Disruption of Gprasp2 down-regulates Hedgehog signaling and leads to apoptosis in auditory cells

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GPRASP2 is implicated in nervous system diseases, tumors and immune inflammation. In our previous study, G protein-coupled receptor associated sorting protein 2 (GPRASP2) was identified as a novel causal gene for X-linked recessive syndromic hearing loss (SHL). However, the role of GPRASP2 in auditory function has not been elucidated. The Gprasp2-knockout (KO) mouse HEI-OC1 auditory cells were constructed using CRISPR/Cas9-mediated gene editing. RNA-sequencing (RNA-seq) was used to investigate the differentially expressed genes (DEGs) and DEGs-enriched signaling pathways, which was verified by Western blot. Flow cytometry assay was used to examine cell apoptosis. The cytological pathology was evaluated by laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Mitochondrial damage was observed in Gprasp2-KO HEI-OC1 cells. RNA-seq analysis suggested that Gprasp2-KO was implicated in the apoptosis process, which could be mediated by Hedgehog (Hh) signaling pathway. The key molecules in Hh signaling pathway (Smo, Gli1, Gli2) were detected to be down-regulated in Gprasp2-KO HEI-OC1 cells. The differential expression of apoptosis molecules (Bcl2, Bax, Caspase-3/cleaved-Caspase-3) indicated that Gprasp2-KO induced apoptosis in HEI-OC1 cells. The treatment of smoothened agonist (Purmorphamine, PUR) activated the Hh-Gli signaling pathway and reduced apoptosis in Gprasp2-KO HEI-OC1 cells. This study revealed that Gprasp2-disruption inhibited Hh signaling pathway and led to cell apoptosis in HEI-OC1 cells, which might provide the potential molecular mechanism of GPRASP2 mutation associated with human SHL.