoblot showing that AXL expression is abolished in AXL-/- CCF-STTG1 cells. (B) Secreted apoE levels were measured after 72 h treatment with vehicle control DMSO, 1 μM TO901317 or 3 μM A1. Graphs represent fold-change over respective DMSO control (dashed line) and +/- 95% confidence intervals from N independent experiments indicated in brackets. *** p < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests.  3.5 AXL RTK plays a role in regulating apoE baseline expression in CCF-STTG1 cells While performing the target validation in WT and AXL-/- CCF-STTG1 cells, we noticed that there was a significant reduction of apoE expression baseline at both secreted protein and mRNA levels in AXL-/- CCF-STTG1 cells. To validate the effect of depletion of AXL on apoE baseline levels, siRNA-mediated AXL knockdown was performed. Two siRNAs targeting AXL mRNA with different sequences were used. As shown in Figure 3.9, when AXL was efficiently knocked down by siRNAs, baseline apoE expression was also significantly reduced, mimicking the phenotype of AXL-/- CCF-STT1 cells. These results strongly suggest that AXL protein plays a role in regulating apoE baseline expression in CCF-STTG1 cells. 33   Figure 3.9. AXL knockdown downregulates apoE expression. AXL protein (A), mRNA (B) and APOE mRNA levels were measured by immunoblotting and RT-qPCR after siRNA treatments in CCF-STTG1 cells. Graphs represent fold-change over respective siCtrl (dashed line) and +/- 95% confidence intervals from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to siCtrl using blocked two-way ANOVA post-hoc tests.  3.5.1 Abolishment of Axl expression in mouse does not change brain apoE level We next assessed whether global abolishment of Axl expression in mice affects apoE expression. We measured brain apoE levels in WT and Axl-/- C57BL/6 mice and found that lack of Axl expression does not alter either cortical brain Apoe mRNA level nor CSF apoE level (Figure 3.10).    34   Figure 3.10. Lack of Axl expression does not alter mouse brain apoE level. (A) Cerebral cortex was harvested from WT and Axl-/- C57BL/6 mice and apoe mRNA levels were measured by qRT-PCR. Graph represent expression ratio of Apoe normalized against housekeeping control. Error bars represent standard deviation. (B) Cerebral spinal fluid was harvested followed by measurement of apoE and control albumin protein levels. A.U. = arbitrary unit. Error bars represent standard deviation.  3.6 Compound A1 upregulates AXL intracellular domain expression Given that A1 upregulates apoE via an AXL-dependent pathway, we next proceeded to investigate the MoA and started by assessing how AXL protein level responds to A1 treatment. Intriguingly, in addition to the downregulation of AXL protein expression, we also detected a ~50 kDa band after 72 h A1 treatment in CCF-STTG1 cells (Figure 3.11A).   It is shown that AXL RTK can be cleaved by α-secretase to generate a 55 kDa C-terminal fragment (CTF). CTF can then be cleaved by γ-secretase to generate the AXL intracellular domain (ICD) which is 52 kDa (Figure 3.11B) [133]. Therefore, we hypothesized that the ~50 kDa band we observed is a cleavage product of AXL RTK. To confirm whether this cleavage product is CTF or ICD, we treated CCF-STTG1 cells with 1 μM compound E, a γ-secretase inhibitor, to block the cleavage of CTF to ICD [133]. Compound E treatment resulted in the 35  accumulation of CTF, which appeared to be larger than the band observed with A1 treatment suggesting that A1 increased the level of ICD (Figure 3.11A).   Figure 3.11. A1 increases AXL-ICD expression. (A) CCF-STTG1 cells were treated with DMSO vehicle control, 3 μM A1 or 1 μM compound E for 72 h. Immunoblotting was performed to probe AXL protein. (B) Demonstration of AXL RTK cleavage process [134].  To rule out the possibility of A1 increasing AXL-ICD expression is specific to CCF-STTG1 cells, we also treated HMC3, primary human astrocytes as well as MEF cells and observed similar results, suggesting A1’s ICD effect is not cell type specific.   Figure 3.12. Compound A1 upregulates AXL-ICD expression in other cell types. CCF-STTG1, primary human astrocytes, and HMC3 (A) as well as MEF cells (B) were treated by A1 for 72 h. Immunoblotting was performed to probe AXL protein.  36  3.7 AXL-ICD regulates apoE baseline expression Given that compound A1 increases AXL-ICD level and promotes apoE expression, we hypothesized that AXL-ICD is the driving force for apoE upregulation. To investigate the role of AXL-ICD in apoE homeostasis, various AXL variants, including WT AXL, kinase-dead AXL mutant (K567R), AXL-ICD (aa 473-894), and AXL NTF (aa 1-451) were stably reconstituted in AXL-/- CCF-STTG1 cells by AstraZeneca. We then compared the basal apoE secretion and mRNA levels across different reconstituted lines. Compared to empty vector, the negative control, K567R AXL – reconstituted line had higher basal apoE expression, suggesting that AXL kinase activity is dispensable for regulating apoE expression. Similarly, WT AXL and ICD – reconstituted lines also elevated apoE expression, confirming the critical role of AXL non-canonical signaling in apoE homeostasis. On the contrary, AXL-NTF – reconstituted line did not lead to elevated apoE levels. Taken together, these results strongly indicate that AXL regulates apoE expression via a kinase independent but ICD dependent pathway.  Figure 3.13. Reconstitution of AXL in AXL-/- CCF-STTG1 elevated apoE baseline expression. (A) Representative immunoblot showing reconstitution efficiency of empty vector (EV), intracellular domain (ICD), full-length (FL) and K567R AXL variants into AXL-/- CCF-STTG1 cells. (B) Secreted apoE levels from WT, AXL KO and reconstituted cell lines were measured by apoE ELSIA 72 h post-seeding.  37  3.8 AXL-ICD localizes in nucleus It has been hypothesized that AXL-ICD may translocate to cell nucleus upon production and regulate gene expression [135]. To determine if AXL-ICD localizes in the nucleus, we transfected AXL-/- CCF-STTG1 cells with WT AXL or AXL-ICD, followed by performing immunofluorescence staining for AXL. Indeed, compared to WT AXL, AXL-ICD predominantly localizes in nucleus.   Figure 3.14. AXL-ICD predominantly localizes in nucleus. AXL-/- CCF-STTG1 was transfected with pcDNA3.1 mammalian vector overexpressing either WT AXL or AXL-ICD for 24 h, followed by confocal immunofluorescence analysis.  Scale bar = 5μm. 38  Chapter 4: Discussion Rapid growth of the aging population coupled with high failure rate in AD drug development, which has primarily targeted the amyloid pathway, has resulted in an urgent need to discover alternative therapeutic AD approaches. ApoE plays an important role in AD pathogenesis as it is the most established primary genetic risk factor for LOAD and therefore is one of the most important AD targets to understand. APOE-ε4 carriers have reduced apoE levels and degree of lipidation, accelerated amyloid deposition and up to 14.9 times greater risk of developing AD [136]. In addition, ABCA1 functional capacity may be impaired with neuropathology and cognitive impairment because ABCA1-mediated cholesterol efflux activity is reduced by 30% in patients with mild cognitive impairment and AD patients compared to healthy controls [137]. Therefore, boosting ABCA1 activity may provide therapeutic benefits. Indeed, one recent study has found that an apoE-mimic peptide (CS-6253) increased ABCA1 protein levels leading to increased apoE4 lipidation and improved cognitive function in apoE4-TR mice [138]. Studies in AD mouse models have also revealed that increasing the levels of functional lipidated apoE by synthetic LXR agonists can alleviate amyloid pathology and improve cognitive functions [69], [71], [139]. However, all LXR agonists have hepatotoxicity adverse effects, which are predicted to be more severe in humans compared to mice and preclude them from being evaluated in human clinical trials.   Using a high-throughput phenotypic screen in CCF-STTG1 astrocytoma cells, we have identified a class of compounds known as AXL modulators that are capable of increasing the levels of secreted lipidated apoE. My thesis work focused on A1, which is a representative compound from this class. CCF-STTG1 cells have the genotype of ε3/ε4 while apoE4 only represents <25% 39  of the total secreted apoE pool [136]. ApoE4 is suggested to be more prone to degradation in vivo, which may partly explain its lower abundance in soluble brain lysates. Human apoE-TR mice studies also revealed that apoE4-TR mice not only have lower apoE levels and higher Aβ levels compared to apoE2- and apoE3-TR mice, but also less lipidated apoE [140]. Increasing apoE lipidation, particularly for apoE4, has been raised as a potential therapeutic strategy to enhance amyloid clearance [139]. Notably, compound A1 also increases ABCA1 expression, which translates to enhanced overall ABCA1 activity determined by cholesterol efflux functional assay. ABCA1 plays an essential role in lipidating apoE and indeed, secreted apoE particles after A1 treatment resemble native apoE particles in size shown by native blot. We also confirmed A1’s apoE and ABCA1 activity in primary human astrocytes and a human microglia cell line, HMC3, where we observed a robust induction of both apoE and ABCA1 expression, suggesting the drug effect is not specific to astrocytoma cells. We also isolated primary glial cells from WT C56BL/6 mice and similarly, A1 robustly increased secreted apoE levels and cellular ABCA1 expression, which provides solid support for potential future preclinical mouse studies.  Next, we determined whether the LXR pathway is needed for A1 to exert its drug response. First, we utilized MEF cells that lack both LXRα and LXRβ expression and isogenic MEF cells that were reconstituted with LXRα. Intriguingly, A1 is able to significantly increase apoE mRNA expression regardless of the genotype, strongly indicating A1 does not require LXR to exert drug effect.  Also, the fact that A1’s apoE induction is similar in both MEF cells suggests that the presence of LXR pathway does not further modify drug effect. Indeed, the dual luciferase reporter assay in CCF-STTG1 cells revealed that A1 does not alter LXR pathway. Taken together, A1 increases apoE expression via a completely LXR-independent pathway. Since LXR 40  nuclear receptor is traditionally considered as the master regulator of apoE expression, the discovery of A1 has indicated that apoE can be independently regulated by alternative novel pathways. In fact, another recent study has shown that apoE can also be regulated by pan class I histone deacetylases independent of  LXR activation [141]. Taken together, regulation of apoE expression is far from simple.   Even though A1 raises apoE through an LXR-independent pathway, we examined potential liver adverse effects by measuring SREBP-1c response in HepG2 and primary hepatocyte cells after A1 treatment. Indeed, since LXR is not the drug target, we observed no or minimal SREBP-1c activity. However, further mouse studies are required to determine if A1 has any adverse effects including hepatotoxicity.   AXL RTK is the putative target of compound A1. It is a phagocytosis receptor that mediates important cellular functions. To validate AXL being the drug target, we generated AXL-/- CCF-STTG1 cells by CRISPR-Cas9 method. As expected, knockout of AXL completely abolished induction of apoE by A1, clearly supporting AXL being the drug target. However, lack of AXL also unexpectedly decreased apoE baseline expression dramatically, which mimics the effect of knocking down AXL using siAXL interference. Thus, here we report that AXL plays a role in regulating apoE baseline expression. As a side note, Axl-deficient mice displayed no apoE expression difference in brain from WT background potentially due to species difference or possible compensations. It is also possible that astrocytes in vivo may behave differently and not manifest AXL-apoE axis as observed in vitro. In addition, whether loss of AXL in other apoE-secreting cell types such as microglia and pericyte can lead to similar reduction of apoE baseline 41  expression as in astrocytes still need to be investigated. The total apoE pool in brain may not drastically change if only apoE secretion from astrocytes is reduced.    That A1 increases apoE expression while knocking out or knocking down AXL decreases apoE expression raises the obvious question that how AXL regulates apoE expression. We first examined the AXL protein levels after A1 treatment. Surprisingly, immunoblotting clearly revealed the presence of AXL intracellular domain fragment upon A1 treatment. There is mounting evidence that AXL RTK can be cleaved [133], [135], [142]. Specifically, similar to Notch signaling, AXL can be first cleaved by α-secretase to generate soluble AXL (sAXL) or NTF and CTF, which can undergo further cleavage by γ-secretase to give rise to the ICD. It is not clear to us why A1 is able to increases AXL-ICD level. Since A1 was originally identified as a potent AXL kinase inhibitor, it is possible that inhibiting kinase activity accelerates AXL cleavage [133]. A1 could also bind to ICD and stabilize it, increasing its half-life. Further experiments such as thermal-shifting assay could elucidate A1’s effect on ICD. Hypothetically it is also possible that A1 enhances α-secretase activity and therefore causes elevated ICD levels. However, recent experiments performed by AstraZeneca have revealed that co-treatment of A1 and α-secretase inhibitor, GI254023X, did not affect the levels of ICD (data not shown), suggesting that A1 elevates ICD expression not via altered α-secretase activity.  It has been proposed that AXL-ICD can be quickly turned over, yet may still have important cellular functions. In addition, AXL-ICD may translocate into nucleus to regulate specific gene expression [133]. Indeed, a recent study has identified the nuclear localization signal (NLS) 42  sequence present in ICD and its entrance to nucleus can be impaired by mutating or deleting the NLS [135]. However, the downstream targets of AXL-ICD remain unknown.  My thesis work also confirmed the nuclear localization of ICD by reconstituting AXL-ICD into AXL-/- CCF-STTG1 cells followed by confocal immunofluorescence. Since A1’s ICD effect is not limited to CCF-STTG1 cells but preserved in all other cell types examined, we hypothesized that AXL-ICD may regulate apoE expression. To study the hypothesis, we reconstituted multiple AXL variants into AXL-/- CCF-STTG1 cells by stable transfection and found that except for NTF, all other reconstitutions of ICD-containing AXL variants drastically rescued apoE baseline expression both at mRNA and secreted levels. It is worth noting that we also generated the K567R AXL mutant line, which abrogates AXL kinase activity [143]–[145]. ApoE expression was again significantly elevated, suggesting the dispensable role of AXL kinase activity in apoE homeostasis. Of note, all reconstituted cell lines are polyclonal pools and this heterogeneous property hinders the direct quantitative comparison between the levels of apoE baseline and ICD across different cell lines.   Taken together, it is likely that A1 upregulates apoE expression not via its kinase inhibition but due to its ability to promote AXL-ICD level, which is consistent with the fact that knocking out or knocking down AXL dramatically decreases apoE baseline expression as there is less AXL-ICD.  The role of AXL in neurodegeneration, specifically with respect to microglia function, has been increasingly gaining attention. The levels of AXL has been indicated in several clinical studies to 43  be potential biomarkers for brain damage [124]–[126]. Recent studies have also identified AXL being one of the markers in activated or disease-associated microglia (DAM) along with apoE [128]. My thesis work provides the first evidence to support AXL – apoE crosstalk.  44  Chapter 5: Conclusions Using a novel phenotypic screening strategy, we have discovered that AXL-ICD regulates apoE expression independent of LXR activation. This thesis work has validated AXL modulator A1’s apoE and ABCA1 activity, its LXR independency, and provided preliminary insights into the role of AXL-ICD in apoE homeostasis (Figure 5.1). Given the essential role of apoE as the major lipid transporter in brain and the importance of AXL in neurodegeneration as well as microglia function, manipulation of apoE via modulating AXL could provide novel therapeutic benefits.   Figure 5.1. Proposed mechanism of apoE regulation by AXL-ICD. AXL RTK can undergo sequential cleavages performed by α- and γ- secretases to release ICD, which translocates into nucleus to regulate apoE expression.   45  5.1 Future directions Further experiments are required to elucidate the MoA of AXL modulators and validate AXL-ICD being the driving force for apoE expression: (1). Determine the MoA by which A1 increases AXL-ICD level. My thesis work has provided solid evidence that A1 increases AXL-ICD level. However, whether these elevated ICD levels are due to increased full-length AXL cleavage or stabilized ICD remains unknown. It is not uncommon for a chemical molecule to bind and stabilize the target protein. In fact, a recent study has utilized a macrocyclic inhibitor to stabilize AXL kinase domain for X-ray crystallography [146]. To determine if A1 indeed stabilizes AXL-ICD, a thermal stability experiment could be performed. (2). To further validate the nuclear role of AXL-ICD, an AXL-/- CCF-STTG1 line harboring NLS-mutated ICD could be generated. If apoE is indeed regulated by nuclear AXL-ICD, this line should have reduced apoE baseline expression.  (3).  Since AXL-ICD is hypothesized to drive apoE expression, one obvious question would be to determine if the ICD directly binds to specific DNA sequences by performing chromatin immunoprecipitation, followed by DNA sequencing or RT-PCR to determine if AXL-ICD directly binds to APOE promoter. (4). AXL-ICD could also interact with other protein players and regulate apoE expression indirectly. To identify potential protein binding partners of ICD, immunoprecipitation could be carried out to pull down ICD fragments in ICD-reconstituted AXL-/- CCF-STTG1 cells, followed by mass spectrometry to determine what other proteins are associated with AXL-ICD. 46  (5). Given the emerging role of AXL in microglia function, evaluating the global effect of AXL-ICD could provide more insights into AXL and apoE signaling by performing whole transcriptome sequencing on AXL-/- CCF-STTG1 reconstituted with or without AXL-ICD.  In addition to further mechanistic studies, it is also important to determine whether compound A1 confers isoform-specific apoE effects through in vitro and in vivo studies with the following possible future aims: (1).  Determine if A1 leads to isoform-dependent apoE changes in iPSC-derived human astrocytes with known APOE alleles (APOE-ε3/ε3 vs. APOE-ε4/ε4) and primary murine astrocytes derived from wild-type and APOE2, APOE3, and APOE4 targeted-replacement (TR) mice. (2).  Determine if A1 affects apoE’s ability to modulate Aβ-induced activation of cerebrovascular endothelial cells (EC) in a translational 3D model of bioengineered cerebral vessels composed of barrier-forming EC, smooth muscle cells (SMC), and iPSC-derived human astrocytes with known APOE genotype previously established in our laboratory. (3). Determine if intracerebroventricular administration of A1 leads to acute changes to brain apoE, ABCA1 and amyloid levels in apoE3- and apoE4-TR AD mice.   5.2 Limitations and caveats The major limitation of this thesis work is that the hypothesis was only studied in vitro on either primary cells or immortalized cell lines. 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