Risk Factor For Late-onset Alzheimer's Disease Research Articles

Independent APOE4 knock-in mouse models display reduced brain APOE protein, altered neuroinflammation, and simplification of dendritic spines.

APOE is an immunomodulator in the brain and the major genetic risk factor for late-onset Alzheimer's disease (AD). Targeted replacement APOE mice (APOE-TR) have been a useful tool to study the effects of APOE isoforms on brain neurochemistry and activity prior to and during AD. We use newly available APOE knock-in mice (JAX-APOE) to compare phenotypes associated with APOE4 across models. Similar to APOE4-TR mice, JAX-E4 mouse brains showed 27% lower levels of APOE protein compared with JAX-E3 (p < 0.001). We analyzed several neuroinflammatory molecules that have been associated with APOE genotype. SerpinA3 was much higher in APOE4-TR mice to APOE3-TR mice, but this effect was not seen in JAX-APOE mice. There were higher levels of IL-3 in JAX-E4 brains compared with JAX-E3, but other neuroinflammatory markers (IL6, TNFα) were not affected by APOE genotype. In terms of neuronal structure, basal dendritic spine density in the entorhinal cortex was 39% lower in JAX-E4 mice compared with JAX-E3 mice (p < 0.001), again similar to APOE-TR mice. One-week treatment with ibuprofen significantly increased dendritic spine density in the JAX-E4 mice, consistent with our previous finding in APOE-TR mice. Behaviorally, there was no effect of APOE genotype on Barnes Maze learning and memory in 6-month-old JAX-APOE mice. Overall, the experiments performed in JAX-APOE mice validated findings from APOE-TR mice, identifying particularly strong effects of APOE4 genotype on lower APOE protein levels and simplified neuron structure. These data demonstrate pathways that could promote susceptibility of APOE4 brains to AD pathological changes.

Genetics of Alzheimer Disease.

This article discusses the spectrum of genetic risk in familial and sporadic forms of early- and late-onset Alzheimer disease (AD). Recent work illuminating the complex genetic architecture of AD is discussed in the context of high and low risk and what is known in different populations. A small proportion of AD is autosomal dominant familial AD caused by variants in PSEN1, PSEN2, or APP, although more recently described rare genetic changes can also increase risk substantially over the general population, with odds ratios estimated at 2 to 4. APOE remains the strongest genetic risk factor for late-onset AD, and understanding the biology of APOE has yielded mechanistic insights and leads for therapeutic interventions. Genome-wide studies enabled by rapidly developing technologic advances in sequencing have identified numerous risk factors that have a low impact on risk but are widely shared throughout the population and involve a repertoire of cell pathways, again shining light on potential paths to intervention. Population studies aimed at defining and stratifying genetic AD risk have been informative, although they are not yet widely applicable clinically because the studies were not performed in people with diverse ancestry and ethnicity and thus population-wide data are lacking. The value of genetic information to practitioners in the clinic is distinct from information sought by researchers looking to identify novel therapeutic targets. It is possible to envision a future in which genetic stratification joins other biomarkers to facilitate therapeutic choices and inform prognosis. Genetics already has transformed our understanding of AD pathogenesis and will, no doubt, continue to reveal the complexity of brain biology in health and disease.

BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia

BackgroundThe BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth.MethodsMicroglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches.ResultsHere, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3.ConclusionsOur convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.Graphical

Endogenous and Exogenous Estrogen Exposures: How Women's Reproductive Health Can Drive Brain Aging and Inform Alzheimer's Prevention.

After advanced age, female sex is the major risk factor for late-onset Alzheimer’s disease (AD), the most common cause of dementia affecting over 24 million people worldwide. The prevalence of AD is higher in women than in men, with postmenopausal women accounting for over 60% of all those affected. While most research has focused on gender-combined risk, emerging data indicate sex and gender differences in AD pathophysiology, onset, and progression, which may help account for the higher prevalence in women. Notably, AD-related brain changes develop during a 10–20 year prodromal phase originating in midlife, thus proximate with the hormonal transitions of endocrine aging characteristic of the menopause transition in women. Preclinical evidence for neuroprotective effects of gonadal sex steroid hormones, especially 17β-estradiol, strongly argue for associations between female fertility, reproductive history, and AD risk. The level of gonadal hormones to which the female brain is exposed changes considerably across the lifespan, with relevance to AD risk. However, the neurobiological consequences of hormonal fluctuations, as well as that of hormone therapies, are yet to be fully understood. Epidemiological studies have yielded contrasting results of protective, deleterious and null effects of estrogen exposure on dementia risk. In contrast, brain imaging studies provide encouraging evidence for positive associations between greater cumulative lifetime estrogen exposure and lower AD risk in women, whereas estrogen deprivation is associated with negative consequences on brain structure, function, and biochemistry. Herein, we review the existing literature and evaluate the strength of observed associations between female-specific reproductive health factors and AD risk in women, with a focus on the role of endogenous and exogenous estrogen exposures as a key underlying mechanism. Chief among these variables are reproductive lifespan, menopause status, type of menopause (spontaneous vs. induced), number of pregnancies, and exposure to hormonal therapy, including hormonal contraceptives, hormonal therapy for menopause, and anti-estrogen treatment. As aging is the greatest risk factor for AD followed by female sex, understanding sex-specific biological pathways through which reproductive history modulates brain aging is crucial to inform preventative and therapeutic strategies for AD.

APOEɛ4 Allele Moderates the Association Between Basal Forebrain Nuclei Volumes and Allocentric Navigation in Older Adults Without Dementia.

Cholinergic deficit and medial temporal lobe (MTL) atrophy are hallmarks of Alzheimer's disease (AD) leading to early allocentric spatial navigation (aSN) impairment. APOEɛ4 allele (E4) is a major genetic risk factor for late-onset AD and contributes to cholinergic dysfunction. Basal forebrain (BF) nuclei, the major source of acetylcholine, project into multiple brain regions and, along with MTL and prefrontal cortex (PFC), are involved in aSN processing. We aimed to determine different contributions of individual BF nuclei atrophy to aSN in E4 positive and E4 negative older adults without dementia and assess whether they operate on aSN through MTL and PFC or independently from these structures. 120 participants (60 E4 positive, 60 E4 negative) from the Czech Brain Aging Study underwent structural MRI and aSN testing in real-space arena setting. Hippocampal and BF nuclei volumes and entorhinal cortex and PFC thickness were obtained. Associations between brain regions involved in aSN were assessed using MANOVA and complex model of mutual relationships was built using structural equation modelling (SEM). Path analysis based on SEM modeling revealed that BF Ch1-2, Ch4p, and Ch4ai nuclei volumes were indirectly associated with aSN performance through MTL (pch1-2 = 0.039; pch4p = 0.042) and PFC (pch4ai = 0.044). In the E4 negative group, aSN was indirectly associated with Ch1-2 nuclei volumes (p = 0.015), while in the E4 positive group, there was indirect effect of Ch4p nucleus (p = 0.035). Our findings suggest that in older adults without dementia, BF nuclei affect aSN processing indirectly, through MTL and PFC, and that APOE E4 moderates these associations.

The neuronal-specific isoform of BIN1 regulates \u03b2-secretase cleavage of APP and A\u03b2 generation in a RIN3-dependent manner

Genome-wide association studies have identified BIN1 (Bridging integrator 1) and RIN3 (Ras and Rab interactor 3) as genetic risk factors for late-onset Alzheimer’s disease (LOAD). The neuronal isoform of BIN1 (BIN1V1), but not the non-neuronal isoform (BIN1V9), has been shown to regulate tau-pathology and Aβ generation via RAB5-mediated endocytosis in neurons. BIN1 directly interacts with RIN3 to initiate RAB5-mediated endocytosis, which is essential for β-secretase (BACE1)-mediated β-secretase cleavage of β-amyloid precursor protein (APP) to generate Amyloid-β (Aβ), the key component of senile plaques in AD. Understanding the regulatory roles of BIN1 (neuronal BIN1V1) and RIN3 in β-secretase mediated cleavage of APP and Aβ generation is key to developing novel therapeutics to delay or prevent AD progression. Neuronal and non-neuronal isoforms of BIN1 (BIN1V1 and BIN1V9, respectively) were introduced with RIN3 into an in vitro cell-based system to test RIN3-dependent effects of neuronal BIN1V1 and non-neuronal BIN1V9 on β-secretase-mediated cleavage of APP and Aβ generation. Confocal microscopy was performed to examine RIN3-dependent subcellular localization of BIN1V1 and BIN1V9. Western blot analysis was performed to assess the effects of RIN3 and BIN1V1/BIN1V9 on β-secretase mediated processing of APP. We enriched cells expressing BIN1V1 without or with RIN3 via FACS to measure Aβ generation using Aβ ELISA assay, and to evaluate APP internalization by chasing biotinylated or antibody-labeled cell surface APP. Neuronal BIN1V1 containing the CLAP domain and non-neuronal BIN1V9 lacking the CLAP domain are the major isoforms present in the brain. Employing confocal microscopy, we showed that RIN3 differentially regulates the recruitment of both BIN1V1 and BIN1V9 into RAB5-endosomes. We further showed that BIN1V1, but not BIN1V9, downregulates β-secretase (BACE1)-mediated processing of APP in a RIN3-dependent manner. Overexpression of BIN1V1 also attenuated Aβ generation in a RIN3-dependent manner. Using cell-based internalization assays, we show BIN1V1, but not BIN1V9, delays the endocytosis of APP, but not of BACE1, into early endosomes, thereby spatially and temporally separating these two proteins into different cellular compartments, resulting in reduced cleavage of APP by BACE1 and reduced Aβ generation—all in a RIN3-dependent manner. Finally, we show that RIN3 sequesters BIN1V1 in RAB5-positive early endosomes, likely via the CLAP-domain, resulting in attenuated β-secretase processing of APP and Aβ generation by delaying endocytosis of APP. Our findings provide new mechanistic data on how two AD-associated molecules, RIN3 and BIN1 (neuronal BIN1V1), interact to govern Aβ production, implicating these two proteins as potential therapeutic targets for the prevention and treatment of AD.

Aging, Cellular Senescence, and Alzheimer's Disease.

Aging is the greatest risk factor for late-onset Alzheimer’s disease (LOAD), which accounts for >95% of Alzheimer’s disease (AD) cases. The mechanism underlying the aging-related susceptibility to LOAD is unknown. Cellular senescence, a state of permanent cell growth arrest, is believed to contribute importantly to aging and aging-related diseases, including AD. Senescent astrocytes, microglia, endothelial cells, and neurons have been detected in the brain of AD patients and AD animal models. Removing senescent cells genetically or pharmacologically ameliorates β-amyloid (Aβ) peptide and tau-protein-induced neuropathologies, and improves memory in AD model mice, suggesting a pivotal role of cellular senescence in AD pathophysiology. Nonetheless, although accumulated evidence supports the role of cellular senescence in aging and AD, the mechanisms that promote cell senescence and how senescent cells contribute to AD neuropathophysiology remain largely unknown. This review summarizes recent advances in this field. We believe that the removal of senescent cells represents a promising approach toward the effective treatment of aging-related diseases, such as AD.

Selective reduction of astrocyte apoE3 and apoE4 strongly reduces A\u03b2 accumulation and plaque-related pathology in a mouse model of amyloidosis

BackgroundOne of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide into amyloid plaques. The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset AD and has been shown to influence the accumulation of Aβ in the brain in an isoform-dependent manner. ApoE can be produced by different cell types in the brain, with astrocytes being the largest producer of apoE, although reactive microglia also express high levels of apoE. While studies have shown that altering apoE levels in the brain can influence the development of Aβ plaque pathology, it is not fully known how apoE produced by specific cell types, such as astrocytes, contributes to amyloid pathology.MethodsWe utilized APOE knock-in mice capable of having APOE selectively removed from astrocytes in a tamoxifen-inducible manner and crossed them with the APP/PS1-21 mouse model of amyloidosis. We analyzed the changes to Aβ plaque levels and assessed the impact on cellular responses to Aβ plaques when astrocytic APOE is removed.ResultsTamoxifen administration was capable of strongly reducing apoE levels in the brain by markedly reducing astrocyte apoE, while microglial apoE expression remained. Reduction of astrocytic apoE3 and apoE4 led to a large decrease in Aβ plaque deposition and less compact plaques. While overall Iba1+ microglia were unchanged in the cortex after reducing astrocyte apoE, the expression of the disease-associated microglial markers Clec7a and apoE were lower around amyloid plaques, indicating decreased microglial activation. Additionally, astrocyte GFAP levels are unchanged around amyloid plaques, but overall GFAP levels are reduced in the cortex of female apoE4 mice after a reduction in astrocytic apoE. Finally, while the amount of neuritic dystrophy around remaining individual plaques was increased with the removal of astrocytic apoE, the overall amount of cortical amyloid-associated neuritic dystrophy was significantly decreased.ConclusionThis study reveals an important role of astrocytic apoE3 and apoE4 on the deposition and accumulation of Aβ plaques as well as on certain Aβ-associated downstream effects.

APOE4 genotype or ovarian hormone loss influence open field exploration in an EFAD mouse model of Alzheimer's disease

Anxiety is a prominent and debilitating symptom in Alzheimer's disease (AD) patients. Carriers of APOE4, the greatest genetic risk factor for late-onset AD, may experience increased anxiety relative to carriers of other APOE genotypes. However, whether APOE4 genotype interacts with other AD risk factors to promote anxiety-like behaviors is less clear. Here, we used open field exploration to assess anxiety-like behavior in an EFAD mouse model of AD that expresses five familial AD mutations (5xFAD) and human APOE3 or APOE4. We first examined whether APOE4 genotype exacerbates anxiety-like exploratory behavior in the open field relative to APOE3 genotype in a sex-specific manner among six-month-old male and female E3FAD (APOE3+/+/5xFAD+/−) and E4FAD mice (APOE4+/+/5xFAD+/−). Next, we determined whether circulating ovarian hormone loss influences exploratory behavior in the open field among female E3FAD and E4FADs. APOE4 genotype was associated with decreased time in the center of the open field, particularly among female EFADs. Furthermore, ovariectomy (OVX) decreased time in the center of the open field among female E3FADs to levels similar to intact and OVXed E4FAD females. Our results suggest that APOE4 genotype increased anxiety-like behavior in the open field, and that ovarian hormones may protect against an anxiety-like phenotype in female E3FAD, but not E4FAD mice.

APOE ε2 resilience for Alzheimer's disease is mediated by plasma lipid species: Analysis of three independent cohort studies.

The apolipoprotein E (APOE) genotype is the strongest genetic risk factor for late-onset Alzheimer's disease. However, its effect on lipid metabolic pathways, and their mediating effect on disease risk, is poorly understood. We performed lipidomic analysis on three independent cohorts (the Australian Imaging, Biomarkers and Lifestyle [AIBL] flagship study, n=1087; the Alzheimer's Disease Neuroimaging Initiative [ADNI] 1 study, n=819; and the Busselton Health Study [BHS], n=4384), and we defined associations between APOE ε2 and ε4 and 569 plasma/serum lipid species. Mediation analysis defined the proportion of the treatment effect of the APOE genotype mediated by plasma/serum lipid species. A total of 237 and 104 lipid species were associated with APOE ε2 and ε4, respectively. Of these 68 (ε2) and 24 (ε4) were associated with prevalent Alzheimer's disease. Individual lipid species or lipidomic models of APOE genotypes mediated up to 30% and 10% of APOE ε2 and ε4 treatment effect, respectively. Plasma lipid species mediate the treatment effect of APOE genotypes on Alzheimer's disease and as such represent a potential therapeutic target.

Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins.

ABSTRACTThe human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.

The detrimental effects of APOE4 on risk for Alzheimer's disease may result from altered dendritic spine density, synaptic proteins, and estrogen receptor alpha

Women carriers of APOE4, the greatest genetic risk factor for late-onset Alzheimer's disease (AD), are at highest risk of developing AD, yet factors underlying interactions between APOE4 and sex are not well characterized. Here, we examined how sex and APOE3 or APOE4 genotypes modulate object and spatial memory, dendritic spine density and branching, and protein expression in 6-month-old male and female E3FAD and E4FAD mice (APOE+/+/5xFAD+/−). APOE4 negatively impacted object recognition and spatial memory, with male E3FADs exhibiting the best memory across 2 object-based tasks. In both sexes, APOE4 reduced basal dendritic spine density in the medial prefrontal cortex and dorsal hippocampus. APOE4 reduced dorsal hippocampal levels of PDS-95, synaptophysin, and phospho-CREB, yet increased levels of ERα. E4FAD females exhibited strikingly increased GFAP levels, in addition to the lowest levels of PSD-95 and pCREB. Overall, our results suggest that APOE4 negatively impacts object memory, dendritic spine density, and levels of hippocampal synaptic proteins and ERα. However, the general lack of sex differences or sex by genotype interactions suggests that the sex-specific effects of APOE4 on AD risk may be related to factors unexplored in the present study.

Using Drosophila to Identify Naturally-Occurring Modifiers of Alzheimer's Disease

Despite significant progress in identifying risk factors for late-onset Alzheimer’s Disease (LOAD), much of the variance in disease pathogenesis remains unexplained, likely due to the contribution of many genes of small effect size. Model organisms such as Drosophila Melanogaster exhibit conservation in both disease-causing genes and cellular processes implicated in Alzheimer’s Disease (AD), offering a genetically tractable model that can be statistically leveraged to identify causal variants. Here, we combine a Drosophila model of AD with the Drosophila Genetic Reference Panel (DGRP), a model of natural variation consisting of over 200 fully sequenced, isogenic lines derived from a wild-caught population. Expression of two proteins closely associated with AD pathogenesis, A□42 and Tau, in the Drosophila eye results in a “rough eye” phenotype, an easily quantifiable phenotype caused by degeneration of the ommatidial array. By quantifying the degree of A□42- and Tau-mediated degeneration across 164 lines of the DGRP and using a gene-based approach to map associations, we have identified and validated a subset of naturally occurring modifiers of degeneration in Drosophila. Enrichment analysis reveals that the set of genes identified in our screen show significant enrichment for genes identified as significant or suggestive (4x10-6>p>2x10-11) in human GWAS studies. The results presented here provide proof-of-principal for an approach that combines the strengths of forward genetic screens in model organisms with the power of human GWAS studies to identify and validate potential risk factors that have been difficult to detect in human studies alone.

Metabolomic Profile Differences Between Demented and Non-Demented APOE4 Carriers in the Long Life Family Study

Rejection of care (RoC) by persons living with dementia (PLWD) has yet to be measured in the hospital setting. Elderspeak communication (i.e., baby talk or infantilization) is an established antecedent to RoC in nursing home dementia care. The purpose of this study was to determine the impact of elderspeak communication by nursing staff on RoC by hospitalized PLWD. Eighty-eight care encounters between 16 PLWD and 53 nursing staff were observed for RoC using the Resistiveness to Care scale in one Midwestern hospital. Audio-recordings of the care encounters were transcribed verbatim and coded for semantic, pragmatic, and prosodic features of elderspeak. Over one-quarter (28.7%) of the duration of nursing staff speech towards PLWD constituted elderspeak and nearly all (96.6%) of the 88 care encounters included some elderspeak. Almost half of the observations (48.9%) included RoC behaviors by PLWD. Rejection of care was modeled as present or absent using a GEE method. Characteristics of the PLWD (e.g., pain, delirium) and the observation (e.g., environmental simulation) were evaluated as potential covariates. After adjusting for pain, length of stay, and gender, a 15-percentage point decrease in the proportion of elderspeak communication by nursing staff reduced the odds of RoC by 62% (OR=0.38, 95% CI=0.21-0.71, p=.002,) and a one unit decrease in pain reduced the odds of RoC by 63% (OR=0.37, 95% CI=0.22-0.63, p<.001). This study identified that pain and elderspeak are two modifiable factors of RoC. Person-centered interventions are needed that address communication practices and approaches to pain management for hospitalized PLWD.

APOE-e4 alters the microglial phenotype in the presence of Alzheimer's disease pathology compared to APOE-e3.

APOE is the largest genetic risk factor for late-onset Alzheimer's disease (AD) with the APOE-e4 allele conferring an increased risk for AD compared to the APOE- e3 allele. While APOE in the brain is a lipid transporter produced by astrocytes, research has shown that APOE can impact diverse biochemical pathways. Interestingly, APOE isoforms have also been shown to modulate levels of neuroinflammation in the brain, yet the exact impact on inflammation remains unknown. As microglia are considered to be a primary source of brain inflammation, this study explores how APOE isoforms impact microglial morphology and activation state in human brains. Two methods were used in this study to assess the impact of APOE isoforms on microglia in human brain sections. First, NanoString Digital Spatial Profiling (DSP) was used to investigate protein levels of inflammatory markers specific for microglia on fixed tissue in the hippocampus with AD pathology in both APOE- e3 (n=7) and APOE- e4 (n=8) subjects. Immunohistochemistry (IHC) was then performed using samples from APOE- e3/3 (n=33 control n=23 AD), APOE- e4/4 (n=19 AD), and APOE-e3/4 (n=15 control) subjects from the University of Kentucky AD Research Center. IBA-1 IHC was used to investigate microglial activation and morphology differences between APOE isoforms. Using both DSP and IHC, the results indicate that APOE isoforms play a role in the microglial changes seen in the presence of AD pathology- both in activation state and morphology. The DSP experiments showed significant changes in microglial markers throughout the hippocampal region between APOE- e3 and APOE- e4. Of interest, in APOE-e3 subjects, we show an alteration of the microglial phenotype based on pathology level, however, in APOE-e4 subjects, this change due to pathology is reduced. Additionally, in the IHC experiments we found changes in microglial morphology and activation that parallel those seen in DSP. The findings from this study suggest that with APOE- e3, microglia are able to respond in the presence of AD pathology. By contrast, APOE- e4 microglia cannot produce an appropriate response to AD pathology. Overall, these findings help highlight the role of the APOE isoforms in AD and neuroinflammation.

Early effects of amyloid-β on structural and vascular brain changes in mid-life cognitively unimpaired individuals.

The apolipoprotein E (APOE) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). It has been shown that APOE ε4 allele (ε4) relates to early pathological accumulation of cerebral amyloid-β (Aβ), leading to decade earlier age-of-onset of AD. However vascular and anatomical brain changes in the presence of Aβ and ε4 gene have not yet been fully examined in middle age. We aim to investigate how ε4 and Aβ level influence MR neuroimaging biomarkers such as white-matter hyperintensities and hippocampus volume in mid-life (45-65 year old) cognitively unimpaired (CU) individuals. 149 middle age (44 to 66 years of age) CU adults (age 58.2±5.4, 115 Females, years of education 13.5±2.8) underwent 3T MRI and amyloid PET scans using 18 F-Florbetaben as part of the PISA study. Of this ε4 enriched cohort (N=78 ε4 carriers), 12 were classified as Aβ+ (Centiloid≥20). 5 participants with ε2ε4 genotypes were excluded due to the protective effect of the ε2 allele. Multivariable general linear models were implemented to study the association of ε4 and Aβ with structural neuroimaging markers including white-matter hyperintensity volume (WMHV) and Hippocampus volume (HV). Both volumes were normalised using total intracranial volume, and age, gender and years-of-education were used as independent covariates. There was no age difference between ε4+ and ε4- groups (Kruskal-Wallis test, p-value=0.8), but Aβ+ participants were older compared to Aβ- (p-value=0.005). There was no HV differences between ε4+ and ε4- groups when controlling for Centiloid (p-value=0.55). When comparing HV differences between groups based on joint ε4 and Aβ, Aβ+ε4+ had significantly lower HV compared to Aβ-ε4+ (p-value=0.01) and Aβ-ε4- (p-value=0.021) as shown in Figure 1. For WMHV differences, a trend towards significance exists for Aβ+ε4+ when compared to and Aβ+ε4- and Aβ-ε4- (p-value=0.069) as shown in Figure 2. In this study, we found that early signs of hippocampus degeneration may occur in the middle age in the presence of both high Aβ level and APOE ε4 allele. Future studies will investigate the effective age spread for middle-aged preclinical AD population which will be beneficial for participant recruitment for new clinical trials.

Transcriptomic Changes Highly Similar to Alzheimer's Disease Are Observed in a Subpopulation of Individuals During Normal Brain Aging.

Aging is a major risk factor for late-onset Alzheimer’s disease (LOAD). How aging contributes to the development of LOAD remains elusive. In this study, we examined multiple large-scale transcriptomic datasets from both normal aging and LOAD brains to understand the molecular interconnection between aging and LOAD. We found that shared gene expression changes between aging and LOAD are mostly seen in the hippocampal and several cortical regions. In the hippocampus, the expression of phosphoprotein, alternative splicing and cytoskeleton genes are commonly changed in both aging and AD, while synapse, ion transport, and synaptic vesicle genes are commonly down-regulated. Aging-specific changes are associated with acetylation and methylation, while LOAD-specific changes are more related to glycoprotein (both up- and down-regulations), inflammatory response (up-regulation), myelin sheath and lipoprotein (down-regulation). We also found that normal aging brain transcriptomes from relatively young donors (45–70 years old) clustered into several subgroups and some subgroups showed gene expression changes highly similar to those seen in LOAD brains. Using brain transcriptomic datasets from another cohort of older individuals (>70 years), we found that samples from cognitively normal older individuals clustered with the “healthy aging” subgroup while AD samples mainly clustered with the “AD similar” subgroups. This may imply that individuals in the healthy aging subgroup will likely remain cognitively normal when they become older and vice versa. In summary, our results suggest that on the transcriptome level, aging and LOAD have strong interconnections in some brain regions in a subpopulation of cognitively normal aging individuals. This supports the theory that the initiation of LOAD occurs decades earlier than the manifestation of clinical phenotype and it may be essential to closely study the “normal brain aging” to identify the very early molecular events that may lead to LOAD development.

APOE*4-stratified genome-wide association study of Alzheimer's disease in over 350,000 individuals.

APOE*4 is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and is highly pleiotropic, such that it may be considered as a biological factor that can affect overall genetic risk for AD. To advance our understanding of the genetic architecture of AD, we sought to perform the largest APOE*4-stratified genome-wide association study (GWAS) of AD. Twenty-five publicly available AD GWAS datasets provided case-control diagnoses for phase-1 samples (imputed to the HRC r1.1 reference panel). The UK Biobank provided subjects with family history of AD status, transformed into an AD phenotype as described previously (Jansen et al., 2019) for phase-2 samples. Linear mixed model regressions were performed on case-control status (LMM-BOLT v.2.3.4), adjusting for age (age-at-onset in cases; age-at-last-exam in controls), sex, APOE*4 and APOE*2 dosage, the first 12 genetic principal components, array/batch, cohort in phase-1, and assessment center in phase-2. In phase-3, phase-1 and phase-2 findings were combined using multivariate genome-wide meta-analysis (Jansen et al., 2019). APOE*4+ heterogeneity tests were evaluated per phase and meta-analyzed in phase-3. Participant demographics are in Table 1. Combining results from both APOE*4-stratified analyses, 106 lead variants across 98 loci passed suggestive significance (p<10-5 ; Figure 1). Although most variants reached only suggestive significance, 28 loci were previously reported at genome-wide significance in large-scale GWAS of AD (Kunkle et al., 2019, Jansen et al., 2019, Bellenguez et al., 2020), supporting that we identified potentially relevant AD loci. APOE*4-stratified effects were observed for 28 variants/loci covered across both phase-1 and phase-2 (NAPOE4+ =19; NAPOE4- =9; Table 2), and 25 variants/loci seen only in phase-2 (NAPOE4+ =17; NAPOE4- =8; Table 3). Notably, a genome-wide significant APOE*4+ heterogeneity effect was observed for the USP17L13 locus (a regulator of deubiquitination), while PPP1R12A, BRINP1, PCBD1, and SESN2 loci passed suggestive significance. Our findings revealed novel AD risk loci/genes and characterized which of these associated with AD risk differentially across APOE*4 status. This contributes highly to personalized genetic medicine and paves the way towards new potential AD drug targets. Ongoing work is adding samples for phase-1 analyses (imputing data to TOPMed) and pursuing both multi-omics and AD endophenotype validation efforts for variant prioritization.

Modelling the role of APOE4 in blood-brain barrier dysfunctions with isogenic IPSC-derived brain microvascular endothelial cells and pericytes.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer's Disease, and is known to affect the function of multiple neuronal and glial cell types. Recent research has suggested that blood-brain barrier (BBB) dysfunction is commonly observed among AD patients. However, it remains unclear whether and how APOE4 directly contributes to blood-brain barrier dysfunction. Isogenic iPSC lines with different APOE isoforms were differentiated into brain microvascular endothelial cell-like cells (BMECs) and neural crest-derived pericyte-like cells (PCs). Isogenic BMECs with different APOE isoforms were compared for their tight junction integrity, efflux transporter activity and amyloid clearance capabilities. Isogenic PCs with different APOE isoforms were compared for their APOE secretion levels and amyloid uptake capabilities. APOE4, APOE3 and APOE2 BMECs exhibited similar levels of tight junction protein expression, efflux transporter activities and trans-endothelial electrical resistance. However, we found that the presence of APOE ε4 protein, when compared with APOE ε2 and APOE ε3, led to reduced BMEC clearance of amyloid in a Transwell model. Compared to APOE3 and APOE2 PCs, APOE4 PCs demonstrated similar levels of APOE secretion, but had lower amyloid uptake capabilities. Our findings reveal that although APOE4 did not directly affect BMEC barrier properties in our hPSC-derived model, it plays a potentially important role in amyloid clearance by both BMECs and PCs.

Effects of APOE genotype and estrogen status on brain transcriptome in female mice.

The predominant genetic risk factor for late-onset Alzheimer's disease (AD) is the ε4 allele of apolipoprotein E (APOE4). APOE4-related risk of developing AD is modified by sex, with women showing greater risk than men. Regardless of APOE genotype, women exhibit a higher prevalence and lifetime risk of AD. Extensive human and animal model literatures indicate that women's risk for AD is also affected by the normal, age-related depletion of estrogens at menopause. For example, in mouse models, estrogen depletion by ovariectomy accelerates the onset and progression of AD-like neuropathology and cognitive impairment, whereas estrogen-based hormone therapies can partially prevent and or reverse these effects. Potential interactions between APOE4 genotype and estrogen status have not been well elucidated. To provide more insight into these issues, we conducted transcriptional analysis on hippocampi from adult female mice across APOE3 and APOE4 genotypes in the presence or absence of estradiol. At 18-22 weeks of age, female mice homozygous for knock-in of human APOE3 or APOE4 were bilaterally ovariectomized (OVX) or sham-OVX and immediately implanted with a subcutaneous silastic capsule containing either vehicle or 0.5% 17β-estradiol (E2). Four weeks following surgery and hormone treatment, hippocampi were harvested and processed for polyA-enriched RNA-seq. Transcripts were mapped to a cDNA library of the mouse genome using kallisto (0.46.1). DEseq2 (1.28.1) normalized fold-changes were then used to estimate differential gene expression among groups. RNA-seq analyses indicate significant differences in hippocampal transcriptional profiles across treatment groups. In particular, we found group differences in pathways related to synaptic integrity and plasticity, lipid metabolism and glucose metabolism. Interestingly, the strongest group differences were associated with APOE genotype whereas estrogen status yielded generally modest effects. Our results suggest that APOE genotype impacts the transcriptome in the female hippocampus to a larger degree than estrogen status. These findings provide novel insight into the independent and interactive neural effects of APOE4 and estrogen, which may provide opportunities for identification of therapeutic targets for AD.