Autism-linked Cullin3 germline haploinsufficiency severely impacts mouse brain development and cortical neurogenesis through RhoA signaling

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for Neurodevelopmental Disorders such as Autism Spectrum Disorder (ASD) and Developmental Delay (DD). This study identifies the impact of the ASD-associated de novo Cul3-mutation on brain anatomy, behavior, molecular, cellular, and circuit-level mechanisms during early neocortical development. We report that Cul3 mutant mice have microcephaly and severe brain structure defects. This mouse model exhibits social and cognitive deficits and hyperactivity behavior. Spatiotemporal transcriptomic and proteomic profiling of Cul3 mouse brain implicates neurogenesis and cytoskeletal defects as key drivers of Cul3 functional effect. We show that Cul3 is critical for neuron growth and development. Cultured cortical neurons from the mutant mice have reduced dendritic length, actin cytosketon defects, reduced network activity, and increased cell death. At the molecular level, we implicate upregulation of small GTPase RhoA, involved in regulation of cytoskeletal dynamics, and neurite outgrowth as one of the pathways impacted by the de novo Cul3 mutations in ASD. Treatment with small molecule RhoA inhibitor Rhosin rescues dendrite length phenotype and implicates RhoA pathway in ASD. Bulk RNA-seq of 108 samples containing Cul3+/- heterozygous and WT mice were performed at different developmental time periods- embryonic(E17.5), Postnatal (P7) and adult (4-6 weeks)E3泛素连接酶（E3-ubiquitin ligase）Cullin3（Cul3）是自闭症谱系障碍（Autism Spectrum Disorder, ASD）与发育迟缓（Developmental Delay, DD）等神经发育障碍的高置信度风险基因。本研究阐明了自闭症相关的新发Cul3突变在新皮层早期发育过程中，对大脑解剖结构、行为、分子、细胞及环路层面机制的影响。我们发现，Cul3突变小鼠表现出小头畸形与严重脑结构缺陷。该小鼠模型呈现社交与认知缺陷以及多动行为。对Cul3突变小鼠大脑的时空转录组与蛋白质组分析显示，神经发生与细胞骨架缺陷是介导Cul3功能异常的关键驱动因素。本研究证实Cul3对神经元生长与发育至关重要：取自突变小鼠的体外培养皮层神经元存在树突长度缩短、肌动蛋白细胞骨架异常、神经网络活动降低以及细胞死亡增加的表型。在分子层面，我们发现参与调控细胞骨架动态与神经突生长的小GTP酶RhoA的上调，是自闭症相关新发Cul3突变影响的核心通路之一。使用小分子RhoA抑制剂Rhosin进行干预，可挽救树突长度异常表型，证实RhoA通路与自闭症发病机制相关。本研究对108份样本（包含Cul3+/-杂合子与野生型（Wild Type, WT）小鼠）开展了批量RNA测序（Bulk RNA-seq），样本采集自三个不同发育阶段：胚胎期（E17.5）、出生后（P7）以及成年期（4-6周龄）。