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INTRODUCTION: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. METHODS: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. RESULTS: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). DISCUSSION: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development.

BackgroundGenetic and experimental evidence strongly implicates myeloid cells in the etiology of AD and suggests that AD-associated alleles and genes may modulate disease risk by altering the transcriptional and cellular responses of macrophages (like microglia) to damage of lipid-rich tissues (like the brain). Specifically, recent single-cell/nucleus RNA sequencing (sc/nRNA-seq) studies identified a transcriptionally distinct state of subsets of macrophages in aging or degenerating brains (usually referred to as disease- associated microglia or DAM) and in other diseased lipid-rich tissues (e.g., obese adipose tissue, fatty liver, and atherosclerotic plaques). We collectively refer to these subpopulations as lipid-associated macrophages or LAMs. Importantly, this particular activation state is characterized by increased expression of genes involved in the phagocytic clearance of lipid-rich cellular debris (efferocytosis), including several AD risk genes.MethodsWe used sc/nRNA-seq data from human and mouse microglia from healthy and diseased brains and macrophages from other lipid-rich tissues to reconstruct gene regulatory networks and identify transcriptional regulators whose regulons are enriched for LAM response genes (LAM TFs) across species. We then used gene knock- down/knock-out strategies to validate some of these LAM TFs in human THP-1 macrophages and iPSC-derived microgliain vitro, as well as mouse microgliain vivo.ResultsWe nominate 11 strong candidate LAM TFs shared across human and mouse networks (BHLHE41,HIF1A,ID2,JUNB,MAF,MAFB,MEF2A,MEF2C,NACA, POU2F2andSPI1). We also demonstrate a strong enrichment of AD risk alleles in the cistrome ofBHLHE41(and its close homologBHLHE40), thus implicating its regulon in the modulation of disease susceptibility. Loss or reduction ofBHLHE40/41expression in human THP-1 macrophages and iPSC-derived microglia, as well as loss ofBhlhe40/41in mouse microglia led to increased expression of LAM response genes, specifically those involved in cholesterol clearance and lysosomal processing, with a concomitant increase in cholesterol efflux and storage, as well as lysosomal mass and degradative capacity.ConclusionsTaken together, this study nominates transcriptional regulators of the LAM response, experimentally validates BHLHE40/41 in human and mouse macrophages/microglia, and provides novel targets for therapeutic modulation of macrophage/microglia function in AD and other disorders of lipid-rich tissues.Graphical abstract

Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.

Genome-wide association studies and functional genomics studies have linked specific cell types, genes, and pathways to Alzheimer's disease (AD) risk. In particular, AD risk alleles primarily affect the abundance or structure, and thus the activity, of genes expressed in macrophages, strongly implicating microglia (the brain-resident macrophages) in the etiology of AD. These genes converge on pathways (endocytosis/phagocytosis, cholesterol metabolism, and immune response) with critical roles in core macrophage functions such as efferocytosis. Here, we review these pathways, highlighting relevant genes identified in the latest AD genetics and genomics studies, and describe how they may contribute to AD pathogenesis. Investigating the functional impact of AD-associated variants and genes in microglia is essential for elucidating disease risk mechanisms and developing effective therapeutic approaches.

The impact of Apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer’s disease (AD), on human brain cellular function remains unclear. Here we investigated the effects of APOE4 on brain cell types derived from population and isogenic human induced pluripotent stem cells (hiPSCs), post-mortem brain and APOE targeted replacement (APOE-TR) mice. Population and isogenic models demonstrate that APOE4 local haplotype rather than a single risk allele contributes to risk. Global transcriptomic analyses reveal human specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation and lipid biosynthesis in astrocytes co-cultured with neurons that recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk.

Genetic association studies have identified 44 common genome-wide significant risk loci for late-onset Alzheimer’s disease (LOAD). However, LOAD genetic architecture and prediction are unclear. Here we estimate the optimal P-threshold (Poptimal) of a genetic risk score (GRS) for prediction of LOAD in three independent datasets comprising 676 cases and 35,675 family history proxy cases. We show that the discriminative ability of GRS in LOAD prediction is maximised when selecting a small number of SNPs. Both simulation results and direct estimation indicate that the number of causal common SNPs for LOAD may be less than 100, suggesting LOAD is more oligogenic than polygenic. The best GRS explains approximately 75% of SNP-heritability, and individuals in the top decile of GRS have ten-fold increased odds when compared to those in the bottom decile. In addition, 14 variants are identified that contribute to both LOAD risk and age at onset of LOAD., Despite the identification of genetic risk loci for late-onset Alzheimer’s disease (LOAD), the genetic architecture and prediction remains unclear. Here, the authors use genetic risk scores for prediction of LOAD across three datasets and show evidence suggesting oligogenic variant architecture for this disease.

Objective The objective of this study was to better understand the pathophysiology and underlying genetic mechanisms behind two abdominal aortic aneurysm (AAA) subtypes using computed tomographic imaging in combination with whole genome sequencing. Methods Patients with a known AAA and European ancestry were included in this investigation and underwent genetic and image analysis. Patients with AAAs and indications of descending thoracic aortic pathology (aortic dissection, penetrating aortic ulcers, intramural hematoma, atheromas, ulcerative plaque, and intramural ulceration, and intimal flaps/tears) were classified as having thoracic aortic disease, grouped together, and compared with patients with an AAA and a normal descending thoracic aorta. Whole genome sequencing was then performed on the 93 patients who had imaging features consistent with thoracic aortic disease and the 126 patients with a normal descending thoracic aorta. Results The results of this study suggest one variant-level, four gene-level, and one gene set-level associations in patients with thoracic aortic disease who also had an AAA. The variant rs79508780 located in TSEN54 achieved study-wide significance (P = 1.71E-06). BATF3 and SMLR1 were significantly associated and EFCAB3 and TAF4 were reached suggestive assocation with a diseased descending thoracic aorta (P = 5.23E-26, P = 1.86E-25, P = 1.54E-05, and P = 8.31E-05, respectively). Gene sets were also compiled using MSigDB and trait-based index single nucleotide variation from major genome-wide association studies. GO_DNA_DOUBLE_STRAND_BREAK_PROCESSING, a gene set related to double-stranded DNA break repair, was significantly associated with thoracic aortic disease in AAA patients (P = 1.80E-06). Conclusions This pilot study provides further evidence that an AAA may be the end result of multiple degenerative pathways. Genetic variations in vitamin D signaling, cholesterol metabolism, extracellular matrix breakdown, and double-stranded DNA break repair pathways were associated with European patients who had an AAA and thoracic aortic disease. Additionally, this study provides support for the application of a radiogenomic approach for the investigation of other potential pathologies that could lead to the development of an AAA or influence future management decisions. (JVS–Vascular Science.) Clinical Relevance In this study, we provide evidence that abdominal aortic aneurysms (AAAs) may be a result of multiple pathophysiologies rather than a single disease. We have identified genetic variants involved in vitamin D signaling, cholesterol metabolism, extracellular matrix breakdown, and double-stranded DNA break repair associated with structural defects in the aortic wall in patients with AAAs who are of European descent. Patients with AAAs and structural defects in the thoracic aorta have been previously linked to differential behavior after endovascular aneurysm repair. These patients with wall defects exhibited greater sac regression, a marker of surgical success, after endovascular aneurysm repair. Our study demonstrates the usefulness of a radiogenomic approach for elucidating mechanisms behind the formation and future behavior of AAAs that could aid surgeons in making future procedural and management decisions.

Primary age-related tauopathy (PART) is a neurodegenerative tauopathy with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) deposition in plaques. The pathogenesis of PART is unknown, but evidence suggests it is associated with genes that promote tau pathology as well as others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n=647) using Braak neurofibrillary tangle stage as a quantitative trait adjusting for sex, age, genotyping platform, and principal components. We found significant associations with some candidate loci associated with AD and progressive supranuclear palsy, a primary tauopathy (SLC24A4, MS4A6A, HS3ST1, MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brain from tauopathies containing isoforms with four microtubule-binding domain repeats (4R) and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation revealed a direct and specific binding of JADE1 protein to tau containing four (4R) and no N-terminal inserts (0N4R) in post-mortem human PART brain tissue. Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a mediator of neurofibrillary degeneration.

Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer’s disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility., This study integrates Alzheimer’s disease (AD) GWAS data with myeloid cell genomics, and reports that myeloid active enhancers are most burdened by AD risk alleles. The authors also nominate candidate causal regulatory elements, variants and genes that likely modulate the risk for AD.

Genome-wide association study (GWAS) summary statistics are a fundamental resource for a variety of research applications 1–6. Yet despite their widespread utility, no common storage format has been widely adopted, hindering tool development and data sharing, analysis and integration. Existing tabular formats 7,8 often ambiguously or incompletely store information about genetic variants and their associations, and also lack essential metadata increasing the possibility of errors in data interpretation and post-GWAS analyses. Additionally, data in these formats are typically not indexed, requiring the whole file to be read which is computationally inefficient. To address these issues, we propose an adaptation of the variant call format9 (GWAS-VCF) and have produced a suite of open-source tools for using this format in downstream analyses. Simulation studies determine GWAS-VCF is 9-46x faster than tabular alternatives when extracting variant(s) by genomic position. Our results demonstrate the GWAS-VCF provides a robust and performant solution for sharing, analysis and integration of GWAS data. We provide open access to over 10,000 complete GWAS summary datasets converted to this format (available from: https://gwas.mrcieu.ac.uk).

More than forty loci contribute to genetic risk for Alzheimer's disease (AD). These risk alleles are enriched in myeloid cell enhancers suggesting that microglia, the brain-resident macrophages, contribute to AD risk. We have previously identified SPI1/PU.1, a master regulator of myeloid cell development in the brain and periphery, as a genetic risk factor for AD. Higher expression of SPI1 is associated with increased risk for AD, while lower expression is protective. To investigate the molecular and cellular phenotypes associated with higher and lower expression of PU.1 in microglia, we used stable overexpression and knock-down of PU.1 in BV2, an immortalized mouse microglial cell line. Transcriptome analysis suggests that reduced PU.1 expression suppresses expression of homeostatic genes similar to the disease-associated microglia response to amyloid plaques in mouse models of AD. Moreover, PU.1 knock-down resulted in activation of protein translation, antioxidant action and cholesterol/lipid metabolism pathways with a concomitant decrease of pro-inflammatory gene expression. PU.1 overexpression upregulated and knock-down downregulated phagocytic uptake in BV2 cells independent of the nature of the engulfed material. However, cells with reduced PU.1 expression retained their ability to internalize myelin similar to control albeit with a delay, which aligns with their anti-inflammatory profile. Here we identified several microglial responses that are modulated by PU.1 expression levels and propose that risk association of PU.1 to AD is driven by increased pro-inflammatory response due to increased viability of cells under cytotoxic conditions. In contrast, low expression of PU.1 leads to increased cell death under cytotoxic conditions accompanied by reduced pro-inflammatory signaling that decreased A1 reactive astrocytes signature supporting the protective effect of SPI1 genotype in AD. These findings inform future in vivo validation studies and design of small molecule screens for therapeutic discovery in AD.

Alzheimer’s disease (AD) is the most common type of dementia, affecting millions of people worldwide; however, no disease-modifying treatments are currently available. Genome-wide association studies (GWASs) have identified more than 40 loci associated with AD risk. However, most of the disease-associated variants reside in non-coding regions of the genome, making it difficult to elucidate how they affect disease susceptibility. Nonetheless, identification of the regulatory elements, genes, pathways and cell type/tissue(s) impacted by these variants to modulate AD risk is critical to our understanding of disease pathogenesis and ability to develop effective therapeutics. In this review, we provide an overview of the methods and approaches used in the field to identify the functional effects of AD risk variants in the causal path to disease risk modification as well as describe the most recent findings. We first discuss efforts in cell type/tissue prioritization followed by recent progress in candidate causal variant and gene nomination. We discuss statistical methods for fine-mapping as well as approaches that integrate multiple levels of evidence, such as epigenomic and transcriptomic data, to identify causal variants and risk mechanisms of AD-associated loci. Additionally, we discuss experimental approaches and data resources that will be needed to validate and further elucidate the effects of these variants and genes on biological pathways, cellular phenotypes and disease risk. Finally, we discuss future steps that need to be taken to ensure that AD GWAS functional mapping efforts lead to novel findings and bring us closer to finding effective treatments for this devastating disease.

Identification of causal variants and genes underlying genome-wide association study (GWAS) loci is essential to understand the biology of alcohol use disorder (AUD) and drinks per week (DPW). Multi-omics integration approaches have shown potential for fine mapping complex loci to obtain biological insights to disease mechanisms. In this study, we use multi-omics approaches, to fine-map AUD and DPW associations at single SNP resolution to demonstrate that rs56030824 on chromosome 11 significantly reduces SPI1 mRNA expression in myeloid cells and lowers risk for AUD and DPW. Our analysis also identifies MAPT as a candidate causal gene specifically associated with DPW. Genes prioritized in this study show overlap with causal genes associated with neurodegenerative disorders. Multi-omics integration analyses highlight, genetic similarities and differences between alcohol intake and disordered drinking, suggesting molecular heterogeneity that might inform future targeted functional and cross-species studies., Alcohol use disorder and drinks per week both have been studied genetically and have different correlations with psychiatric diseases. Here the authors integrate multi-omics data to identify unique and shared variants, genes and pathways for alcohol use disorder and drinks per week.

SignificanceIdentification of causal variants and genes underlying genome-wide association study (GWAS) loci is essential to understanding the biology of alcohol use disorder (AUD).MethodsIntegration of “multi-omics” data is often necessary to nominate candidate causal variants and genes and prioritize them for follow up studies. Here, we used Mendelian randomization to integrate AUD and drinks per week (DPW) GWAS summary statistics with the gene expression and methylation quantitative trait loci (eQTLs and mQTLs) in the largest brain and myeloid datasets. We also used AUD-related single cell epigenetic data to nominate candidate causal variants and genes associated with DPW and AUD.ResultsOur multi-omics integration analyses prioritized unique as well as shared genes and pathways among AUD and DPW. The GWAS variants associated with both AUD and DPW showed significant enrichment in the promoter regions of fetal and adult brains. The integration of GWAS SNPs with mQTLs from fetal brain prioritized variants on chromosome 11 in both AUD and DPW GWASs. The co-localized variants were found to be overlapping with promoter marks for SPI1, specifically in human microglia, the myeloid cells of the brain. The co-localized SNPs were also strongly associated with SPI1 mRNA expression in myeloid cells from peripheral blood. The prioritized variant at this locus is predicted to alter the binding site for a transcription factor, RXRA, a key player in the regulation of myeloid cell function. Our analysis also identified MAPT as a candidate causal gene specifically associated with DPW. mRNA expression of MAPT was also correlated with daily amounts of alcohol intake in post-mortem brains (frontal cortex) from alcoholics and controls (N = 92). Results may be queried and visualized in an online public resource of these integrative analysis (https://lcad.shinyapps.io/alc_multiomics/). These results highlight overlap between causal genes for neurodegenerative diseases, alcohol use disorder and alcohol consumption.In conclusionintegrating GWAS summary statistics with multi-omics datasets from multiple sources identified biological similarities and differences between typical alcohol intake and disordered drinking highlighting molecular heterogeneity that might inform future targeted functional and cross-species studies. Interestingly, overlap was also observed with causal genes for neurodegenerative diseases.

Objective The purpose of this study was to infer causal relationships between 22 previously reported risk factors for Alzheimer's disease (AD) and the "AD phenome": AD, AD age of onset (AAOS), hippocampal volume, cortical surface area and thickness, cerebrospinal fluid (CSF) levels of amyloid-β (Aβ42 ), tau, and ptau181 , and the neuropathological burden of neuritic plaques, neurofibrillary tangles (NFTs), and vascular brain injury (VBI). Methods Polygenic risk scores (PRS) for the 22 risk factors were computed in 26,431 AD cases/controls and the association with AD was evaluated using logistic regression. Two-sample Mendelian randomization (MR) was used to infer the causal effect of risk factors on the AD phenome. Results PRS for increased education and diastolic blood pressure were associated with reduced risk for AD. MR indicated that only education was causally associated with reduced risk of AD, delayed AAOS, and increased cortical surface area and thickness. Total- and LDL-cholesterol levels were causally associated with increased neuritic plaque burden, although the effects were driven by single nucleotide polymorphisms (SNPs) within the APOE locus. Diastolic blood pressure and pulse pressure are causally associated with increased risk of VBI. Furthermore, total cholesterol was associated with decreased hippocampal volume; smoking initiation with decreased cortical thickness; type 2 diabetes with an earlier AAOS; and sleep duration with increased cortical thickness. Interpretation Our comprehensive examination of the genetic evidence for the causal relationships between previously reported risk factors in AD using PRS and MR supports a causal role for education, blood pressure, cholesterol levels, smoking, and diabetes with the AD phenome. ANN NEUROL 2021;89:54-65.

Alzheimer's disease (AD) is a debilitating, chronic neurodegenerative disease. Genetic studies involving genome-wide association studies (GWAS) and meta-analysis have discovered numerous genomic loci associated with AD; however, the causal genes and variants remain unidentified in most loci. Integration of GWAS signals with epigenomic annotations has demonstrated that AD risk variants are enriched in myeloid-specific enhancers, implicating myeloid cells in AD etiology. AD risk variants in these regulatory elements modify disease susceptibility by regulating the expression of genes that play crucial roles in microglial phagocytosis. Several of these AD risk genes are specifically expressed in myeloid cells, whereas others are ubiquitously expressed but are regulated by AD risk variants within myeloid enhancers in a cell type-specific manner. We discuss the impact of established AD risk variants on microglial phagocytosis and debris processing via the endolysosomal system.

The Alzheimer’s Disease Sequencing Project (ADSP) undertook whole exome sequencing in 5,740 late-onset Alzheimer disease (AD) cases and 5,096 cognitively normal controls primarily of European ancestry (EA), among whom 218 cases and 177 controls were Caribbean Hispanic (CH). An age-, sex- and APOE based risk score and family history were used to select cases most likely to harbor novel AD risk variants and controls least likely to develop AD by age 85 years. We tested ~1.5 million single nucleotide variants (SNVs) and 50,000 insertion-deletion polymorphisms (indels) for association to AD, using multiple models considering individual variants as well as gene-based tests aggregating rare, predicted functional, and loss of function variants. Sixteen single variants and 19 genes that met criteria for significant or suggestive associations after multiple-testing correction were evaluated for replication in four independent samples; three with whole exome sequencing (2,778 cases, 7,262 controls) and one with genome-wide genotyping imputed to the Haplotype Reference Consortium panel (9,343 cases, 11,527 controls). The top findings in the discovery sample were also followed-up in the ADSP whole-genome sequenced family-based dataset (197 members of 42 EA families and 501 members of 157 CH families). We identified novel and predicted functional genetic variants in genes previously associated with AD. We also detected associations in three novel genes: IGHG3 (p = 9.8 × 10−7), an immunoglobulin gene whose antibodies interact with β-amyloid, a long non-coding RNA AC099552.4 (p = 1.2 × 10−7), and a zinc-finger protein ZNF655 (gene-based p = 5.0 × 10−6). The latter two suggest an important role for transcriptional regulation in AD pathogenesis.

Motivation Annotation of genomic variants is an increasingly important and complex part of the analysis of sequence-based genomic analyses. Computational predictions of variant function are routinely incorporated into gene-based analyses of rare-variants, though to date most studies use limited information for assessing variant function that is often agnostic of the disease being studied. Results In this work, we outline an annotation process motivated by the Alzheimer’s Disease Sequencing Project, illustrate the impact of including tissue-specific transcript sets and sources of gene regulatory information and assess the potential impact of changing genomic builds on the annotation process. While these factors only impact a small proportion of total variant annotations (∼5%), they influence the potential analysis of a large fraction of genes (∼25%). Availability and implementation Individual variant annotations are available via the NIAGADS GenomicsDB, at https://www.niagads.org/genomics/ tools-and-software/databases/genomics-database. Annotations are also available for bulk download at https://www.niagads.org/datasets. Annotation processing software is available at http://www.icompbio.net/resources/software-and-downloads/. Supplementary information Supplementary data are available at Bioinformatics online.

A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.

Recent methylome-wide association studies (MWAS) in humans have solidified the concept that aberrant DNA methylation is associated with Alzheimer's disease (AD). We summarize these findings to improve the understanding of mechanisms governing DNA methylation pertinent to transcriptional regulation, with an emphasis of AD-associated promoter DNA hypermethylation, which establishes an epigenetic barrier for transcriptional activation. By considering brain cell type specific expression profiles that have been published only for non-demented individuals, we detail functional activities of selected neuron, microglia, and astrocyte-enriched genes (AGAP2, DUSP6 and GPR37L1, respectively), which are DNA hypermethylated at promoters in AD. We highlight future directions in MWAS including experimental confirmation, functional relevance to AD, cell type-specific temporal characterization, and mechanism investigation.

Genome-wide association studies (GWAS) have identified more than thirty loci associated with Alzheimer’s disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown thus impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS signals with myeloid epigenomic and transcriptomic datasets using novel analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We nominate candidate AD risk enhancers and identify their target causal genes (including AP4E1, AP4M1, APBB3, BIN1, CD2AP, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, SPPL2A, TP53INP1, ZKSCAN1, and ZYX) in sixteen loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify disease susceptibility by regulating causal gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it experimentally in human induced pluripotent stem cell (hiPSC)-derived microglia. Combined, these results strongly implicate dysfunction of the myeloid endolysosomal system in the etiology of AD.

Background Potentially modifiable risk factors have been associated with Alzheimer’s disease (AD). However, the causality of these risk factors on AD is unclear. Using Mendelian randomization we evaluated the causal effect of potentially modifiable risk factors on AD and its associated endophenotypes to inform the development of lifestyle interventions that could reduce risk of developing AD. Methods and Findings Genetic instruments for the exposures were selected from genome wide association studies (GWAS) for traits previously linked to AD in observational studies including alcohol intake, physical activity, lipid traits, blood pressure traits, type 2 diabetes, body-mass index (BMI), depression, sleep, social isolation, smoking, oily fish intake, and educational attainment. The outcomes included AD status, AD age of onset survival (AAOS), hippocampal volume, CSF levels of Aβ 42 , tau, and ptau 181 , and neuropathological burden of neuritic plaques, neurofibrillary tangles, and vascular brain injury (VBI). MR estimates were calculated using an inverse variance weighted approach. Genetically predicted educational attainment (OR [CI]: 0.7 [0.63, 0.78]), diastolic blood pressure (DBP) (OR [CI]: 0.99 [0.98, 0.99]), systolic blood pressure (SBP) (OR [CI]: 0.99 [0.99, 1]) and physical activity (OR [CI]: 2.5 [1.47, 4.23]) were causally associated with AD risk. Genetically predicted BMI (HR [CI]: 1.13 [1.07, 1.2]) and educational attainment (HR [CI]: 0.74 [0.68, 0.82]) were causally associated with AAOS. Genetically predicted alcohol consumption (β [CI]: −0.15 [−0.25, −0.05]), broad depressive symptoms (β [CI]: 0.5 [0.2, 0.8]), major depressive disorder (β [CI]: 0.12 [0.05, 0.18]) and physical activity were causally associated with CSF Aβ 42 (β [CI]: 0.25 [0.1, 0.39]). Genetically predicted DBP was causally associated with CSF total Tau (β [CI]: −0.005 [−0.007, −0.002]). Increased risk of VBI were observed for genetically predicted DBP (OR [CI]: 1.05 [1.02, 1.08]), SBP (OR [CI]: 1.06 [1.03, 1.1]), and pulse pressure (OR [CI]: 1.03 [1.01, 1.05]). Increased risk of neuritic plaque burden were observed for genetically predicted LDL-cholesterol (OR [CI]: 1.87 [1.3, 2.69]) and total cholesterol (OR [CI]: 2.03 [1.44, 2.85]). Genetically predicted insomnia symptoms (β [CI]: −0.2 [−0.34, −0.06]) and total cholesterol were associated (β [CI]: −0.06 [−0.1, −0.03]) with hippocampal volume. Potential limitations include weak instrument bias, non-homogenous samples and other implicit limitations of MR analysis. Conclusions Demonstration of a causal relationship between blood pressure, cholesterol levels, BMI, depression, insomnia symptoms, physical activity and educational attainment on the AD phenome strongly support public health programs to educate the public about these preventable causes of AD.

Apolipoprotein E (APOE) e4 is the strongest genetic risk factor for Alzheimer's disease (AD). Although its association with AD is well-established, the impact of APOE e4 on human brain cell function remains unclear. Here we investigated the effects of APOE e4 on brain cell types derived from human induced pluripotent stem cells and human APOE targeted replacement mice. Global transcriptomic profiles showed that APOE e4 is associated with dysregulation of cholesterol homeostasis in human but not mouse glia. Elevated matrisome signaling in APOE e4 mixed neuron/astrocyte cultures parallels altered pathways uncovered in cell-type deconvoluted APOE e4 glia and AD post-mortem brains. In vitro study showed that isogenic APOE e4 is associated with increased lysosomal cholesterol levels and decreased cholesterol efflux, demonstrating decoupled lipid metabolism. APOE e4 glia also secrete higher levels of proinflammatory chemokines/cytokines, indicative of glial activation. Thus, APOE e4 induces human glia-specific dysregulation that may initiate AD risk.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 A, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.

Microglia play a pivotal role in maintenance of brain homeostasis, but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and Alzheimer’s disease (AD) and in microglia surrounding neuritic β-amyloid (Aβ) -plaques in human AD brains. The APOE pathway mediated a switch from a homeostatic to neurodegenerative microglia phenotype following phagocytosis of apoptotic neurons. Triggering receptor expressed on myeloid cells 2 (TREM2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia led to a loss in their tolerogenic function. Taken together, our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target to restore homeostatic microglia.

A genome-wide survival analysis of 14,406 Alzheimer’s disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and fourteen novel loci associated with age at onset. LD score regression of 220 cell types implicated regulation of myeloid gene expression in AD risk. In particular, the minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability is enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affect the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.

Background Phosphodiesterase 10A (PDE10A) is expressed at high levels in the striatum and has been proposed both as a biomarker for Huntington's disease pathology and as a target for intervention. Objective PDE10A radiotracers have been successfully used to measure changes in binding density in Huntington's disease patients, but little is known about PDE10A binding in mouse models that are used extensively to model pathology and test therapeutic interventions. Methods Our study investigated changes in PDE10A binding using the selective tracer 3H-7980 at specific ages of two Huntington's disease transgenic mouse models: R6/2, a short-lived model carrying exon-1 of mutant HTT and BACHD, a longer-lived model carrying full-length mutant HTT. PDE10A binding was compared to binding of known markers of striatal atrophy in Huntington's disease, e.g. dopamine transporter (DAT) and dopamine receptors D1 and D2. Results We found that in the R6/2 model at 6 weeks of age, mice showed high variability of binding, however binding of all ligands was significantly decreased at 8 and 12 weeks of age. In contrast, no changes were detectable in the BACHD model at 8, 10 or 12 month of age. Conclusions These findings suggest that radiotracer binding of PDE10A, DAT, D1 and D2 receptor in the R6/2 model may be a good indicator of striatal pathological changes that are observed in Huntington's disease patients, and that the first 12 months in the BACHD model may be more reflective of early stages of the disease.

NeuroD/BETA2 (referred to as NeuroD hereafter) is a basic helix–loop–helix (bHLH) transcription factor that is required for the development and survival of a subset of neurons and pancreatic endocrine cells in mice. Gain-of-function analyses demonstrated that NeuroD can (i) convert epidermal fate into neuronal fate when overexpressed in Xenopus embryos, and (ii) activate the insulin promoter in pancreatic beta cell lines in response to glucose stimulation. In glucose-stimulated INS-1 pancreatic beta cells, mutations of S259, S266, and S274 to alanines inhibited the ability of NeuroD to activate the insulin promoter. Phosphorylation of those serine residues by ERK1/2 was required for NeuroD activity in that assay. To determine whether the same residues are implicated in the neurogenic activity of NeuroD, we mutated the conserved S259, S266, and S274 of Xenopus NeuroD to alanines (S259A, S266A, and S274A), and performed an ectopic neurogenesis assay in Xenopus embryos. In contrast to what has been observed in the pancreatic beta cell line, the S266A and S274A mutant forms of Xenopus NeuroD displayed significantly increased abilities to form ectopic neurons, while S259A had little effect. In addition, S266A and S274A of Xenopus NeuroD resulted in increased accumulation of protein in the injected embryos while the corresponding mutations on mouse NeuroD did not have the same effect in an insulinoma cell line. Our results demonstrate that the consequence of NeuroD protein modification is context-dependent at both the molecular and functional levels.

NeuroD (ND) is a basic helix–loop–helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

Expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Loss of the normal function of Htt is thought to be an important pathogenetic component of HD. However, the function of wild-type Htt is not well defined. Htt is thought to be a multifunctional protein that plays distinct roles in several biological processes, including synaptic transmission, intracellular transport and neuronal transcription. Here, we show with biochemical and live cell imaging studies that wild-type Htt stimulates the transport of nuclear factor κ light-chain-enhancer of activated B cells (NF-κB) out of dendritic spines (where NF-κB is activated by excitatory synaptic input) and supports a high level of active NF-κB in neuronal nuclei (where NF-κB stimulates the transcription of target genes). We show that this novel function of Htt is impaired by the polyQ expansion and thus may contribute to the etiology of HD.

SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion ofsynGAPwas sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adultsynGAP+/−mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated insynGAP+/−mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 μmNMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion ofsynGAPlacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices fromsynGAP+/−mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton.

Many intractable neurological and mental diseases, including epilepsy, depression, and schizophrenia, are believed to result, in part, from derangements of regulation of synaptic transmission in the brain. For this reason, much effort has been made to discover how the delicate mechanisms of signal transduction at synapses lead to modification of synaptic strength. One fruitful area of research over the last twenty years has been the postsynaptic signaling apparatus in glutamatergic spines (8, 97, 99, 106). Spines contain clusters of receptors and signaling proteins located in a dense submembranous structure that can be seen in the electron microscope and is called the postsynaptic density or PSD (For review of early work see 98). In the 1970’s, Philip Siekevitz (40) and Carl Cotman (42) worked out subcellular fractionation methods to purify tiny disks from brain that contain many of the proteins that are most tightly bound to the PSD. Their purification methods were later used to obtain material from which several ubiquitous PSD proteins were identified and sequenced, including the prototypical scaffold protein, PSD-95 (37, 109, 113), synGAP (35), the shank/proSAP family (20), and densin (6). At the same time, and independently, some of these proteins, and additional new ones from the PSD fraction, were identified by their interactions in yeast two hybrid screens, including GKAP (104), synGAP (107), shank/proSAP (158), and homer (24). Continuing proteomic analyses of the PSD fraction or related “NMDA-receptor complexes” (89, 127, 177, 235) have produced a catalog of putative PSD-associated proteins that comprises a few hundred individual proteins. More recent studies have begun to address the crucial question of the relative abundance of each protein in the PSD fraction and their stoichiometric ratios (126, 127, 177). However, we are still in the early stages of understanding which core protein complexes are present in most excitatory synapses, and which proteins are present only in a subset of synapses, perhaps conferring specialized properties.