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. Overall design: 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.