Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms.

RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders; yet, the role of cell type-specific splicing and transcript-isoform diversity during human brain development has not been systematically investigated. In this work, we leveraged single-molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone and cortical plate regions of the developing human neocortex at tissue and single-cell resolution. We identified 214,516 distinct isoforms, of which 72.6% were novel (not previously annotated in Gencode version 33), and uncovered a substantial contribution of transcript-isoform diversity-regulated by RNA binding proteins-in defining cellular identity in the developing neocortex. We leveraged this comprehensive isoform-centric gene annotation to reprioritize thousands of rare de novo risk variants and elucidate genetic risk mechanisms for neuropsychiatric disorders.

Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms.

RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, yet the role of cell-type-specific splicing or transcript-isoform diversity during human brain development has not been systematically investigated. Here, we leveraged single-molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution. We identified 214,516 unique isoforms, of which 72.6% are novel (unannotated in Gencode-v33), and uncovered a substantial contribution of transcript-isoform diversity, regulated by RNA binding proteins, in defining cellular identity in the developing neocortex. We leveraged this comprehensive isoform-centric gene annotation to re-prioritize thousands of rare de novo risk variants and elucidate genetic risk mechanisms for neuropsychiatric disorders. One-Sentence Summary: A cell-specific atlas of gene isoform expression helps shape our understanding of brain development and disease. Structured Abstract: INTRODUCTION: The development of the human brain is regulated by precise molecular and genetic mechanisms driving spatio-temporal and cell-type-specific transcript expression programs. Alternative splicing, a major mechanism increasing transcript diversity, is highly prevalent in the human brain, influences many aspects of brain development, and has strong links to neuropsychiatric disorders. Despite this, the cell-type-specific transcript-isoform diversity of the developing human brain has not been systematically investigated.RATIONALE: Understanding splicing patterns and isoform diversity across the developing neocortex has translational relevance and can elucidate genetic risk mechanisms in neurodevelopmental disorders. However, short-read sequencing, the prevalent technology for transcriptome profiling, is not well suited to capturing alternative splicing and isoform diversity. To address this, we employed third-generation long-read sequencing, which enables capture and sequencing of complete individual RNA molecules, to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution.RESULTS: We profiled microdissected GZ and CP regions of post-conception week (PCW) 15-17 human neocortex in bulk and at single-cell resolution across six subjects using high-fidelity long-read sequencing (PacBio IsoSeq). We identified 214,516 unique isoforms, of which 72.6% were novel (unannotated in Gencode), and >7,000 novel exons, expanding the proteome by 92,422 putative proteoforms. We uncovered thousands of isoform switches during cortical neurogenesis predicted to impact RNA regulatory domains or protein structure and implicating previously uncharacterized RNA-binding proteins in cellular identity and neuropsychiatric disease. At the single-cell level, early-stage excitatory neurons exhibited the greatest isoform diversity, and isoform-centric single-cell clustering led to the identification of previously uncharacterized cell states. We systematically assessed the contribution of transcriptomic features, and localized cell and spatio-temporal transcript expression signatures across neuropsychiatric disorders, revealing predominant enrichments in dynamic isoform expression and utilization patterns and that the number and complexity of isoforms per gene is strongly predictive of disease. Leveraging this resource, we re-prioritized thousands of rare de novo risk variants associated with autism spectrum disorders (ASD), intellectual disability (ID), and neurodevelopmental disorders (NDDs), more broadly, to potentially more severe consequences and revealed a larger proportion of cryptic splice variants with the expanded transcriptome annotation provided in this study.CONCLUSION: Our study offers a comprehensive landscape of isoform diversity in the human neocortex during development. This extensive cataloging of novel isoforms and splicing events sheds light on the underlying mechanisms of neurodevelopmental disorders and presents an opportunity to explore rare genetic variants linked to these conditions. The implications of our findings extend beyond fundamental neuroscience, as they provide crucial insights into the molecular basis of developmental brain disorders and pave the way for targeted therapeutic interventions. To facilitate exploration of this dataset we developed an online portal ( https://sciso.gandallab.org/ ).

A Single-Cell Transcriptomic Atlas of Human Neocortical Development during Mid-gestation.

We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expression atlas of developing human cortex, providing the first single-cell characterization of previously uncharacterized cell types, including human subplate neurons, comparisons with bulk tissue, and systematic analyses of technical factors. These data permit deconvolution of regulatory networks connecting regulatory elements and transcriptional drivers to single-cell gene expression programs, significantly extending our understanding of human neurogenesis, cortical evolution, and the cellular basis of neuropsychiatric disease. We tie cell-cycle progression with early cell fate decisions during neurogenesis, demonstrating that differentiation occurs on a transcriptomic continuum; rather than only expressing a few transcription factors that drive cell fates, differentiating cells express broad, mixed cell-type transcriptomes before telophase. By mapping neuropsychiatric disease genes to cell types, we implicate dysregulation of specific cell types in ASD, ID, and epilepsy. We developed CoDEx, an online portal to facilitate data access and browsing.

Author Correction: Inhibiting amyloid-ß cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design.

In the version of this Article originally published online, the upper right panel of Fig. 5a was mistakenly a repeat of the lower right panel. This has now been corrected in all versions of the Article.

Inhibiting amyloid-ß cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design.

Inhibiting the interaction between amyloid-ß (Aß) and a neuronal cell surface receptor, LilrB2, has been suggested as a potential route for treating Alzheimer's disease. Supporting this approach, Alzheimer's-like symptoms are reduced in mouse models following genetic depletion of the LilrB2 homologue. In its pathogenic, oligomeric state, Aß binds to LilrB2, triggering a pathway to synaptic loss. Here we identify the LilrB2 binding moieties of Aß (16KLVFFA21) and identify its binding site on LilrB2 from a crystal structure of LilrB2 immunoglobulin domains D1D2 complexed to small molecules that mimic phenylalanine residues. In this structure, we observed two pockets that can accommodate the phenylalanine side chains of KLVFFA. These pockets were confirmed to be 16KLVFFA21 binding sites by mutagenesis. Rosetta docking revealed a plausible geometry for the Aß-LilrB2 complex and assisted with the structure-guided selection of small molecule inhibitors. These molecules inhibit Aß-LilrB2 interactions in vitro and on the cell surface and reduce Aß cytotoxicity, which suggests these inhibitors are potential therapeutic leads against Alzheimer's disease.

Rbfox1 Regulates Synaptic Transmission through the Inhibitory Neuron-Specific vSNARE Vamp1.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Splicing Activation by Rbfox Requires Self-Aggregation through Its Tyrosine-Rich Domain.

Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.

The neurogenetics of alternative splicing.

Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.