Binte Zehra, Nesrin Mohamed, Richa Tambi, Muhammad Faizan, Dharana Satsangi, Alexander D Giddey, Gilles Bru-Mercier, Ahmad Farhat, Nesreen K Al-Jezawi, Muhammad Kumail, Nasna Nassir, Bipin Balan, Noor Kosaji, Mariam Eldesouky, Talal Al Yazeedi, Shuhd BinEshaq, Suhana Shiyas, Nidhina Vinod, Saif S Alqassim, Awab Ahmed, Mohammad Amiruddin Hashmi, Nelson C Soares, Marc Woodbury-Smith, Stefan S Du Plessis, Dimitri J Stavropoulos, Stephen W Scherer, Alawi Alsheikh-Ali, Reem Khalil, Mauro Pessia, Maria Cristina D'Adamo, Bakhrom K Berdiev, Mohammed Uddin
Heterozygous loss-of-function variants in Neurabin I (PPP1R9A), responsible for encoding a cytoskeletal scaffolding protein essential for synaptic plasticity, are recurrently associated with neurodevelopmental and neuropsychiatric disorders, yet their direct effects on human neuronal maturation remain unclear. Here, we establish the first comprehensive human mechanistic model of PPP1R9A haploinsufficiency using an isogenic CRISPR/Cas9-engineered iPSC system differentiated into cortical neurons to define dosage-dependent functional consequences. PPP1R9A+/- neurons exhibited pronounced hyperspinogenesis and increased neuritic complexity, indicative of aberrant structural maturation; however, whole-cell patch-clamp recordings revealed impaired intrinsic excitability, including reduced action potential firing, altered waveform properties, and defective axo-somatic coupling, uncovering a striking dissociation between neuronal morphology and function. Long-read single-cell transcriptomics and quantitative proteomics identified coordinated downregulation of ion channel and synaptic transmission pathways, including genes essential for sodium channel function and glutamatergic signaling, together with disruption of synaptic vesicle cycling, axon guidance, and neurodevelopmental programs. Pseudotime trajectory analysis further demonstrated delayed neuronal differentiation, with mutant neurons accumulating at intermediate developmental states rather than acquiring mature cortical identities. Importantly, molecular rescue experiments confirmed causality, as restoration of full-length PPP1R9A expression robustly normalized transcriptional and synaptic signaling programs, whereas allele-specific antisense oligonucleotide-mediated suppression of the mutant transcript achieved only partial rescue. Taken together, these findings establish PPP1R9A haploinsufficiency as a driver of impaired molecular, electrophysiological, and developmental maturation in human cortical neurons, providing a human-specific mechanistic framework linking reduced Neurabin I dosage to neurodevelopmental and psychiatric disease risk.