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.

Single-cell genomics and regulatory networks for 388 human brains.

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multiomics datasets into a resource comprising >2.8 million nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550,000 cell type-specific regulatory elements and >1.4 million single-cell expression quantitative trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.

Single-cell genomics and regulatory networks for 388 human brains.

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.

Deep sequencing of proteotoxicity modifier genes uncovers a Presenilin-2/beta-amyloid-actin genetic risk module shared among alpha-synucleinopathies.

Whether neurodegenerative diseases linked to misfolding of the same protein share genetic risk drivers or whether different protein-aggregation pathologies in neurodegeneration are mechanistically related remains uncertain. Conventional genetic analyses are underpowered to address these questions. Through careful selection of patients based on protein aggregation phenotype (rather than clinical diagnosis) we can increase statistical power to detect associated variants in a targeted set of genes that modify proteotoxicities. Genetic modifiers of alpha-synuclein (ɑS) and beta-amyloid (Aß) cytotoxicity in yeast are enriched in risk factors for Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. Here, along with known AD/PD risk genes, we deeply sequenced exomes of 430 ɑS/Aß modifier genes in patients across alpha-synucleinopathies (PD, Lewy body dementia and multiple system atrophy). Beyond known PD genes GBA1 and LRRK2, rare variants AD genes (CD33, CR1 and PSEN2) and Aß toxicity modifiers involved in RhoA/actin cytoskeleton regulation (ARGHEF1, ARHGEF28, MICAL3, PASK, PKN2, PSEN2) were shared risk factors across synucleinopathies. Actin pathology occurred in iPSC synucleinopathy models and RhoA downregulation exacerbated ɑS pathology. Even in sporadic PD, the expression of these genes was altered across CNS cell types. Genome-wide CRISPR screens revealed the essentiality of PSEN2 in both human cortical and dopaminergic neurons, and PSEN2 mutation carriers exhibited diffuse brainstem and cortical synucleinopathy independent of AD pathology. PSEN2 contributes to a common-risk signal in PD GWAS and regulates ɑS expression in neurons. Our results identify convergent mechanisms across synucleinopathies, some shared with AD.

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

Understanding how genetic variation exerts its effects on the human brain in health and disease has been greatly informed by functional genomic characterization. Studies over the last decade have demonstrated robust evidence of convergent transcriptional and epigenetic profiles in post-mortem cerebral cortex from individuals with Autism Spectrum Disorder (ASD). Here, we perform deep single nuclear (sn) RNAseq to elucidate changes in cell composition, cellular transcriptomes and putative candidate drivers associated with ASD, which we corroborate using snATAC-seq and spatial profiling. We find changes in cell state composition representing transitions from homeostatic to reactive profiles in microglia and astrocytes, a pattern extending to oligodendrocytes and blood brain barrier cells. We identify profound changes in differential expression involving thousands of genes across neuronal and glial subtypes, of which a substantial portion can be accounted for by specific transcription factor networks that are significantly enriched in common and rare genetic risk for ASD. These data, which are available as part of the PsychENCODE consortium, provide robust causal anchors and resultant molecular phenotypes for understanding ASD changes in human brain.

Transcriptome analyses reveal tau isoform-driven changes in transposable element and gene expression.

Alternative splicing of the gene MAPT produces several isoforms of tau protein. Overexpression of these isoforms is characteristic of tauopathies, which are currently untreatable neurodegenerative diseases. Though non-canonical functions of tau have drawn interest, the role of tau isoforms in these diseases has not been fully examined and may reveal new details of tau-driven pathology. In particular, tau has been shown to promote activation of transposable elements-highly regulated nucleotide sequences that replicate throughout the genome and can promote immunologic responses and cellular stress. This study examined tau isoforms' roles in promoting cell damage and dysregulation of genes and transposable elements at a family-specific and locus-specific level. We performed immunofluorescence, Western blot and cytotoxicity assays, along with paired-end RNA sequencing on differentiated SH-SY5Y cells infected with lentiviral constructs of tau isoforms and treated with amyloid-beta oligomers. Our transcriptomic findings were validated using publicly available RNA-sequencing data from Alzheimer's disease, progressive supranuclear palsy and control human samples from the Accelerating Medicine's Partnership for AD (AMP-AD). Significance for biochemical assays was determined using Wilcoxon ranked-sum tests and false discovery rate. Transcriptome analysis was conducted through DESeq2 and the TEToolkit suite available from the Hammell lab at Cold Spring Harbor Laboratory. Our analyses show overexpression of different tau isoforms and their interactions with amyloid-beta in SH-SY5Y cells result in isoform-specific changes in the transcriptome, with locus-specific transposable element dysregulation patterns paralleling those seen in patients with Alzheimer's disease and progressive supranuclear palsy. Locus-level transposable element expression showed increased dysregulation of L1 and Alu sites, which have been shown to drive pathology in other neurological diseases. We also demonstrated differences in rates of cell death in SH-SY5Y cells depending on tau isoform overexpression. These results demonstrate the importance of examining tau isoforms' role in neurodegeneration and of further examining transposable element dysregulation in tauopathies and its role in activating the innate immune system.

Mutant three-repeat tau expression initiates retinal ganglion cell death through Caspase-2.

The microtubule-associated protein tau is implicated in multiple degenerative diseases including retinal diseases such as glaucoma; however, the way tau initiates retinopathy is unclear. Previous retinal assessments in mouse models of tauopathy suggest that mutations in four-repeat (4R) tau are associated with disease-induced retinal dysfunction, while shifting tau isoform ratio to favor three-repeat (3R) tau production enhanced photoreceptor function. To further understand how alterations in tau expression impact the retina, we analyzed the retinas of transgenic mice overexpressing mutant 3R tau (m3R tau-Tg), a model known to exhibit Pick's Disease pathology in the brain. Analysis of retinal cross-sections from young (3 month) and adult (9 month) mice detected asymmetric 3R tau immunoreactivity in m3R tau-Tg retina, concentrated in the retinal ganglion and amacrine cells of the dorsal retinal periphery. Accumulation of hyperphosphorylated tau was detected specifically in the detergent insoluble fraction of the adult m3R tau-Tg retina. RNA-seq analysis highlighted biological pathways associated with tauopathy that were uniquely altered in m3R tau-Tg retina. The upregulation of transcript encoding apoptotic protease caspase-2 coincided with increased immunostaining in predominantly 3R tau positive retinal regions. In adult m3R tau-Tg, the dorsal peripheral retina of the adult m3R tau-Tg exhibited decreased cell density in the ganglion cell layer (GCL) and reduced thickness of the inner plexiform layer (IPL) compared to the ventral peripheral retina. Together, these data indicate that mutant 3R tau may mediate toxicity in retinal ganglion cells (RGC) by promoting caspase-2 expression which results in RGC degeneration. The m3R tau-Tg line has the potential to be used to assess tau-mediated RGC degeneration and test novel therapeutics for degenerative diseases such as glaucoma.