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Abstract Determining the genetic architecture of Alzheimer’s disease pathologies can enhance mechanistic understanding and inform precision medicine strategies. Here, we perform a genome-wide association study of cortical tau quantified by positron emission tomography in 3046 participants from 12 independent studies. The CYP1B1-RMDN2 locus is associated with tau deposition. The most significant signal is at rs2113389, explaining 4.3% of the variation in cortical tau, while APOE4 rs429358 accounts for 3.6%. rs2113389 is associated with higher tau and faster cognitive decline. Additive effects, but no interactions, are observed between rs2113389 and diagnosis, APOE4, and amyloid beta positivity. CYP1B1 expression is upregulated in AD. rs2113389 is associated with higher CYP1B1 expression and methylation levels. Mouse model studies provide additional functional evidence for a relationship between CYP1B1 and tau deposition but not amyloid beta. These results provide insight into the genetic basis of cerebral tau deposition and support novel pathways for therapeutic development in AD.

Abstract Background The APOE gene is the strongest genetic risk factor for late-onset Alzheimer’s Disease (LOAD). However, the gene regulatory mechanisms at this locus remain incompletely characterized. Methods To identify novel AD-linked functional elements within the APOE locus, we integrated SNP variants with multi-omics data from human postmortem brains including 2,179 RNA-seq samples from 3 brain regions and two ancestries (European and African), 667 DNA methylation samples, and ChIP-seq samples. Additionally, we plotted the expression trajectory of APOE transcripts in human brains during development. Results We identified an AD-linked APOE transcript (jxn1.2.2) particularly observed in the dorsolateral prefrontal cortex (DLPFC). The APOE jxn1.2.2 transcript is associated with brain neuropathological features, cognitive impairment, and the presence of the APOE4 allele in DLPFC. We prioritized two independent functional SNPs (rs157580 and rs439401) significantly associated with jxn1.2.2 transcript abundance and DNA methylation levels. These SNPs are located within active chromatin regions and affect brain-related transcription factor-binding affinities. The two SNPs shared effects on the jxn1.2.2 transcript between European and African ethnic groups. Conclusion The novel APOE functional elements provide potential therapeutic targets with mechanistic insight into the disease etiology.

Abstract Multiple reference panels of a given tissue or multiple tissues often exist, and multiple regression methods could be used for training gene expression imputation models for transcriptome-wide association studies (TWAS). To leverage expression imputation models (i.e., base models) trained with multiple reference panels, regression methods, and tissues, we develop a Stacked Regression based TWAS (SR-TWAS) tool which can obtain optimal linear combinations of base models for a given validation transcriptomic dataset. Both simulation and real studies show that SR-TWAS improves power, due to increased training sample sizes and borrowed strength across multiple regression methods and tissues. Leveraging base models across multiple reference panels, tissues, and regression methods, our real studies identify 6 independent significant risk genes for Alzheimer’s disease (AD) dementia for supplementary motor area tissue and 9 independent significant risk genes for Parkinson’s disease (PD) for substantia nigra tissue. Relevant biological interpretations are found for these significant risk genes.

Abstract The emergence of single nucleus RNA sequencing (snRNA-seq) offers to revolutionize the study of Alzheimer’s disease (AD). Integration with complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link cell subpopulations and molecular networks with a broader disease-relevant context. We report snRNA-seq profiles from superior frontal gyrus samples from 101 well characterized subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in hematological parameters. We observed an AD-associated CD83(+) microglial subtype with unique molecular networks and which is associated with immunoglobulin IgG4 production in the transverse colon. Our major observations were replicated in two additional, independent snRNA-seq data sets. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal disease biology.

Abstract Accumulation of amyloid-β (Aβ) and tau tangles are hallmarks of Alzheimer’s disease. Aβ is extracellular while tau tangles are typically intracellular, and it is unknown how these two proteinopathies are connected. Here, we use data of 1206 elders and test that RNA expression levels of GPER1, a transmembrane protein, modify the association of Aβ with tau tangles. GPER1 RNA expression is related to more tau tangles (p = 0.001). Moreover, GPER1 expression modifies the association of immunohistochemistry-derived Aβ load with tau tangles (p = 0.044). Similarly, GPER1 expression modifies the association between Aβ proteoforms and tau tangles: total Aβ protein (p = 0.030) and Aβ38 peptide (p = 0.002). Using single nuclei RNA-seq indicates that GPER1 RNA expression in astrocytes modifies the relation of Aβ load with tau tangles (p = 0.002), but not GPER1 in excitatory neurons or endothelial cells. We conclude that GPER1 may be a link between Aβ and tau tangles driven mainly by astrocytic GPER1 expression.

The cortical epigenetic clock was developed in brain tissue as a biomarker of brain aging. As one way to identify mechanisms underlying aging, we conducted a GWAS of cortical age. We leveraged postmortem cortex tissue and genotyping array data from 694 participants of the Rush Memory and Aging Project and Religious Orders Study (ROSMAP; 11000,000 SNPs), and meta-analysed ROSMAP with 522 participants of Brains for Dementia Research (5,000,000 overlapping SNPs). We confirmed results using eQTL (cortical bulk and single nucleus gene expression), cortical protein levels (ROSMAP), and phenome-wide association studies (clinical/neuropathologic phenotypes, ROSMAP). In the meta-analysis, the strongest association was rs4244620 (p = 1.29 × 10−7), which also exhibited FDR-significant cis-eQTL effects for CD46 in bulk and single nucleus (microglia, astrocyte, oligodendrocyte, neuron) cortical gene expression. Additionally, rs4244620 was nominally associated with lower cognition, faster slopes of cognitive decline, and greater Parkinsonian signs (n ~ 1700 ROSMAP with SNP/phenotypic data; all p ≤ 0.04). In ROSMAP alone, the top SNP was rs4721030 (p = 8.64 × 10−8) annotated to TMEM106B and THSD7A. Further, in ROSMAP (n = 849), TMEM106B and THSD7A protein levels in cortex were related to many phenotypes, including greater AD pathology and lower cognition (all p ≤ 0.0007). Overall, we identified converging evidence of CD46 and possibly TMEM106B/THSD7A for potential roles in cortical epigenetic clock age.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in nonhuman species and recently demonstrated to occur in rare instances from one human generation to the next. Here, we investigated numtogenesis dynamics in humans in 2 ways. First, we quantified Numts in 1,187 postmortem brain and blood samples from different individuals. Compared to circulating immune cells (n = 389), postmitotic brain tissue (n = 798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples, we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex (DLPFC) compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, an increase in the number of brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment (NCI) who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures whole-genome sequencing design in a human fibroblast model that recapitulates several molecular hallmarks of aging. These longitudinal experiments revealed a gradual accumulation of 1 Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven by cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human postmitotic tissues produces functionally relevant human Numts over timescales shorter than previously assumed.

Abstract Changes in high-affinity nicotinic acetylcholine receptors are intricately connected to neuropathology in Alzheimer’s Disease (AD). Protective and cognitive-enhancing roles for the nicotinic α5 subunit have been identified, but this gene has not been closely examined in the context of human aging and dementia. Therefore, we investigate the nicotinic α5 gene CHRNA5 and the impact of relevant single nucleotide polymorphisms (SNPs) in prefrontal cortex from 922 individuals with matched genotypic and post-mortem RNA sequencing in the Religious Orders Study and Memory and Aging Project (ROS/MAP). We find that a genotype robustly linked to increased expression of CHRNA5 (rs1979905A2) predicts significantly reduced cortical β-amyloid load. Intriguingly, co-expression analysis suggests CHRNA5 has a distinct cellular expression profile compared to other nicotinic receptor genes. Consistent with this prediction, single nucleus RNA sequencing from 22 individuals reveals CHRNA5 expression is disproportionately elevated in chandelier neurons, a distinct subtype of inhibitory neuron known for its role in excitatory/inhibitory (E/I) balance. We show that chandelier neurons are enriched in amyloid-binding proteins compared to basket cells, the other major subtype of PVALB-positive interneurons. Consistent with the hypothesis that nicotinic receptors in chandelier cells normally protect against β-amyloid, cell-type proportion analysis from 549 individuals reveals these neurons show amyloid-associated vulnerability only in individuals with impaired function/trafficking of nicotinic α5-containing receptors due to homozygosity of the missense CHRNA5 SNP (rs16969968A2). Taken together, these findings suggest that CHRNA5 and its nicotinic α5 subunit exert a neuroprotective role in aging and Alzheimer’s disease centered on chandelier interneurons.

Abstract Microglia and neuroinflammation play an important role in the development and progression of Alzheimer’s disease (AD). Inositol polyphosphate-5-phosphatase D (INPP5D/SHIP1) is a myeloid-expressed gene genetically-associated with AD. Through unbiased analyses of RNA and protein profiles in INPP5D-disrupted iPSC-derived human microglia, we find that reduction in INPP5D activity is associated with molecular profiles consistent with disrupted autophagy and inflammasome activation. These findings are validated through targeted pharmacological experiments which demonstrate that reduced INPP5D activity induces the formation of the NLRP3 inflammasome, cleavage of CASP1, and secretion of IL-1β and IL-18. Further, in-depth analyses of human brain tissue across hundreds of individuals using a multi-analytic approach provides evidence that a reduction in function of INPP5D in microglia results in inflammasome activation in AD. These findings provide insights into the molecular mechanisms underlying microglia-mediated processes in AD and highlight the inflammasome as a potential therapeutic target for modulating INPP5D-mediated vulnerability to AD.

Abstract Many of the Alzheimer’s disease (AD) risk genes are specifically expressed in microglia and astrocytes, but how and when the genetic risk localizing to these cell types contributes to AD pathophysiology remains unclear. Here, we derive cell-type-specific AD polygenic risk scores (ADPRS) from two extensively characterized datasets and uncover the impact of cell-type-specific genetic risk on AD endophenotypes. In an autopsy dataset spanning all stages of AD (n = 1457), the astrocytic ADPRS affected diffuse and neuritic plaques (amyloid-β), while microglial ADPRS affected neuritic plaques, microglial activation, neurofibrillary tangles (tau), and cognitive decline. In an independent neuroimaging dataset of cognitively unimpaired elderly (n = 2921), astrocytic ADPRS was associated with amyloid-β, and microglial ADPRS was associated with amyloid-β and tau, connecting cell-type-specific genetic risk with AD pathology even before symptom onset. Together, our study provides human genetic evidence implicating multiple glial cell types in AD pathophysiology, starting from the preclinical stage.

Abstract Background Insulin resistance (IR) is a major risk factor for Alzheimer’s disease (AD) dementia. The mechanisms by which IR predisposes to AD are not well-understood. Epigenetic studies may help identify molecular signatures of IR associated with AD, thus improving our understanding of the biological and regulatory mechanisms linking IR and AD. Methods We conducted an epigenome-wide association study of IR, quantified using the homeostatic model assessment of IR (HOMA-IR) and adjusted for body mass index, in 3,167 participants from the Framingham Heart Study (FHS) without type 2 diabetes at the time of blood draw used for methylation measurement. We identified DNA methylation markers associated with IR at the genome-wide level accounting for multiple testing (P

Abstract Telomere length (TL) attrition, epigenetic age acceleration, and mitochondrial DNA copy number (mtDNAcn) decline are established hallmarks of aging. Each has been individually associated with Alzheimer’s dementia, cognitive function, and pathologic Alzheimer’s disease (AD). Epigenetic age and mtDNAcn have been studied in brain tissue directly but prior work on TL in brain is limited to small sample sizes and most studies have examined leukocyte TL. Importantly, TL, epigenetic age clocks, and mtDNAcn have not been studied jointly in brain tissue from an AD cohort. We examined dorsolateral prefrontal cortex (DLPFC) tissue from N = 367 participants of the Religious Orders Study (ROS) or the Rush Memory and Aging Project (MAP). TL and mtDNAcn were estimated from whole genome sequencing (WGS) data and cortical clock age was computed on 347 CpG sites. We examined dementia, MCI, and level of and change in cognition, pathologic AD, and three quantitative AD traits, as well as measures of other neurodegenerative diseases and cerebrovascular diseases (CVD). We previously showed that mtDNAcn from DLPFC brain tissue was associated with clinical and pathologic features of AD. Here, we show that those associations are independent of TL. We found TL to be associated with β-amyloid levels (beta = − 0.15, p = 0.023), hippocampal sclerosis (OR = 0.56, p = 0.0015) and cerebral atherosclerosis (OR = 1.44, p = 0.0007). We found strong associations between mtDNAcn and clinical measures of AD. The strongest associations with pathologic measures of AD were with cortical clock and there were associations of mtDNAcn with global AD pathology and tau tangles. Of the other pathologic traits, mtDNAcn was associated with hippocampal sclerosis, macroscopic infarctions and CAA and cortical clock was associated with Lewy bodies. Multi-modal age acceleration, accelerated aging on both mtDNAcn and cortical clock, had greater effect size than a single measure alone. These findings highlight for the first time that age acceleration determined on multiple genomic measures, mtDNAcn and cortical clock may have a larger effect on AD/AD related disorders (ADRD) pathogenesis than single measures.

Cognitive impairment in the elderly features complex molecular pathophysiology extending beyond the hallmark pathologies of traditional disease classification. Molecular subtyping using large-scale -omic strategies can help resolve this biological heterogeneity. Using quantitative mass spectrometry, we measured ∼8000 proteins across >600 dorsolateral prefrontal cortex tissues with clinical diagnoses of no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer's disease (AD) dementia. Unbiased classification of MCI and AD cases based on individual proteomic profiles resolved three classes with expression differences across numerous cell types and biological ontologies. Two classes displayed molecular signatures atypical of AD neurodegeneration, such as elevated synaptic and decreased inflammatory markers. In one class, these atypical proteomic features were associated with clinical and pathological hallmarks of cognitive resilience. We were able to replicate these classes and their clinicopathological phenotypes across two additional tissue cohorts. These results promise to better define the molecular heterogeneity of cognitive impairment and meaningfully impact its diagnostic and therapeutic precision.

Abstract Background Genetic variants within nearly 1000 loci are known to contribute to modulation of blood lipid levels. However, the biological pathways underlying these associations are frequently unknown, limiting understanding of these findings and hindering downstream translational efforts such as drug target discovery. Results To expand our understanding of the underlying biological pathways and mechanisms controlling blood lipid levels, we leverage a large multi-ancestry meta-analysis (N = 1,654,960) of blood lipids to prioritize putative causal genes for 2286 lipid associations using six gene prediction approaches. Using phenome-wide association (PheWAS) scans, we identify relationships of genetically predicted lipid levels to other diseases and conditions. We confirm known pleiotropic associations with cardiovascular phenotypes and determine novel associations, notably with cholelithiasis risk. We perform sex-stratified GWAS meta-analysis of lipid levels and show that 3–5% of autosomal lipid-associated loci demonstrate sex-biased effects. Finally, we report 21 novel lipid loci identified on the X chromosome. Many of the sex-biased autosomal and X chromosome lipid loci show pleiotropic associations with sex hormones, emphasizing the role of hormone regulation in lipid metabolism. Conclusions Taken together, our findings provide insights into the biological mechanisms through which associated variants lead to altered lipid levels and potentially cardiovascular disease risk.

Summary: SORL1 is implicated in the pathogenesis of Alzheimer’s disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain. Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor β (TGF-β)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.

IntroductionMonitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations.MethodsWe evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique.ResultsOverall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson’s ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5% vs. 68.0% in SFC and MC, respectively; p20) of antibody reagents.

Abstract In advancing age, some individuals maintain a stable cognitive performance over time, while others experience a rapid decline. Such variation in cognitive trajectory is only partially explained by common neurodegenerative pathologies. Hence, we aimed to identify new molecular processes underlying variation in cognitive trajectory using brain microRNA profile followed by an integrative analysis with brain transcriptome and proteome. Individual cognitive trajectories were derived from longitudinally assessed cognitive-test scores of older-adult brain donors from four longitudinal cohorts. Postmortem brain microRNA profiles, transcriptomes, and proteomes were derived from the dorsolateral prefrontal cortex. The global microRNA association study of cognitive trajectory was performed in a discovery (n = 454) and replication cohort (n = 134), followed by a meta-analysis that identified 6 microRNAs. Among these, miR-132-3p and miR-29a-3p were most significantly associated with cognitive trajectory. They explain 18.2% and 2.0% of the variance of cognitive trajectory, respectively, and act independently of the eight measured neurodegenerative pathologies. Furthermore, integrative transcriptomic and proteomic analyses revealed that miR-132-3p was significantly associated with 24 of the 47 modules of co-expressed genes of the transcriptome, miR-29a-3p with 3 modules, and identified 84 and 214 downstream targets of miR-132-3p and miR-29a-3p, respectively, in cognitive trajectory. This is the first global microRNA study of cognitive trajectory to our knowledge. We identified miR-29a-3p and miR-132-3p as novel and robust contributors to cognitive trajectory independently of the eight known cerebral pathologies. Our findings lay a foundation for future studies investigating mechanisms and developing interventions to enhance cognitive stability in advanced age.

Identifying ancestry-specific molecular profiles of late-onset Alzheimer's Disease (LOAD) in brain tissue is crucial to understand novel mechanisms and develop effective interventions in non-European, high-risk populations. We performed gene differential expression (DE) and consensus network-based analyses in RNA-sequencing data of postmortem brain tissue from 39 Caribbean Hispanics (CH). To identify ancestry-concordant and -discordant expression profiles, we compared our results to those from two independent non-Hispanic White (NHW) samples (n = 731). In CH, we identified 2802 significant DE genes, including several LOAD known-loci. DE effects were highly concordant across ethnicities, with 373 genes transcriptome-wide significant in all three cohorts. Cross-ancestry meta-analysis found NPNT to be the top DE gene. We replicated over 82% of meta-analyses genome-wide signals in single-nucleus RNA-seq data (including NPNT and LOAD known-genes SORL1, FBXL7, CLU, ABCA7). Increasing representation in genetic studies will allow for deeper understanding of ancestry-specific mechanisms and improving precision treatment options in understudied groups.

Resources reporting RNA editing sites from brain tissue have been published. Here, the authors provide an atlas of RNA editing events found in the aged and Alzheimer’s disease human brain tissue resulting in changes at protein level.

Integrating functional data with GWAS loci can help interpret the function of genetic variants associated with disease. Here the authors map cis and trans methylation QTL in CD4 + T cells from patients and colocalize with GWAS loci in order to interpret genetic variants associated with multiple sclerosis.

Abstract Background Mitochondrial dysfunction is a feature of neurodegenerative diseases, including Alzheimer’s disease (AD). Changes in the mitochondrial DNA copy number (mtDNAcn) and increased mitochondrial DNA mutation burden have both been associated with neurodegenerative diseases and cognitive decline. This study aims to systematically identify which common brain pathologies in the aged human brain are associated with mitochondrial recalibrations and to disentangle the relationship between these pathologies, mtDNAcn, mtDNA heteroplasmy, aging, neuronal loss, and cognitive function. Methods Whole-genome sequencing data from n = 1361 human brain samples from 5 different regions were used to quantify mtDNAcn as well as heteroplasmic mtDNA point mutations and small indels. Brain samples were assessed for 10 common pathologies. Annual cognitive test results were used to assess cognitive function proximal to death. For a subset of samples, neuronal proportions were estimated from RNA-seq profiles, and mass spectrometry was used to quantify the mitochondrial protein content of the tissue. Results mtDNAcn was 7–14% lower in AD relative to control participants. When accounting for all 10 common neuropathologies, only tau was significantly associated with lower mtDNAcn in the dorsolateral prefrontal cortex. In the posterior cingulate cortex, TDP-43 pathology demonstrated a distinct association with mtDNAcn. No changes were observed in the cerebellum, which is affected late by pathologies. Neither age nor gender was associated with mtDNAcn in the studied brain regions when adjusting for pathologies. Mitochondrial content and mtDNAcn independently explained variance in cognitive function unaccounted by pathologies, implicating complex mitochondrial recalibrations in cognitive decline. In contrast, mtDNA heteroplasmy levels increased by 1.5% per year of life in the cortical regions, but displayed no association with any of the pathologies or cognitive function. Conclusions We studied mtDNA quantity and quality in relation to mixed pathologies of aging and showed that tau and not amyloid-β is primarily associated with reduced mtDNAcn. In the posterior cingulate cortex, the association of TDP-43 with low mtDNAcn points to a vulnerability of this region in limbic-predominant age-related TDP-43 encephalopathy. While we found low mtDNAcn in brain regions affected by pathologies, the absence of associations with mtDNA heteroplasmy burden indicates that mtDNA point mutations and small indels are unlikely to be involved in the pathogenesis of late-onset neurodegenerative diseases.

Abstract Cognitive decline is associated with both normal aging and early pathologies leading to dementia. Here we used quantitative profiling of metabolites involved in the regulation of inflammation, vascular function, neuronal function and energy metabolism, including oxylipins, endocannabinoids, bile acids, and steroid hormones to identify metabolic biomarkers of mild cognitive impairment (MCI). Serum samples (n = 212) were obtained from subjects with or without MCI opportunistically collected with incomplete fasting state information. To maximize power and stratify the analysis of metabolite associations with MCI by the fasting state, we developed an algorithm to predict subject fasting state when unknown (n = 73). In non-fasted subjects, linoleic acid and palmitoleoyl ethanolamide levels were positively associated with perceptual speed. In fasted subjects, soluble epoxide hydrolase activity and tauro-alpha-muricholic acid levels were negatively associated with perceptual speed. Other cognitive domains showed associations with bile acid metabolism, but only in the non-fasted state. Importantly, this study shows unique associations between serum metabolites and cognitive function in the fasted and non-fasted states and provides a fasting state prediction algorithm based on measurable metabolites.

The molecular basis of Alzheimer’s Disease has been obscured by heterogeneity and scarcity of brain gene expression data, which limit effectiveness in complex models. Here, the authors introduce a multi-task deep learning framework to learn generalizable and nuanced relationships between gene expression and neuropathology.

Summary: Existing methods for integrating functional annotations in genome-wide association studies (GWASs) to fine-map and prioritize potential causal variants are limited to using non-overlapped categorical annotations or limited by the computation burden of modeling genome-wide variants. To overcome these limitations, we propose a scalable Bayesian functional GWAS method to account for multivariate quantitative functional annotations (BFGWAS_QUANT), accompanied by a scalable computation algorithm enabling joint modeling of genome-wide variants. Simulation studies validated the performance of BFGWAS_QUANT for accurately quantifying annotation enrichment and improving GWAS power. Applying BFGWAS_QUANT to study five Alzheimer disease (AD)-related phenotypes using individual-level GWAS data (n = ∼1,000), we found that histone modification annotations have higher enrichment than expression quantitative trait locus (eQTL) annotations for all considered phenotypes, with the highest enrichment in H3K27me3 (polycomb regression). We also found that cis-eQTLs in microglia had higher enrichment than eQTLs of bulk brain frontal cortex tissue for all considered phenotypes. A similar enrichment pattern was also identified using the International Genomics of Alzheimer’s Project (IGAP) summary-level GWAS data of AD (n = ∼54,000). The strongest known APOE E4 risk allele was identified for all five phenotypes, and the APOE locus was validated using the IGAP data. BFGWAS_QUANT fine-mapped 32 significant variants from 1,073 genome-wide significant variants in the IGAP data. We also demonstrated that the polygenic risk scores (PRSs) using effect size estimates by BFGWAS_QUANT had a similar prediction accuracy as other methods assuming a sparse causal model. Overall, BFGWAS_QUANT is a useful GWAS tool for quantifying annotation enrichment and prioritizing potential causal variants.

Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations.

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual “expert-based”) gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.

Array-based epigenome-wide association studies only test about 2% of the CpG sites in the genome. Here, the authors describe EWASplus, a supervised machine learning strategy that extends EWAS coverage to the entire genome, and use it to identify novel brain CpGs associated with Alzheimer’s disease.

Although epigenome-wide association studies of Alzheimer’s disease have highlighted neuropathology-associated DNA methylation differences, previous studies have been limited in sample size and brain region used. Here, the authors combine data from six DNA methylomic studies of Alzheimer’s disease (N = 1453 unique individuals) to identify differentially methylated loci across cortex.

Cortical neuron loss is a pathological hallmark of late-onset Alzheimer’s disease (AD). However, it remains unclear which neuronal subtypes beyond broad excitatory and inhibitory classes are most vulnerable. Here, we analyzed cell subtype proportion differences in AD compared to non-AD controls using 1037 post-mortem brain samples from six neocortical regions. We identified the strongest associations of AD with fewer somatostatin (SST) inhibitory neurons (β = −0.48, pbonf = 8.98 × 10–9) and intra-telencephalic (IT) excitatory neurons (β = -0.45, pbonf = 4.32 × 10–7). Replication in three AD case-control single-nucleus RNAseq datasets most strongly supported the bulk tissue association of fewer SST neurons in AD. In depth analyses of cell type proportions with specific AD-related neuropathological and cognitive phenotypes revealed fewer SST neurons with greater brain-wide post-mortem tau and beta amyloid, as well as a faster rate of antemortem cognitive decline. In contrast, greater IT neuron proportions were associated with a slower rate of cognitive decline as well as greater residual cognition–a measure of cognitive resilience–but not canonical AD neuropathology. Our findings implicate somatostatin inhibitory and intra-telencephalic excitatory neuron subclasses in the pathogenesis of AD and in cognitive resilience to AD pathology, respectively.

Abstract Motor resilience proteins may be a high value therapeutic target that offset the negative effects of pathologies on motor function. This study sought to identify cortical proteins associated with motor decline unexplained by brain pathologies that provide motor resilience. We studied 1226 older decedents with annual motor testing, postmortem brain pathologies and quantified 226 proteotypic peptides in prefrontal cortex. Twenty peptides remained associated with motor decline in models controlling for ten brain pathologies (FDR

Abstract Objective To report initial results of a planned multicenter year‐long prospective study examining the risk and impact of COVID‐19 among persons with neuroinflammatory disorders (NID), particularly multiple sclerosis (MS). Methods In April 2020, we deployed online questionnaires to individuals in their home environment to assess the prevalence and potential risk factors of suspected COVID‐19 in persons with NID (PwNID) and change in their neurological care. Results Our cohort included 1115 participants (630 NID, 98% MS; 485 reference) as of 30 April 2020. 202 (18%) participants, residing in areas with high COVID‐19 case prevalence, met the April 2020 CDC symptom criteria for suspected COVID‐19, but only 4% of all participants received testing given testing shortages. Among all participants, those with suspected COVID‐19 were younger, more racially diverse, and reported more depression and liver disease. PwNID had the same rate of suspected COVID‐19 as the reference group. Early changes in disease management included telemedicine visits in 21% and treatment changes in 9% of PwNID. After adjusting for potential confounders, increasing neurological disability was associated with a greater likelihood of suspected COVID‐19 (ORadj = 1.45, 1.17–1.84). Interpretations Our study of real‐time, patient‐reported experience during the COVID‐19 pandemic complements physician‐reported MS case registries which capture an excess of severe cases. Overall, PwNID seem to have a risk of suspected COVID‐19 similar to the reference population.

Abstract Background Single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies only explain part of the heritability of Alzheimer’s disease (AD). Epistasis has been considered as one of the main causes of “missing heritability” in AD. Methods We performed genome-wide epistasis screening (N = 10,389) for the clinical diagnosis of AD using three popularly adopted methods. Subsequent analyses were performed to eliminate spurious associations caused by possible confounding factors. Then, candidate genetic interactions were examined for their co-expression in the brains of AD patients and analyzed for their association with intermediate AD phenotypes. Moreover, a new approach was developed to compile the epistasis risk factors into an epistasis risk score (ERS) based on multifactor dimensional reduction. Two independent datasets were used to evaluate the feasibility of ERSs in AD risk prediction. Results We identified 2 candidate genetic interactions with P FDR

Abstract Microglial dysfunction has been proposed as one of the many cellular mechanisms that can contribute to the development of Alzheimer’s disease (AD). Here, using a transcriptional network map of the human frontal cortex, we identify five modules of co-expressed genes related to microglia and assess their role in the neuropathologic features of AD in 540 subjects from two cohort studies of brain aging. Two of these transcriptional programs—modules 113 and 114—relate to the accumulation of β-amyloid, while module 5 relates to tau pathology. We replicate these associations in brain epigenomic data and in two independent datasets. In terms of tau, we propose that module 5, a marker of activated microglia, may lead to tau accumulation and subsequent cognitive decline. We validate our model further by showing that three representative module 5 genes (ACADVL, TRABD, and VASP) encode proteins that are upregulated in activated microglia in AD.

Muscle weakness has been associated with morbidity and mortality in older people. Here, the authors have investigated this trait further by performing a genome-wide meta-analysis of grip strength and Mendelian randomization to discover causal relationships between muscle weakness and other diseases.

White matter hyperintensities (WMH) are a common brain-imaging feature of cerebral small vessel disease. Here, the authors carry out a GWAS and followup analyses for WMH-volume, implicating several variants with potential for risk stratification and drug targeting.

Follicular-helper T cells (TFH) are an essential arm of the adaptive immune system. Although TFH were first discovered through their ability to contribute to antibody affinity maturation through co-stimulatory interactions with B cells, new light has been shed on their ability to remain a complex and functionally plastic cell type. Due to a lack sample availability, however, many studies have been limited to characterizing TFH in mice or non-canonical tissue types, such as peripheral blood. Such constraints have resulted in a limited, and sometimes contradictory, understanding of this fundamental cell type. One subset of TFH receiving attention in chronic infection are CXCR3-expressing TFH cells (CXCR3+TFH) due to their abnormal accumulation in secondary lymphoid tissues. Their function and clonal relationship with other TFH subsets in lymphoid tissues during infection, however, remains largely unclear. We thus systematically investigated this and other subsets of TFH within untreated HIV-infected human lymph nodes using Mass CyTOF and a combination of RNA and TCR repertoire sequencing. We show an inflation of the CXCR3+TFH compartment during HIV infection that correlates with a lower HIV burden. Deeper analysis into this population revealed a functional shift of CXCR3+TFH away from germinal center TFH (GC-TFH), including the altered expression of several important transcription factors and cytokines. CXCR3+TFH also upregulated cell migration transcriptional programs and were clonally related to peripheral TFH populations. In combination, these data suggest that CXCR3+TFH have a greater tendency to enter circulation than their CXCR3- counterparts, potentially functioning through distinct modalities that may lead to enhanced defense.

Mitochondrial respiratory chain (RC) function requires the stoichiometric interaction among dozens of proteins but their co-regulation has not been defined in the human brain. Here, using quantitative proteomics across three independent cohorts we systematically characterized the co-regulation patterns of mitochondrial RC proteins in the human dorsolateral prefrontal cortex (DLPFC). Whereas the abundance of RC protein subunits that physically assemble into stable complexes were correlated, indicating their co-regulation, RC assembly factors exhibited modest co-regulation. Within complex I, nuclear DNA-encoded subunits exhibited >2.5-times higher co-regulation than mitochondrial (mt)DNA-encoded subunits. Moreover, mtDNA copy number was unrelated to mtDNA-encoded subunits abundance, suggesting that mtDNA content is not limiting. Alzheimer's disease (AD) brains exhibited reduced abundance of complex I RC subunits, an effect largely driven by a 2–4% overall lower mitochondrial protein content. These findings provide foundational knowledge to identify molecular mechanisms contributing to age- and disease-related erosion of mitochondrial function in the human brain.

The limited understanding of the temporal molecular changes in late-onset Alzheimer’s disease hinder the development of therapeutic treatment. The authors use manifold learning to develop a molecular model for disease progression from RNASeq data from human postmortem brain samples.

Imbalance of microglial phenotypes in the aging brain might underlie their involvement in late onset neurodegenerative diseases. Here we report the population structure of microglia in the aged human brain and the reduction of a particular microglia subset in individuals with Alzheimer’s disease .

Abstract Epidemiological studies have suggested differences in the rate of multiple sclerosis (MS) in individuals of European ancestry compared to African ancestry, motivating genetic scans to identify variants that could contribute to such patterns. In a whole-genome scan in 899 African-American cases and 1155 African-American controls, we confirm that African-Americans who inherit segments of the genome of European ancestry at a chromosome 1 locus are at increased risk for MS [logarithm of odds (LOD) = 9.8], although the signal weakens when adding an additional 406 cases, reflecting heterogeneity in the two sets of cases [logarithm of odds (LOD) = 2.7]. The association in the 899 individuals can be fully explained by two variants previously associated with MS in European ancestry individuals. These variants tag a MS susceptibility haplotype associated with decreased CD58 gene expression (odds ratio of 1.37; frequency of 84% in Europeans and 22% in West Africans for the tagging variant) as well as another haplotype near the FCRL3 gene (odds ratio of 1.07; frequency of 49% in Europeans and 8% in West Africans). Controlling for all other genetic and environmental factors, the two variants predict a 1.44-fold higher rate of MS in European-Americans compared to African-Americans.

Abstract Background Tau neurofibrillary tangle pathology characterizes Alzheimer’s disease and other neurodegenerative tauopathies. Brain gene expression profiles can reveal mechanisms; however, few studies have systematically examined both the transcriptome and proteome or differentiated Tau- versus age-dependent changes. Methods Paired, longitudinal RNA-sequencing and mass-spectrometry were performed in a Drosophila model of tauopathy, based on pan-neuronal expression of human wildtype Tau (TauWT) or a mutant form causing frontotemporal dementia (TauR406W). Tau-induced, differentially expressed transcripts and proteins were examined cross-sectionally or using linear regression and adjusting for age. Hierarchical clustering was performed to highlight network perturbations, and we examined overlaps with human brain gene expression profiles in tauopathy. Results TauWT induced 1514 and 213 differentially expressed transcripts and proteins, respectively. TauR406W had a substantially greater impact, causing changes in 5494 transcripts and 697 proteins. There was a ~ 70% overlap between age- and Tau-induced changes and our analyses reveal pervasive bi-directional interactions. Strikingly, 42% of Tau-induced transcripts were discordant in the proteome, showing opposite direction of change. Tau-responsive gene expression networks strongly implicate innate immune activation. Cross-species analyses pinpoint human brain gene perturbations specifically triggered by Tau pathology and/or aging, and further differentiate between disease amplifying and protective changes. Conclusions Our results comprise a powerful, cross-species functional genomics resource for tauopathy, revealing Tau-mediated disruption of gene expression, including dynamic, age-dependent interactions between the brain transcriptome and proteome.

Abstract Background Identified as an Alzheimer’s disease (AD) susceptibility gene by genome wide-association studies, BIN1 has 10 isoforms that are expressed in the Central Nervous System (CNS). The distribution of these isoforms in different cell types, as well as their role in AD pathology still remains unclear. Methods Utilizing antibodies targeting specific BIN1 epitopes in human post-mortem tissue and analyzing mRNA expression data from purified microglia, we identified three isoforms expressed in neurons and astrocytes (isoforms 1, 2 and 3) and four isoforms expressed in microglia (isoforms 6, 9, 10 and 12). The abundance of selected peptides, which correspond to groups of BIN1 protein isoforms, was measured in dorsolateral prefrontal cortex, and their relation to neuropathological features of AD was assessed. Results Peptides contained in exon 7 of BIN1’s N-BAR domain were found to be significantly associated with AD-related traits and, particularly, tau tangles. Decreased expression of BIN1 isoforms containing exon 7 is associated with greater accumulation of tangles and subsequent cognitive decline, with astrocytic rather than neuronal BIN1 being the more likely culprit. These effects are independent of the BIN1 AD risk variant. Conclusions Exploring the molecular mechanisms of specific BIN1 isoforms expressed by astrocytes may open new avenues for modulating the accumulation of Tau pathology in AD.

APOE is the major genetic risk factor for Alzheimer’s disease. In a large number of neuropathologically confirmed cases and controls, the impact of different APOE genotypes on Alzheimer’s dementia risk was greater than previously thought and APOE2 homozygotes had an exceptionally low risk.

The Major Histocompatibility Complex (MHC) makes the largest genetic contribution to multiple sclerosis (MS) susceptibility, with 32 independent effects across the region explaining 20% of the heritability in European populations. Variation is high across populations with allele frequency differences and population-specific risk alleles identified. We sought to identify MHC-specific MS susceptibility variants and assess the effect of ancestral risk modification within 2652 Latinx and Hispanic individuals as well as 2435 Black and African American individuals. We have identified several novel susceptibility alleles which are rare in European populations including HLA-B*53:01, and we have utilized the differing linkage disequilibrium patterns inherent to these populations to identify an independent role for HLA-DRB1*15:01 and HLA-DQB1*06:02 on MS risk. We found a decrease in Native American ancestry in MS cases vs controls across the MHC, peaking near the previously identified MICB locus with a decrease of ~5.5% in Hispanics and ~0.4% in African Americans. We have identified several susceptibility variants, including within the MICB gene region, which show global ancestry risk modification and indicate ancestral differences which may be due in part to correlated environmental factors. We have also identified several susceptibility variants for which MS risk is modified by local ancestry and indicate true ancestral genetic differences; including HLA-DQB1*06:02 for which MS risk for European allele carriers is almost two times the risk for African allele carriers. These results validate the importance of investigating MS susceptibility at an ancestral level and offer insight into the epidemiology of MS phenotypic diversity.

Some individuals maintain cognitive health despite neuropathology. Targets impacting “cognitive resilience” may provide interventions for preventing dementia without decreasing neuropathology. Neuroticism represents the tendency to experience negative emotions, and is related to worse cognitive resilience. Exploring proteins associated with cognitive resilience risk factors, such as neuroticism, could yield new protein targets. We used 355 postmortem prefrontal cortex from two cohorts to measure 8356 proteins. We identified (i) proteins associated with both neuroticism and cognitive resilience, and (ii) proteins statistically mediating relations of neuroticism to cognitive resilience. We found two proteins, 40S ribosomal proteinS3 (RPS3) and branched chain keto acid dehydrogenase E1, subunit beta (BCKDHB), ranked in the top 1% of smallest p-values in parallel linear regression models of neuroticism to protein levels, and protein levels to cognitive decline resilience. In mediation models, RPS3 and BCKDHB accounted for 25% (p = 0.005) of the relation of neuroticism to cognitive resilience. Our sample size is modest, thus results may be due to chance (p-values did not meet Bonferroni significance) and will require further confirmation; however, investigating biologic mediators of associations of risk factors to cognitive resilience may help discover targets to promote cognitive resilience and reduce dementia.

The majority of genetic variants detected in genome wide association studies (GWAS) exert their effects on phenotypes through gene regulation. Motivated by this observation, we propose a multi-omic integration method that models the cascading effects of genetic variants from epigenome to transcriptome and eventually to the phenome in identifying target genes influenced by risk alleles. This cascading epigenomic analysis for GWAS, which we refer to as CEWAS, comprises two types of models: one for linking cis genetic effects to epigenomic variation and another for linking cis epigenomic variation to gene expression. Applying these models in cascade to GWAS summary statistics generates gene level statistics that reflect genetically-driven epigenomic effects. We show on sixteen brain-related GWAS that CEWAS provides higher gene detection rate than related methods, and finds disease relevant genes and gene sets that point toward less explored biological processes. CEWAS thus presents a novel means for exploring the regulatory landscape of GWAS variants in uncovering disease mechanisms.

Epigenetic clocks are calculated by combining DNA methylation states across select CpG sites to estimate biologic age, and have been noted as the most successful markers of biologic aging to date. Yet, limited research has considered epigenetic clocks calculated in brain tissue. We used DNA methylation states in dorsolateral prefrontal cortex specimens from 721 older participants of the Religious Orders Study and Rush Memory and Aging Project, to calculate DNA methylation age using four established epigenetic clocks: Hannum, Horvath, PhenoAge, GrimAge, and a new Cortical clock. The four established clocks were trained in blood samples (Hannum, PhenoAge, GrimAge) or using 51 human tissue and cell types (Horvath); the recent Cortical clock is the first trained in postmortem cortical tissue. Participants were recruited beginning in 1994 (Religious Orders Study) and 1997 (Memory and Aging Project), and followed annually with questionnaires and clinical evaluations; brain specimens were obtained for 80–90% of participants. Mean age at death was 88.0 (SD 6.7) years. We used linear regression, logistic regression, and linear mixed models, to examine relations of epigenetic clock ages to neuropathologic and clinical aging phenotypes, controlling for chronologic age, sex, education, and depressive symptomatology.Hannum, Horvath, PhenoAge and Cortical clock ages were related to pathologic diagnosis of Alzheimer's disease (AD), as well as to Aβ load (a hallmark pathology of Alzheimer's disease). However, associations were substantially stronger for the Cortical than other clocks; for example, each standard deviation (SD) increase in Hannum, Horvath, and PhenoAge clock age was related to approximately 30% greater likelihood of pathologic AD (all p

The past 10 years have seen a rapid advance in our ability to profile the epigenome from human pathologic material, opening up new study designs to investigate the role of epigenomic features in human disease. Moderate to large scale studies have now been conducted in the target tissue of neurodegenerative diseases, the brain, and, through the use of rigorous statistical methodologies, have laid a foundation of validated observations and successful study designs that inform our perspective on the role of the epigenome in these diseases, generate new hypotheses, and guide our path forward for a second generation of studies. It is clear that sampling the epigenome is not redundant with other “omic” profiling of the same tissue and that it can serve as an important vehicle for the integration of the effect of multiple environmental exposures on risk of disease. In some cases, change in the epigenome may thus have a causal impact on disease, but we now have evidence that such changes may also mediate some of the effect of tau proteinopathy and that other changes may moderate the impact of genetic risk factors. Thus, the epigenome may be involved at multiple different stages of the sequence of events that leads to human neurodegeneration, and we review the study designs that may begin to guide the development of a more comprehensive perspective on the aging brain's epigenome.