Loss-of-function Mutations of CUL3, a High Confidence Gene for Psychiatric Disorders, Lead to Aberrant...

Both rare, high risk, loss-of-function mutations and common, low risk, genetic variants in the CUL3 gene are strongly associated with neuropsychiatric disorders. Network analyses of neuropsychiatric risk genes have shown high CUL3 expression in the prenatal human brain and an enrichment in neural precursor cells (NPCs) and cortical neurons. The role of CUL3 in human neurodevelopment however, is poorly understood. In the present study, we used CRISPR/Cas9 nickase to knockout CUL3 in human induced pluripotent stem cells (iPSCs). iPSCs were subsequently differentiated into cortical glutamatergic neurons using two different protocols and tested for structural/functional alterations. Immunocytochemical analysis and transcriptomic profiling revealed that pluripotency of heterozygous CUL3 knockout (KO) iPSCs remained unchanged compared to isogenic control iPSCs. Following small molecule-mediated differentiation into cortical glutamatergic neurons however, we detected a significant delay in transition from proliferating radial glia cells/NPCs to postmitotic neurons in CUL3 KO cultures. Notably, direct neural conversion of CUL3 KO iPSCs by lentiviral expression of Neurogenin-2 massively attenuated the neurodevelopmental delay. However, both optogenetic and electrical stimulation of induced neurons revealed decreased excitability in Cullin-3 deficient cultures, while basal synaptic transmission remained unchanged. Analysis of target gene expression pointed to alterations in FGF signaling in CUL3 KO NPCs, which is required for NPC proliferation and self-renewal, while RhoA and Notch signaling appeared unaffected. Our data provide first evidence for a major role of Cullin-3 in neuronal differentiation, and for neurodevelopmental deficits underlying neuropsychiatric disorders associated with CUL3 mutations.