Cell type-resolved long-read transcriptomics of the human cortex reveals pervasive alternative splicing and disease-relevant isoforms

Abstract

Alternative splicing generates extraordinary transcriptomic complexity in the human brain, yet the full-length isoform landscape across human cortical cell types remains uncharted. Combining fluorescence-activated nuclei sorting with long- and short-read RNA sequencing, we generated isoform-resolved transcriptomes for five major lineages of the adult human prefrontal and orbitofrontal cortex: GABAergic neurons, glutamatergic neurons, oligodendrocytes, astrocytes, and microglia. We cataloged over 220,000 full-length isoforms, [~]35-56% previously unannotated; novel transcripts were longer, more exon-rich, and predominantly protein-coding. Contrary to the neuron-centric view of cortical complexity, glial lineages, particularly oligodendrocytes and microglia, emerged as the most isoform-diverse populations in the cortex. Differential transcript usage and dominant isoform switching defined cell identity, with [~]59-62% of differentially regulated transcripts absent from current annotations. Critically, pathogenic variants were enriched >2-fold at novel splice boundaries within disease genes including POGZ, TARDBP, and PLP1, establishing isoform selection as a primary axis of cortical identity and exposing a layer of pathogenic variation invisible to canonical gene annotations.