GPR75 knockdown alleviates mitochondrial dysfunction via AMPK in diabetic retinal ganglion cells

Background Mitochondrial dysfunction plays a crucial role in retinal ganglion cells (RGCs) injury, the early pathogenesis of diabetic retinopathy (DR). G protein-coupled receptor 75 (GPR75), an orphan receptor, is a novel regulator of metabolic diseases. However, the role and mechanisms of GPR75 underlying diabetic RGCs mitochondrial dysfunction has not been reported. Methods High glucose (HG)-treated RGCs and streptozotocin (STZ)-induced C57BL/6 diabetic mice were used in the present study. GPR75-knockdown adeno-associated virus (AAV), GPR75-overexpression (OE) plasmid, and AMPK-activator AICAR were utilized to investigate the role of GPR75 in DR. Retinal thickness and cell number were assessed with optical coherence tomography (OCT) and hematoxylin and eosin (HE) staining. Mitochondrial damage, reactive oxygen species (ROS) levels, and ATP production in the retina and RGCs were assessed with transmission electron microscopy (TEM), Mito-Tracker Red staining, dihydroethidium (DHE) staining, and ATP assay kits. We also assess the expression of GPR75, AMPK, p-AMPK, pyroptosis (NLRP3, Cleaved-Caspase-1, IL-1β, IL-18, GSDMD, N-GSDMD), apoptosis (Cleaved-Caspase-3, Cytochrome C, Bax, Bcl-2) and mitochondrial homeostasis (OPA1, NDUFS3, DRP1). The interaction between GPR75 and AMPK was detected through co-immunoprecipitation (CO-IP) and double immunofluorescence staining. Results Compared with control retina and RGCs, diabetic retina and HG-treated RGCs exhibited increased GPR75 expression and AMPK dephosphorylation accompanied by pyroptosis, apoptosis, and a decrease in retinal thickness and RGCs number. Moreover, we observed increased DRP1 expression, decreased expression of OPA1 and NDUFS3, reduced ATP production, abnormal mitochondrial morphology and quantity, and increased ROS accumulation in diabetic retina and HG-treated RGCs, indicating mitochondrial dysfunction. What’s more, GPR75-knockdown reversed these phenomena. Mechanistically, the upregulation of GPR75 inhibits AMPK, leading to mitochondrial dysfunction with increased ROS accumulation, ultimately resulting in RGCs pyroptosis and apoptosis. Additionally, double immunofluorescence demonstrated the presence of both GPR75 and AMPK located in RGCs, and CO-IP revealed an interaction between GPR75 and AMPK in RGCs. Notably, AICAR counteracted the effects of GPR75-OE on pyroptosis, apoptosis and mitochondrial dysfunction in RGCs. Conclusions GPR75 induces mitochondrial dysfunction by interacting with AMPK and inhibiting its phosphorylation, which contribute to RGCs pyroptosis and apoptosis in DR. These findings suggest that GPR75 can serve as a therapeutic target in DR treatment.