Molecular cascades and cell type–specific signatures in ASD revealed by single-cell genomics

Genomic profiling in postmortem brain from autistic individuals has consistently revealed convergent molecular changes. What drives these changes and how they relate to genetic susceptibility in this complex condition are not well understood. We performed deep single-nucleus RNA sequencing (snRNA-seq) to examine cell composition and transcriptomics, identifying dysregulation of cell type–specific gene regulatory networks (GRNs) in autism spectrum disorder (ASD), which we corroborated using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) and spatial transcriptomics. Transcriptomic changes were primarily cell type specific, involving multiple cell types, most prominently interhemispheric and callosal-projecting neurons, interneurons within superficial laminae, and distinct glial reactive states involving oligodendrocytes, microglia, and astrocytes. Autism-associated GRN drivers and their targets were enriched in rare and common genetic risk variants, connecting autism genetic susceptibility and cellular and circuit alterations in the human brain. INTRODUCTION Historically, psychiatric disorders have been distinguished from neurological disorders by the absence of the associated histological pathology observed in neurological conditions. But, over the past 15 years, epigenetic and transcriptional profiling of postmortem brain samples from multiple psychiatric conditions, including autism spectrum disorder (ASD), have revealed robust underlying molecular differences. In ASD, this reflects up-regulation of immune signaling genes, down-regulation of neuronal markers and synaptic genes, and a blunting of gene expression signatures of cortical regional identity. How a genetically complex condition such as ASD converges on shared transcriptional alterations remains a mystery. This gap is further amplified by the lack of biological insights into the differences in laminar and cell type–specific pathways affected in ASD and the underlying gene regulatory mechanisms. RATIONALE We reasoned that deep molecular profiling at the single-cell level would inform our understanding of changes in cortical cell types and circuits and, when integrated with genetic risk factors, enable identification of candidate drivers of pathways altered in ASD. Further, knowledge of these pathways at a cellular level would inform mechanistically driven therapeutic development. RESULTS We conducted single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) in a large ASD and control (CTL) cohort, as a core component of the PsychENCODE Consortium (https://www.psychencode.org/), to identify cell type–specific changes and the cellular regulatory networks perturbed by genetic risk factors. Our approach (on average >10,000 cells per individual and >1860 genes per cell) enabled identification of all 26 major cortical cell types, validated with published cortical cell atlases. We also identified cell states, primarily activated forms of microglia (MG), oligodendrocytes (ODCs), astrocytes (ASTROs), and blood-brain barrier cells, observed predominantly in ASD. Changes in cell composition in ASD were subtle, involving increases in activated microglia and astrocyte states, which are very rarely observed in controls. In contrast to the minor changes in cell composition, the changes observed in gene expression in ASD were substantial: 2166 down-regulated and 1319 up-regulated genes across 35 cell types, most of which were cell type specific. Through integration of snRNA-seq, snATAC-seq, and spatial transcriptomics, we identified regulatory networks driving cell type–specific transcriptional changes and their location within cortical laminae. These analyses demonstrate concentration of activated microglia, astrocytes, and somatostatin (SST) interneurons in superficial cortical laminae, in conjunction with profound down-regulation of synaptic gene expression and up-regulation of stress-response and proinflammatory pathways in inter- and intrahemispheric projection neurons. A large proportion of these changes could be ascribed to specific transcriptional drivers, and both the drivers and their targets were enriched in genes harboring common and rare genetic risk for ASD. CONCLUSION These analyses refine our knowledge of cellular and circuit alterations in the brain in ASD. By identifying and validating transcriptional drivers enriched in rare and common genetic risk variants, we have discovered a link between autism genetic susceptibility and molecular and cellular circuits and pathways, providing a roadmap for understanding cellular interactions and therapeutic development in ASD. Single-cell genomics reveals cell type–specific and laminar changes in ASD. These changes prominently affect layers 2 and 3 interhemispheric and callosal-projecting excitatory (Ex) neurons, superficial SST interneurons, and reactive glial states in the frontal cortex (FC). By defining gene regulatory networks (GRNs; red, up-regulated; blue, down-regulated) and integrating them with ASD genetic risk variants, we discerned candidate drivers of the transcriptional changes and genetic susceptibility acting in specific cell types. OPCs, oligodendrocyte progenitor cells; INTs, inhibitory neurons; WM, white matter; DEGs, differentially expressed genes. [Created with Biorender.com]