ABSTRACT: Neurodevelopmental disorders (NDDs) are often associated with mutations that disrupt synaptic function and neuronal differentiation, but the mechanisms linking these genetic alterations to cellular phenotypes remain elusive. Here, we investigate the role of Neurabin I (PPP1R9A), a scaffolding protein critical for synaptic plasticity, in human neurons derived from induced pluripotent stem cells (iPSCs) with CRISPR/Cas9-mediated heterozygous mutation (Neurabin I+/-). We observed that, although mutant neurons exhibit dense and elongated neuritic extensions, they fail to form connections and display disrupted action potentials, as observed in electrophysiological studies. Employing a multi-omics approach that integrates long-read transcriptomics, including single-cell and bulk RNA sequencing, and proteomics, we revealed profound transcriptional, structural, and functional deficits associated with Neurabin I haploinsufficiency. We observed that Neurabin I+/- iNeurons revealed downregulation of key neurogenesis genes and ion channel components, while pathway enrichment highlighted dysregulation in synaptic signaling and axon guidance. Long-read sequencing provided isoform-specific insights, revealing altered expression of Neurabin I isoforms, particularly in cortical excitatory neuron subtypes, implicating isoform-specific regulatory mechanisms in the pathophysiology of NDDs. Proteomic profiling identified differentially expressed proteins enriched in pathways related to synaptic vesicle cycle, neurotransmitter release, and neurodegeneration, further substantiating the functional impact of Neurabin I deficiency. Furthermore, single-cell RNA sequencing delineated distinct neuronal differentiation trajectories, showing that Neurabin I+/- neurons exhibit stalled progression and accumulate in intermediate states, suggesting impaired maturation. Gene-based molecular rescue experiments using Neurabin I overexpression (OE) and antisense oligonucleotides (ASO) partially restored neuronal morphology, transcriptomic profiles, and synaptic protein expression, demonstrating the therapeutic potential of targeting Neurabin I in NDDs. This comprehensive multi-omics study elucidates the critical role of Neurabin I in neuronal differentiation and synaptic function, providing a mechanistic link between Neurabin I mutations and neurodevelopmental pathologies, with implications for therapeutic intervention.
