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.

CUL3 基因中罕见的高风险功能丧失突变和常见的低风险遗传变异都与神经精神疾病密切相关。神经精神风险基因的网络分析显示，CUL3 在产前人类大脑中的高表达以及神经前体细胞 (NPC) 和皮层神经元的富集。然而，人们对 CUL3 在人类神经发育中的作用知之甚少。在本研究中，我们使用 CRISPR/Cas9 切口酶敲除人类诱导多能干细胞 (iPSC) 中的 CUL3。随后使用两种不同的方案将 iPSC 分化为皮质谷氨酸能神经元，并测试其结构/功能改变。免疫细胞化学分析和转录组分析显示，与同基因对照 iPSC 相比，杂合 CUL3 敲除 (KO) iPSC 的多能性保持不变。然而，在小分子介导的向皮质谷氨酸能神经元分化之后，我们检测到在 CUL3 KO 培养物中从增殖的放射状胶质细胞/NPC 到有丝分裂后神经元的转变存在显着延迟。值得注意的是，通过 Neurogenin-2 的慢病毒表达对 CUL3 KO iPSCs 的直接神经转化大大减轻了神经发育迟缓。然而，诱导神经元的光遗传学和电刺激均显示 Cullin-3 缺陷培养物中的兴奋性降低，而基础突触传递保持不变。对靶基因表达的分析表明 CUL3 KO NPC 中 FGF 信号的改变，这是 NPC 增殖和自我更新所必需的，而 RhoA 和 Notch 信号似乎不受影响。我们的数据为 Cullin-3 在神经元分化中的主要作用以及与 CUL3 突变相关的神经精神疾病的神经发育缺陷提供了第一个证据。