Investigation Of OSBPL2 Deficits On The Cholesterol-homeostasis Deregulation And Oxidative Damage In Auditory Cells

Background: Oxysterol binding like protein(OSBP)and OSBP-related proteins(ORPs)are highly-conserved and evolutionarily-close analogues,which were extensively expressed in almost all eukaryotes.Oxysterol binding protein like 2(OSBPL2)is one of the OSBP/ORP family members and participates in multiple bioprocesses,including cell lipid metabolism,vesicle trafficking,cell growth regulation and signal transduction.In our previous work,OSBPL2 was identified as a novel causative gene for autosomal dominant non-syndromic hearing loss(DFNA67).However,the pathogenesis of the OSBPL2 deficits in ADNSHL remained unclear.Objective:Based on Osbpl2-knockout(KO)OC1 auditory cells and Osbpl2b-KO zebrafish models,the potential effect of OSBPL2 deficits on the cellular function and physiopathology of inner ear was investigated in vivo and in vitro.Methods:(1)The Osbpl2-KO and wild-type(WT)OC1 cells were subjected to RNA sequencing,and bioinformatics analysis was used to predict the effect of OSBPL2 deficits on cell function and signaling pathway.(2)The expressions of AMPKassociated effectors(p-AMPK,AMPK,p-ACC,ACC,HMGCS1,HMGCR)were detected by Western blot and q RT-PCR.(3)AMPK-knockdown/overexpression OC1 cells were used to investigate the regulatory role of AMPK signaling pathway on cellular cholesterol biosynthesis and expression of related genes.(4)The proteomic/interactomic analysis and co-immunoprecipitation(Co-IP)assay were performed to investigate the role of OSBPL2 on the regulation of AMPK signaling pathway.(5)The effect of Osbpl2/osbpl2b-KO on intracellular cholesterol levels and mitochondrial damage in OC1 cells and zebrafish inner ear were investigated by total cholesterol(TC)and reactive oxygen species(ROS)assay,the mitochondrial membrane potentials determination and subcellular characterization.Results:(1)Gene ontology(GO)analysis showed that Osbpl2-KO affected biological processes related to cholesterol synthesis.KEGG pathway analysis indicated that AMPK signaling was the most significant pathway.(2)Western blot analysis showed that Osbpl2/osbpl2b-KO inhibited AMPK signaling pathway and increased cholesterol synthesis.(3)AMPK-knockdown/overexpression in OC1 cells revealed that the AMPK signaling pathway was implicated in cholesterol biosynthesis,which could be promoted by inhibiting AMPK activity.(4)The proteomic/interactomic analysis and co-immunoprecipitation(Co-IP)assay indicate that ATIC,the key activator of AMPK,formed the complex with OSBPL2,and AMPK activity was modulated by the OSBPL2-ATIC interaction.(5)Osbpl2/osbpl2b-KO promoted cholesterol biosynthesis and increased TC and ROS levels in OC1 cells and zebrafish inner ear,which might be responsible for abnormal mitochondrial membrane potentials and mitochondrial damages in OC1 cells.Conclusions: The present work uncovered the regulatory role of OSBPL2 on cholesterol biosynthesis via AMPK signaling pathway.OSBPL2 deficits inhibited AMPK activity and led to increased TC and ROS levels in OC1 cells and zebrafish inner ear,and caused oxidative mitochondrial damage.This work laid the theoretical and experimental basis to elucidate the potential pathogenesis of OSBPL2 deficits in ADNSHL.