Functional Research On Phospholipase ? Subunit TMEM30A And Associated Genes

The phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane in eukaryotes.Phospholipid asymmetry in the plasma membrane is established by phospholipid flippases—which belong to the P4-ATPase family—using energy from ATP hydrolysis.Mutations of P4-ATPase have been associated with many diseases,indicating the critical roles in vivo.Most P4-ATPases require a ?-subunit to form a functional flippase complex in membrane.TMEM30A is the most abundantly expressed TMEM30 protein and interacts with 11 of 14 P4-ATPases,which is enssential for their propor subcellular localization and function.Indeed,considering the the complexity of the tissue distribution of P4-ATPases,further exploring the functions of TMEM30A is advisable.At present,however,little is known about the cellular physiological functions of TMEM30A,and there lacks relevant animal models and in vivo studies.To understand the roles of flippase and their ?subunits TMEM30A,we constructed severval Tmem30a conditional knockout(KO)mouse model to explore the pathogenesis and mechanism of disease phenotypes in retinal cells,cerebellar Purkinje cells and islet ? cells.The functions of TMEM30A and P4-ATPases in these cells were studied from different perspectives,and thus lays a foundation for revealing the pathogenesis of P4-ATPase-related diseases.1.Functional study of TMEM30A in mouse retinaMutations in phospholipid flippase Atp8a2 in mouse could lead to retinal degeneration,while the pathogenic mechanisms remain elusive.We have verified that TMEM30A,the ? subunit of ATP8A2,is broadly expressed in mouse retina.We,therefore,generated several mouse models that Tmem30a was conditional deleted in either photoreceptors or retinal bipolar cells(RBCs)to study its roles in retinal cell survival and function,and ultimately reveal the pathogenic mechanisms of P4-ATPase-related retinopathy.Our data demonstrated that Tmem30a deficiency in mouse cone cells(HKO)leads to loss of photopic electroretinogram(ERG)responses,mislocalization of cone opsin,and death of cone cells.Besides,global KO of Tmem30a in adult mice(GKO)led to a reduced scotopic photoresponse,mislocalization of ATP8A2 to the inner segment and cell body,and caused increased apoptosis in the retina.Mechanistically,embryonic fibroblasts(MEFs)of GKO mouse exhibited diminished PS flippase activity and increased exposure of PS on the cell surface.These data demonstrated novel roles of TMEM30A in phospholipid flipping,protein transport and visual function maintenance of retinal photoreceptors.In addition,to investigate the role of TMEM30A in visual signal transmission,we generated RBC-specific Tmem30a knockout mouse model(PKO).The PKO mice exhibited defect in RBC function and progressive RBC death.PKC? staining of retinal cryosections revealed a remarkable dendritic sprouting of rod bipolar cells during the early degenerative process.Immunostaining analysis of PSD95 and mGluR6 expression demonstrated that PKO RBCs exhibited aberrant dendritic sprouting as a result of impaired visual transmission.Besides,loss of Tmem30a led to reactive gliosis with increased expression of GFAP and CD68,which induces apoptotic cell death.Taken together,our studies reveal the roles of TMEM30A in photoreceptors and RBCs,and demonstrated the importance of TMEM30A for protein transport and visual transmission in retina.2.Functional study of TMEM30A in cerebellar purkinje cells.Mutations in ATP8A2 cause a severe neurological disorder in human characterized by cerebellar ataxia and disequilibrium syndrome.However,the mechanism of the disease remains unclear.To investigate the the pathogenic mechanism and the role of TMEM30A in the cerebellum,we developed Tmem30a Purkinje cell(PC)-specific knockout mouse(PKO).Tmem30a deplation led to an increased expression of GFAP and astrogliosis in regions with PC loss,which accelerated cell death.Mechanistically,we found that TMEM30A is mainly located in vesicle transport-related organelles,such as endoplasmic reticulum(ER),Golgi and endosome,suggesting that TMEM30A may be involved in vesicle transport.We further performed a detailed analysis of the ER stress response.Elevated CHOP and BiP levels in PKO PCs indicated the presence of ER stress,which activates the downstream apoptotic cascades,as shown by elevated protein levels of Claeaved caspase-12 and Claeaved caspase-3.TUNEL analysis verified apoptotic cell death in cerebellum.Taken together,our data demonstrate an essential role of TMEM30A in the cerebellar PCs,and also reveal that TMEM30A is involved in the process of cellular vesicle transport.3.Functional study of TMEM30A in pancreatic ? cells.Mutations in the ATP10A and ATP10D have been associated with diabetes-related disease.However,the pathogenic mechanisms remain elusive.Diabetes results from the dysfunction of pancreatic ? cells.To investigate the pathogenesis of P4-ATPase-related diabetes and study the roles of TMEM30A in insulin secretion,we generated a mouse model which Tmem30a is deleted in pancreatic ? cells(IKO).Here,we demonstrated that IKO mouse exhibited diabetes-like symptoms characterized by impaired insulin secretion,glucose intolerance,insulin resistance,islet hyperplasia,obesity and hepatic lessons.Western blot using isolated islets and MIN6 cells showed that the insulin content in Tmem30a deficient ? cells was significantly decreased,while the ratio of proinsulin to insulin was increased,indicating impaired insulin maturation.Transmission electron microscopy showed a striking reduction in the number of mature insulin-containing granules in ? cells,accompany with distended Golgi apparatus.Insulin Immunostaining further revealed that the insulin was more concentrated in the Golgi apparatus,suggesting defects in insulin release and maturation from Golgi.In addition,Tmem30a deficient ? cells showed defective GLUT2 translocation to the membrane and a reduction of glycosylated mature GLUT2,which in turn disrupts GLUT2-mediated glucose responses,including membrane depolarization and Ca2+influx.Mechanistically,we found that Tmem30a depletion caused down-regulation of clathrin level accompany by abnormal aggregation in Golgi,and the clathrin-mediated endocytosis process was also affected,suggesting that TMEM30A is required for clathrin-dependent vesicle secretory pathways.Clathrin mediates vesicle trafficking between Golgi and membrane,thus blockage of this pathway should affect the secretion of insulin granules and GLUT2 transportation,which finally caused reduced insulin release and subsequent diabetic phenotypes.We further measured the expression of P4-ATPase in mouse ? cells and MIN6 cells,the qPCR data showed that the expression of most P4-ATPases was down-regulated.Besides,immunofluorescence staining on the highly expressed P4-ATPases(ATP8A1,ATP8B2,ATP11A)in ? cells with or without TMEM30A transfection verified that TMEM30A could help them correctly locate in Golgi and partially on the membrane,where they participate in the clathrin-mediated vesicle trafficking.Moreover,the Tmem30a knockdown MIN6 cells exhibited increased PS exposure on the cell surface,indicating impaired PS flippase activity.Taken together,our data demonstrate a novel role of TMEM30A in insulin maturation and glucose metabolic homeostasis,and investigate the mechanisms of TMEM30A involved in vesicle trafficking,which explains the pathogenesis of P4-ATPase-related diabetes.