The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

Precise control of protein ubiquitination is essential for brain development, and hence, disruption of ubiquitin signaling networks can lead to neurological disorders. Mutations of the deubiquitinase USP7 cause the Hao-Fountain syndrome (HAFOUS), characterized by developmental delay, intellectual disability, autism, and aggressive behavior. These neurological and behavioral manifestations indicate important unknown roles of USP7 specificially in the nervous system. Here, we report that conditional deletion of USP7 in excitatory neurons in the mouse forebrain triggers diverse phenotypes including growth delay, sensorimotor deficits, learning and memory impairment, and aggressive behavior, resembling clinical features of HAFOUS. USP7 deletion induces neuronal apoptosis in a manner dependent of the tumor suppressor p53. However, most behavioral abnormalities in USP7 conditional mice persist despite p53 loss. Strikingly, USP7 deletion in the brain perturbs the synaptic proteome and dendritic spine morphogenesis independently of p53. Integrated proteomics and subsequent ubiquitin biochemical analysis identify the RNA splicing factor Ppil4 as a novel neuronal substrate of USP7. Consistent with the role of Ppil4 in RNA splicing, loss of USP7 disrupts splicing program of synaptic genes in the cerebral cortex. Improtantly, knockdown of Ppil4 in cortical neurons impairs dendritic spine morphogenesis, phenocopying the effect of USP7 loss on dendritic spines. These findings reveal a novel USP7-Ppil4 ubiquitin signaling link that regulates neuronal connectivity in the developing brain, with implications for our understanding of the pathogenesis of HAFOUS and other neurodevelopmental disorders. We performed bulk RNA-seq of the cerebral cortex of 5 pairs of Usp7 cKO; Trp53+/- mice and Usp7 WT; Trp53+/- mice at age P18. The 5 pairs of mice were litter- and sex-controlled.蛋白质泛素化（protein ubiquitination）的精准调控对于大脑发育不可或缺，因此泛素信号网络的紊乱可引发神经系统疾病。去泛素化酶USP7（deubiquitinase USP7）的突变会导致郝-方丹综合征（Hao-Fountain syndrome, HAFOUS），该疾病以发育迟缓、智力障碍、孤独症谱系障碍及攻击性行为为核心临床特征。此类神经与行为表型提示，USP7在神经系统中存在尚未被阐明的重要功能。本研究证实，在小鼠前脑兴奋性神经元中条件性敲除USP7会引发多种表型，包括生长迟缓、感觉运动缺陷、学习记忆障碍与攻击性行为，其表现与HAFOUS的临床特征高度相似。USP7缺失可通过依赖肿瘤抑制因子p53（tumor suppressor p53）的途径诱导神经元凋亡，但即便p53缺失，USP7条件性敲除小鼠的多数行为异常仍持续存在。值得注意的是，脑内USP7缺失可在不依赖p53的情况下扰乱突触蛋白质组（synaptic proteome）与树突棘形态发生（dendritic spine morphogenesis）。通过整合蛋白质组学与后续泛素化生化分析，本研究鉴定出RNA剪接因子Ppil4（RNA splicing factor Ppil4）为USP7的新型神经元底物。结合Ppil4在RNA剪接中的功能，USP7缺失会破坏大脑皮层（cerebral cortex）中突触相关基因的剪接程序。至关重要的是，在皮层神经元中敲低Ppil4会损伤树突棘形态发生，其表型与USP7缺失对树突棘的影响完全一致。本研究揭示了一条全新的USP7-Ppil4泛素信号通路，该通路可调控发育中大脑的神经元连接，为理解HAFOUS及其他神经发育障碍的发病机制提供了新的理论依据。我们对P18日龄的5对Usp7条件性敲除（cKO）；Trp53+/-小鼠与Usp7野生型（WT）；Trp53+/-小鼠的大脑皮层开展了批量RNA测序（bulk RNA-seq），该5对小鼠均为同窝且性别匹配的个体。