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The relationship between genetic variation and gene expression in brain cell types and subtypes remains understudied. Here, we generated single-nucleus RNA sequencing data from the neocortex of 424 individuals of advanced age; we assessed the effect of genetic variants on RNA expression in cis(cis-expression quantitative trait loci) for seven cell types and 64 cell subtypes using 1.5 million transcriptomes. This effort identified 10,004 eGenes at the cell type level and 8,099 eGenes at the cell subtype level. Many eGenes are only detected within cell subtypes. A new variant influences APOEexpression only in microglia and is associated with greater cerebral amyloid angiopathy but not Alzheimer’s disease pathology, after adjusting for APOEε4, providing mechanistic insights into both pathologies. Furthermore, only a TMEM106Bvariant affects the proportion of cell subtypes. Integration of these results with genome-wide association studies highlighted the targeted cell type and probable causal gene within Alzheimer’s disease, schizophrenia, educational attainment and Parkinson’s disease loci.

The role of different cell types and their interactions in Alzheimer’s disease (AD) is a complex and open question. Here, we pursued this question by assembling a high-resolution cellular map of the aging frontal cortex using single-nucleus RNA sequencing of 24 individuals with a range of clinicopathologic characteristics. We used this map to infer the neocortical cellular architecture of 638 individuals profiled by bulk RNA sequencing, providing the sample size necessary for identifying statistically robust associations. We uncovered diverse cell populations associated with AD, including a somatostatin inhibitory neuronal subtype and oligodendroglial states. We further identified a network of multicellular communities, each composed of coordinated subpopulations of neuronal, glial and endothelial cells, and we found that two of these communities are altered in AD. Finally, we used mediation analyses to prioritize cellular changes that might contribute to cognitive decline. Thus, our deconstruction of the aging neocortex provides a roadmap for evaluating the cellular microenvironments underlying AD and dementia.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1–4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

The role of non-neuronal cells in Alzheimer’s disease progression has not been fully elucidated. Using single-nucleus RNA sequencing, we identified a population of disease-associated astrocytes in an Alzheimer’s disease mouse model. These disease-associated astrocytes appeared at early disease stages and increased in abundance with disease progression. We discovered that similar astrocytes appeared in aged wild-type mice and in aging human brains, suggesting their linkage to genetic and age-related factors.

Background: In droplet-based single-cell and single-nucleus RNA-seq experiments, not all reads associated with one cell barcode originate from the encapsulated cell. Such background noise is attributed to spillage from cell-free ambient RNA or barcode swapping events. Results: Here, we characterize this background noise exemplified by three scRNA-seq and two snRNA-seq replicates of mouse kidneys. For each experiment, cells from two mouse subspecies are pooled, allowing to identify cross-genotype contaminating molecules and thus profile background noise. Background noise is highly variable across replicates and cells, making up on average 3–35% of the total counts (UMIs) per cell and we find that noise levels are directly proportional to the specificity and detectability of marker genes. In search of the source of background noise, we find multiple lines of evidence that the majority of background molecules originates from ambient RNA. Finally, we use our genotype-based estimates to evaluate the performance of three methods (CellBender, DecontX, SoupX) that are designed to quantify and remove background noise. We find that CellBender provides the most precise estimates of background noise levels and also yields the highest improvement for marker gene detection. By contrast, clustering and classification of cells are fairly robust towards background noise and only small improvements can be achieved by background removal that may come at the cost of distortions in fine structure. Conclusions: Our findings help to better understand the extent, sources and impact of background noise in single-cell experiments and provide guidance on how to deal with it.

Accumulation of tau and amyloid-β are two pathologic hallmarks of Alzheimer’s disease. We conducted an epigenome-wide association study using the histone 3 lysine 9 acetylation (H3K9ac) mark in 669 aged human prefrontal cortices; in contrast with amyloid-β, tau protein burden had a broad effect on the epigenome, affecting 5,990 of 26,384 H3K9ac domains. Tau-related alterations aggregated in large genomic segments reflecting spatial chromatin organization, and the magnitude of these effects correlated with the segment’s nuclear lamina association. Functional relevance of these chromatin changes was demonstrated by (1) consistent transcriptional changes in three independent datasets and (2) similar findings in two mouse models of Alzheimer’s disease. Finally, we found that tau overexpression in induced pluripotent stem cell-derived neurons altered chromatin structure and that these effects could be blocked by a small molecule predicted to reverse the tau effect. Thus, we report broad tau-driven chromatin rearrangements in the aging human brain that may be reversible with heat-shock protein 90 (Hsp90) inhibitors.

There is a need for new therapeutic targets with which to prevent Alzheimer’s disease (AD), a major contributor to aging-related cognitive decline. Here we report the construction and validation of a molecular network of the aging human frontal cortex. Using RNA sequence data from 478 individuals, we first build a molecular network using modules of coexpressed genes and then relate these modules to AD and its neuropathologic and cognitive endophenotypes. We confirm these associations in two independent AD datasets. We also illustrate the use of the network in prioritizing amyloid- and cognition-associated genes for in vitro validation in human neurons and astrocytes. These analyses based on unique cohorts enable us to resolve the role of distinct cortical modules that have a direct effect on the accumulation of AD pathology from those that have a direct effect on cognitive decline, exemplifying a network approach to complex diseases. The authors constructed and validated a molecular network of the aging human cortex from RNA sequencing data from 478 individuals and identified genes that affect cognitive decline or neuropathology in Alzheimer’s disease.

While genetic variants are known to be associated with overall gene abundance in stimulated immune cells, less is known about their effects on alternative isoform usage. By analyzing RNA-seq profiles of monocyte-derived dendritic cells from 243 individuals, we uncovered thousands of unannotated isoforms synthesized in response to influenza infection and type 1 interferon stimulation. We identified more than a thousand quantitative trait loci (QTLs) associated with alternate isoform usage (isoQTLs), many of which are independent of expression QTLs (eQTLs) for the same gene. Compared with eQTLs, isoQTLs are enriched for splice sites and untranslated regions, but depleted of sequences upstream of annotated transcription start sites. Both eQTLs and isoQTLs explain a significant proportion of the disease heritability attributed to common genetic variants. At the ERAP2locus, we shed light on the function of the gene and how two frequent, highly differentiated haplotypes with intermediate frequencies could be maintained by balancing selection. At baseline and following type 1 interferon stimulation, the major haplotype is associated with low ERAP2expression caused by nonsense-mediated decay, while the minor haplotype, known to increase Crohn's disease risk, is associated with high ERAP2expression. In response to influenza infection, we found two uncharacterized isoforms expressed from the major haplotype, likely the result of multiple perfectly linked variants affecting the transcription and splicing at the locus. Thus, genetic variants at a single locus could modulate independent gene regulatory processes in innate immune responses and, in the case of ERAP2, may confer a historical fitness advantage in response to virus.

We report a multi-omic resource generated by applying quantitative trait locus (xQTL) analyses to RNA sequence, DNA methylation and histone acetylation data from the dorsolateral prefrontal cortex of 411 older adults who have all three data types. We identify SNPs significantly associated with gene expression, DNA methylation and histone modification levels. Many of these SNPs influence multiple molecular features, and we demonstrate that SNP effects on RNA expression are fully mediated by epigenetic features in 9% of these loci. Further, we illustrate the utility of our new resource, xQTL Serve, by using it to prioritize the cell type(s) most affected by an xQTL. We also reanalyze published genome wide association studies using an xQTL-weighted analysis approach and identify 18 new schizophrenia and 2 new bipolar susceptibility variants, which is more than double the number of loci that can be discovered with a larger blood-based expression eQTL resource.

IMPORTANCE While numerous genetic susceptibility loci have been identified for clinical Alzheimer disease (AD), it is important to establish whether these variants are risk factors for the underlying disease pathology, including neuritic plaques. OBJECTIVES To investigate whether AD susceptibility loci from genome-wide association studies affect neuritic plaque pathology and to additionally identify novel risk loci for this trait. DESIGN, SETTING, AND PARTICIPANTS Candidate analysis of single-nucleotide polymorphisms and genome-wide association study in a joint clinicopathologic cohort, including 725 deceased subjects from the Religious Orders Study and the Rush Memory and Aging Project (2 prospective, community-based studies), followed by targeted validation in an independent neuroimaging cohort, including 114 subjects from multiple clinical and research centers. MAIN OUTCOMES AND MEASURES A quantitative measure of neuritic plaque pathologic burden, based on assessments of silver-stained tissue averaged from multiple brain regions. Validation based on β-amyloid load by immunocytochemistry, and replication with fibrillar β-amyloid positron emission tomographic imaging with Pittsburgh Compound B or florbetapir. RESULTS Besides the previously reported APOE and CR1 loci, we found that the ABCA7 (rs3764650; P = .02) and CD2AP (rs9349407; P = .03) AD susceptibility loci are associated with neuritic plaque burden. In addition, among the top results of our genome-wide association study, we discovered a novel variant near the amyloid precursor protein gene (APP, rs2829887) that is associated with neuritic plaques (P = 3.3 × 10−6). This polymorphism was associated with postmortem β-amyloid load as well as fibrillar β-amyloid in 2 independent cohorts of adults with normal cognition. CONCLUSIONS AND RELEVANCE These findings enhance understanding of AD risk factors by relating validated susceptibility alleles to increased neuritic plaque pathology and implicate common genetic variation at the APP locus in the earliest, presymptomatic stages of AD.