Modeling and targeting haploinsufficiency in SHINE syndrome

DLG4-related Synaptopathy, or SHINE syndrome, is a neurodevelopmental disorder caused by de novo heterozygous variants in DLG4 gene, encoding the postsynaptic scaffold PSD-95. Although clinical and genetic evidence support haploinsufficiency, the consequences of pathogenic DLG4 variants in human neurons remain poorly defined. Here, we model three mutations spanning distinct protein domains: a frameshift, nonsense and a missense mutation using iPSC-derived excitatory neurons. Molecular analysis of mature neurons reveals shared PSD-95 deficiency irrespective of transcript levels, together with reduced mature spine density. High-density microelectrode array recordings further reveal convergent and mutationspecific electrophysiological signatures at both single-neuron and network levels, as mutant cultures display genotype-dependent shifts in extracellular waveform states associated with altered firing dynamics. Importantly, restoration of PSD-95 levels using an adeno-associated viral vector (AAV9) harboring human

DLG4 cDNA and driven by the human neuronal Synapsin I promoter (AAV9-hSynI-DLG4) rescues PSD95 abundance and associated cellular and electrophysiological deficits. Together, these findings establish DLG4 haploinsufficiency as a shared consequence of pathogenic DLG4 variants, while revealing additional variant-associated effects on neuronal structure and activity, rescued by AAV9-mediated neuronal restoration.