The Mechanism Of GJA8-Related Cataract And Autophagy And The Exploration Of Medicine Application

PurposeGJA8 is a gap junction protein in vertebrate lenses,which specifically expresses in lens.Mutations in GJA8 can cause cataract in humans.The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions,resulting in defects in intercellular communication among lens cells.However,we found that ablation of GJA8 in zebrafish led to severe defects in organelle degradation,causing an abnormal organelle-free zone(OFZ),which is an important cause of cataractogenesis in developing lens.Among all the pathways to maintain lens transparency,autophagy is important for intracellular quality control in the lens,but its role in organelle degradation in the lens remains disputable.In this study,we aimed to explore the mechanism between autophagy and organelle degradation during the development of lens,and hoped to find a new clue for GJA8-related cataractogenesis and possible medicine application.MethodsWe used frozen section and confocal microscope to explore the difference between wildtype and gja8b(a homolog of mammalian GJA8)mutant zebrafish during lens development.Next,we use human cell lines,via plasmid transfections,immunofluorescence,co-immunoprecipitations and immunoblots,to fully explore the mechanism of GJA8-related cataract and autophagy.To explore the possible applications for cataract,we tried the autophagy inducer to rescue the cataract-like phenotype in the lens of gja8 b mutant zebrafish.ResultsIn this study,we observed that ablation of GJA8 in zebrafish led to severe defects in organelle degradation,and the abnormal formation of OFZ,causing cataract phenotype.Intriguingly,we found that the cataract phenotype is related with deficient autophagy,such as decline in the expression of ATG proteins and the abnormal morphology and number of autophagy vesicles in the gja8 b mutant lens.Further studies in cells revealed that overexpression of GJA8 can stimulate autophagy in HLE cells and regulating autophagy via its direct interacting with ATGs.In vivo treatment of zebrafish with rapamycin,an autophagy activator,stimulated autophagy in the lens and relieved the defects in organelle degradation,resulting in the mitigation of cataracts in gja8 b mutant zebrafish.Conversely,inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects,suggesting the important roles of autophagy in GJA8-related cataract.ConclusionIn this study,we firstly revealed the direct relationship between organelle degradation in the lens with autophagy,which is also an unrecognized cataractogenesis mechanism caused by ablation of GJA8,and found a new biological function of GJA8 as an autophagy regulator.More importantly,we did imply that stimulation of autophagy may be a potential clinical treatment for cataract caused by GJA8 mutations.