Aging of the human brain is characterized by widespread changes in gene expression regulated in part by microRNAs (miRNAs). We present a lifespan miRNA atlas of the human dorsolateral prefrontal cortex generated from small RNA sequencing of 113 postmortem samples spanning 18 to 100 years of age. Differential expression analysis revealed progressive age-associated remodeling of miRNA expression, with the strongest differences observed between old and young individuals. Among the significantly altered miRNAs, miR-34a-5p emerged as one of the most robustly upregulated miRNAs in the aged cortex, alongside additional aging-associated miRNAs including miR-155-5p, miR-132-3p, miR-212-3p, miR-449a, and members of the miR-302 family. This atlas provides a resource for investigating miRNA dysregulation and small RNA regulatory networks during human cortical aging.

Gene signatures of Alzheimer’s disease (AD) brains reflect the output of a complex interplay of genetic, epigenetic, epi-transcriptomic, and post-transcriptional regulations. To nominate candidate factors modulating these signatures, we developed a machine learning model to integrate cellular and molecular features explaining differential gene expression in AD. Among the features tested, YTHDF proteins, the canonical readers of N6-methyladenosine (m6A) RNA modification, are among the most influential predictors of AD gene signatures. Protein modules containing YTHDFs were downregulated in human AD brains, and knockdown or pharmacological inhibition of YTHDFs in iPSC-derived 2D and 3D neuronal models recapitulated key AD-associated gene signatures. Furthermore, eCLIP-seq revealed altered YTHDF binding to transcripts in AD brains, at both m6A-dependent and m6A-independent sites. Together, these results support an important role for YTHDF proteins in modulating AD-associated gene signatures in the human brain.

The dorsolateral prefrontal cortex (dlPFC) controls many cognitive and emotional processes that are disrupted in schizophrenia (SCZ). However, the spatial location of molecular changes associated with SCZ within the dlPFC remains poorly characterized. dlPFC cell types are spatially organized across six layers into microcircuits that mediate dlPFC function. While SCZ has been linked to regionally-defined cell types, spatially-resolved transcriptomics (SRT) can more directly map molecular associations of disease. We integrated protein detection for perineuronal nets, neurons and vasculature with SRT to investigate how gene expression varies across different cellular microenvironments in the human dlPFC from neurotypical control (n=31) and SCZ (n=32) brain donors. We mapped transcriptional alterations in synaptic, neuroimmune and metabolic pathways to neuropil and glia-enriched domains including the white matter. Integrative analyses linked laminar alterations predominantly to non-neuronal populations, and in situ profiling further resolved these changes at cellular resolution, indicating intracellular transcriptional changes that may underpin SCZ-associated alterations. By mapping SCZ-associated ligand-receptor pairs, we curated altered patterns of cell-cell communication that may coordinate local signaling dynamics across specialized tissue microenvironments. Finally, analyses that integrate SCZ genetic risk further highlight the significance of neuron-glia interactions, suggesting that neuronal genetic liability may signal through non-neuronal alterations across cortical domains. This multimodal atlas provides a spatial-anatomical framework for linking genetic risk to transcriptional phenotypes in the human cortex, delineating SCZ-associated gene expression landscapes across layers, single cells, and microenvironments. The data is available as a browsable PsychENCODE resource to support broad utilization that can inform mechanistic studies investigating SCZ pathology and risk.

The human prefrontal cortex (PFC), whose laminar organization is essential for cognitive function, is among the first regions to show age-related functional decline1,2. Single-cell sequencing studies revealed cell type-dependent aging effects but lacked spatial specificity3-6. Spatial transcriptomics (ST) advanced our molecular understanding of the human PFC7, yet whether aging-driven changes differ across PFC layers remains unclear. Here, we performed whole-transcriptome ST on postmortem PFC from 37 individuals across the adult lifespan. We mapped cortical layers and revealed aging mechanisms across layers. This represents one of the largest and most comprehensive lifespan ST analysis of the human PFC brain, offering crucial insight into how the brain ages and identifying potential molecular targets to mitigate cognitive aging and extend healthspan.

Amyloid-beta (Aβ) plaques and their associated glial responses are hallmark features of Alzheimer’s disease (AD), yet their interactions within the human brain remain poorly defined. We applied spatial transcriptomics (ST) and immunohistochemistry (IHC) to 78 postmortem brain sections from 21 individuals in the Religious Orders Study and Memory and Aging Project (ROSMAP). We paired ST with histological data and stratified spots into major categories of plaque-glia niches based on Aβ, GFAP, and IBA1 intensity. Leveraging published ROSMAP single-nucleus RNA-seq data, we examined differences in gene expression, cellular composition, and intercellular communication across these niches. Neuronal and glial changes were validated by IHC and quantitative analyses. We further characterized glial responses using gene set enrichment analysis (GSEA) with known mouse glial signatures and human AD-associated microglial states. Finally, we used iPSC-derived multicellular cultures and single-cell RNA sequencing (scRNA-seq) to identify cell types that, upon short-term Aβ exposure, recapitulate the glial responses observed in the human spatial data. Low-Aβ regions, enriched for diffuse plaques, exhibited transcriptomic profiles consistent with greater neuronal loss than high-Aβ regions. High-glia regions showed increased expression of inflammatory and neurodegenerative pathways. Spatial glial responses aligned with established gene modules, including plaque-induced genes (PIGs), oligodendrocyte (OLIG) responses, disease-associated microglia (DAM), disease-associated astrocytes (DAA), and human AD-associated microglial states, indicating that diverse glial phenotypes emerge around plaques and shape the local immune environment. IHC confirmed elevated neuronal apoptosis near low-Aβ plaques and greater CD68 abundance and synaptic loss near glia-high plaques. In vitro, iPSC-derived microglia—but not astrocytes—exposed to Aβ displayed transcriptomic changes that closely mirrored the glial states identified in our ST dataset. Our study provides a comprehensive spatial transcriptomic dataset from human AD brain tissue and bridges spatial gene expression with traditional neuropathology. By integrating ST, snRNA-seq, and human multicellular models, we map cellular states and molecular events within plaque-glia niches. This work offers a spatially resolved framework for dissecting plaque-glia interactions and reveals new insights into the cellular and molecular heterogeneity underlying neurodegenerative pathology.

Background:Long non-coding RNAs (lncRNAs) are critical regulators of physiological and pathological processes, with their dysregulation increasingly implicated in aging and Alzheimer's disease (AD). To investigate the spatial and cellular distribution of lncRNAs in the aging brain, we leveraged published spatial transcriptomics (ST), single-nucleus RNA sequencing (snRNA-seq), and bulk RNA-seq datasets from the dorsolateral prefrontal cortex (DLPFC) of ROSMAP participants with and without pathological AD. Results:LncRNAs exhibited greater subregion-specific expression than mRNAs, with enrichment in antisense and lincRNA biotypes. Subregion-enriched lncRNAs were generally not cell-type specific, and vice versa. Differential expression analysis of ST data identified AD-associated lncRNAs with distinct spatial patterns and moderate overlap with differentially expressed (DE) lncRNAs from bulk RNA-seq. Gene set enrichment revealed their involvement in chromatin remodeling, epigenetic regulation, and RNA metabolism. We also identified AD DE lncRNAs across major brain cell types using snRNA-seq but overlap with ST DE lncRNAs was limited. Among previously reported lncRNAs, OIP5-AS1 was consistently upregulated in AD in all cortical subregions. Antisense oligonucleotide (ASO) knockdown of OIP5-AS1 in iPSC-derived microglia led to upregulation of pro-inflammatory genes and downregulation of DNA replication and repair pathways. Immunoassays confirmed increased secretion of pro-inflammatory cytokines. The knockdown expression pattern was enriched for microglia-specific AD DE genes and microglia states. Conclusions:This study provides a spatial and cellular map of lncRNAs in the aging human cortex and identifies subregion-and cell-type-enriched DE lncRNAs in AD. Our findings implicate OIP5-AS1 in microglial activation, suggesting its potential contribution to AD pathogenesis.

We explore strategies to harness ancestral diversity in PsychENCODE Consortium Genotype-Expression (GEx) reference panels (adult and developing brain) and Psychiatric Genomics Consortium GWAS data to improve genetically regulated expression (GReX) models and their use for Transcriptome-wide association study (TWAS) discoveries, uncovering previously unknown aspects of psychiatric functional genomics. We trained multiple GReX models on rigorously constructed GEx panel subsets, generated by downsampling, segregating, and/or mixing samples of Admixed African and European ancestries, and based on disease status. Ancestry-specific GReX genes were enriched in pathways involving mitochondrial functions, organelle structure, and metabolism. These models were integrated with ancestry-specific GWASs to conduct bipolar disorder, major depressive disorder, posttraumatic stress disorder, and schizophrenia TWAS. TWAS signals obtained by applying AA- and EUR-specific GReX models to an ancestry-specific GWAS were largely concordant, and mismatched-TWAS (e.g. AA-GReX applied to EUR-GWAS) revealed biologically meaningful signals missed by matched-TWAS. Shared signals across the four disorders were more prominent in the developing brain, involving genes such as H4C13, ZSCAN12P1, and FLOT1, and pathways related to megakaryocyte and muscle development, and neurotransmitter regulation. Overall, we demonstrate concordance in shared TWAS signals across GReX models and provide insight into GReX-specific detectable genes and pathways. Genetic models for psychiatric disorders often overlook ancestry diversity. Here, the authors use PsychENCODE and GWAS data to build ancestry-specific GReX models, improving TWAS and revealing novel genes and pathways linked to brain development and psychiatric risk.

The molecular pathology of stress-related disorders remains elusive. Our brain multiregion, multiomic study of posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) included the central nucleus of the amygdala, hippocampal dentate gyrus, and medial prefrontal cortex (mPFC). Genes and exons within the mPFC carried most disease signals replicated across two independent cohorts. Pathways pointed to immune function, neuronal and synaptic regulation, and stress hormones. Multiomic factor and gene network analyses provided the underlying genomic structure. Single nucleus RNA sequencing in dorsolateral PFC revealed dysregulated (stress-related) signals in neuronal and non-neuronal cell types. Analyses of brain-blood intersections in >50,000 UK Biobank participants were conducted along with fine-mapping of the results of PTSD and MDD genome-wide association studies to distinguish risk from disease processes. Our data suggest shared and distinct molecular pathology in both disorders and propose potential therapeutic targets and biomarkers.

Stress-related disorders stem from the interplay of genetic susceptibility and stress exposure, shaping gene and protein expression through epigenetic modifications across the lifespan. Studies on postmortem brains of PTSD and MDD patients, compared to neurotypical controls, reveal genetic overlaps, sex disparities, and immune and interneuron signaling involvement, yet lack integrative analyses. To address this gap, we established a brain multi-omic, multi-region database comprising individuals with PTSD, MDD, and NCs (77/group, n = 231). We analyzed molecular changes across the central nucleus of the amygdala (CeA), medial prefrontal cortex (mPFC), and hippocampal dentate gyrus (DG) at transcriptomic, methylomic, and proteomic levels. Our approach is supplemented by single-nucleus RNA sequencing (snRNA-seq), genetics, and blood proteomics, aiming for a comprehensive systems perspective. Our findings highlight predominant molecularchanges in the mPFC, with differentially expressed genes (DEGs) and exons carrying disease signals. Notably, methylation alterations were concentrated in the DG for PTSD and CeA for MDD. Findings supported by replication analyses across two cohorts (n = 114). We observed a moderate overlap between disorders, with childhood trauma and suicide driving molecular variations, and sex-specificity was more notable in MDD. Pathway analyses link disease-associated molecular signatures to immune mechanisms, metabolism, mitochondria function, and stress hormone signaling, albeit with low concordance across omics. Top upstream regulators include IL1B, GR, STAT3, and TNF. Multi-omic factor and gene network analyses suggest latent factors and modules related to aging, inflammation, vascular processes, and stress.Complementing multi-omics, our snRNA-seq in the dorsolateral PFC reveals dysregulated pathways and upstream regulators in neuronal and non-neuronal cell types, including stress-related genes. Examining brain multi-omics with blood proteins in the large UK Biobank cohort shows significant correlation, overlap, and directional similarity, implying potential blood-based biomarkers. Fine-mapping of PTSD and MDD genome-wide association study results reveals limited overlap between risk and disease processes at the gene and pathway level.Ultimately, prioritized genes with multi-omic, multi-region, or multi-trait associations are implicated in pathways/networks, exhibit cell-type specificity, demonstrate blood biomarker potential, or are linked to genetic risk for PTSD and MDD.In conclusion, our study unveils shared and unique brain multi-omic molecular dysregulations in PTSD and MDD, elucidating distinct cell-type involvement and paving the way for blood-based biomarker development. These insights not only implicate established stress-related pathways but also offer potential therapeutic avenues.

Posttraumatic Stress Disorder (PTSD) and Major Depressive Disorder (MDD) are prevalent and debilitating conditions associated with stress. The precise molecular mechanisms remain unclear. In this study, we employed a multi-omic systems biology approach to pinpoint regions, omics, and cell-types with disease-associated alteration.

The gene signatures of Alzheimer's Disease (AD) brains reflect an output of a complex interplay of genetic, epigenetic, epi-transcriptomic, and post-transcriptional regulations. To identify the most significant factor that shapes the AD brain signature, we developed a machine learning model (DEcode-tree) to integrate cellular and molecular factors explaining differential gene expression in AD. Our model indicates that YTHDF proteins, the canonical readers of N6-methyladenosine RNA modification (m6A), are the most influential predictors of the AD brain signature. We then show that protein modules containing YTHDFs are downregulated in human AD brains, and knocking out YTHDFs in iPSC-derived neural cells recapitulates the AD brain gene signature in vitro . Furthermore, eCLIP-seq analysis revealed that YTHDF proteins influence AD signatures through both m6A-dependent and independent pathways. These results indicate the central role of YTHDF proteins in shaping the gene signature of AD brains.

Emerging evidence shows that the meninges conduct essential immune surveillance and immune defense at the brain border, and the dysfunction of meningeal immunity contributes to aging and neurodegeneration. However, no study exists on the molecular properties of cell types within human leptomeninges. Here, we provide single nuclei profiling of dissected postmortem leptomeninges from aged individuals. We detect diverse cell types, including unique meningeal endothelial, mural, and fibroblast subtypes. For immune cells, we show that most T cells express CD8 and bear characteristics of tissue-resident memory T cells. We also identify distinct subtypes of border-associated macrophages (BAMs) that display differential gene expressions from microglia and express risk genes for Alzheimer's Disease (AD), as nominated by genome-wide association studies (GWAS). We discover cell-type-specific differentially expressed genes in individuals with Alzheimer's dementia, particularly in fibroblasts and BAMs. Indeed, when cultured, leptomeningeal cells display the signature of ex vivo AD fibroblasts upon amyloid-β treatment. We further explore ligand-receptor interactions within the leptomeningeal niche and computationally infer intercellular communications in AD. Thus, our study establishes a molecular map of human leptomeningeal cell types, providing significant insight into the border immune and fibrotic responses in AD.

Emerging evidence shows that the brain border regions act as a functional neuro-immune interface to conduct essential immune surveillance and immune defense 1 . The dysfunction of border immunity contributes to aging, microglial inflammatory response, and neurodegeneration 2-3 . We conducted single nuclei multiome to simultaneously profile gene expression and open chromatin of dissected postmortem human brain borders from aged individuals with and without Alzheimer’s Disease (AD) (n = 32). We further generated border cell models from iPSCs and postmortem leptomeningeal cells to understand the effects of genetic and environmental factors on border cells. We detect diverse cell types at the brain borders, including unique endothelial, mural, immune, and fibroblast subtypes. Border-associated macrophages (BAMs) display differential gene expressions from microglia and express AD risk genes, as nominated by genome-wide association studies (GWAS). We discover cell-type-specific differentially expressed genes in AD individuals, particularly fibroblasts and BAMs. We identify gene regulatory networks and master regulators of specific border cell states. Human cell models of leptomeningeal cells display the signature of ex vivo AD fibroblasts upon amyloid-β treatment, and iPSC-derived choroid plexus models carrying the APOE4 risk alleles demonstrate differentially expressed genes related to immune and fibrotic response. Lastly, we explore ligand-receptor interactions within the border niche and observe increased intercellular activities and altered communication patterns at AD brain borders. Our study establishes a molecular map of human border cell types and reveals the functional effects of AD risk factors on brain border cells, providing significant insight into the border immune and fibrotic responses in AD pathogenesis. References: 1. Alves de Lima, K., Rustenhoven, J. & Kipnis, J. Meningeal Immunity and Its Function in Maintenance of the Central Nervous System in Health and Disease. Annu. Rev. Immunol. 38 , 597-620 (2020). 2. Da Mesquita, S. et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease. Nature 560 , 185-191 (2018). 3. Da Mesquita, S. et al. Meningeal lymphatics affect microglia responses and anti-Aβ immunotherapy. Nature 593 , 255-260 (2021).

INTRODUCTION:The secreted phosphoprotein 1 (SPP1) gene expressed by CD11c+ cells is known to be associated with microglia activation and neuroinflammatory diseases. As most studies rely on mouse models, we investigated these genes and proteins in the cortical brain tissue of older adults and their role in Alzheimer's disease (AD) and related disorders. METHODS:We leveraged protein measurements, single-nuclei, and RNASeq data from the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) of over 1200 samples for association analysis. RESULTS:Expression of SPP1 and its encoded protein osteopontin were associated with faster cognitive decline and greater odds of common neuropathologies. At single-cell resolution,  integrin subunit alpha X (ITGAX) was highly expressed in microglia, where specific subpopulations were associated with AD and cerebral amyloid angiopathy. DISCUSSION:The study provides evidence of SPP1 and ITGAX association with cognitive decline and common neuropathologies identifying a microglial subset associated with disease.

Recent studies have demonstrated that the dorsal raphe nucleus (DRN) is among the first brain regions affected in Alzheimer’s disease. Hence, in this study we conducted the first comprehensive epigenetic analysis of the DRN in AD, targeting both bulk tissue and single isolated cells. The Illumina Infinium MethylationEPIC BeadChip array was used to analyze the bulk tissue, assessing differentially modified positions (DMoPs) and regions (DMoRs) associated with Braak stage. The strongest Braak stage-associated DMoR in TNXB was targeted in a second patient cohort utilizing single laser-capture microdissected serotonin-positive (5-HT+) and -negative (5-HT-) cells isolated from the DRN. Our study revealed previously identified epigenetic loci, including TNXB and PGLYRP1 , and novel loci, including RBMXL2 , CAST , GNAT1 , MALAT1 , and DNAJB13 . Strikingly, we found that the methylation profile of TNXB depends both on disease phenotype and cell type analyzed, emphasizing the significance of single cell(-type) neuroepigenetic studies in AD.