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

The development of the brain relies on precise control of protein ubiquitination and degradation, and hence, deregulation of ubiquitination signaling networks is thought to play an important role in neurological disorders. Mutation or deletion of the deubiquitinase USP7 causes the Hao-Fountain syndrome, characterized by developmental delay, intellectual disability, autism, and aggressive behavior. Although USP7 function has been studied in proliferating cells in the context of cancer biology, the roles and mechanisms of USP7 in post-mitotic neurons have remained largely unknown. Here, we report that conditional deletion of USP7 in excitatory glutamatergic neurons in the mouse forebrain triggers diverse phenotypes in adult mice including sensory and motor deficits, learning and memory impairment, and aggressive behavior, resembling the clinical features of the Hao-Fountain syndrome. In developmental analyses of the cerebral cortex, USP7 deletion induces neuronal apoptosis, which is rescued by loss of the tumor suppressor protein p53, an established downstream mediator of USP7 signaling. However, most of the behavior abnormalities in USP7 conditional mice are not rescued by loss of p53. Strikingly, USP7 deletion in the brain perturbs the synaptic proteome and dendritic spine morphogenesis independently of p53. Through a combination of comprehensive interaction and TMT-proteomics as well as ubiquitin biochemistry assays, we identify the RNA splicing factor Ppil4 as a novel neuronal substrate of USP7. Knockdown of Ppil4 in cortical neurons impairs dendritic spine morphogenesis, phenocopying the effect of conditional knockout of USP7 on dendritic spine morphogenesis. Collectively, our 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 the Hao-Fountain syndrome.