The Role And Mechanism Of Mitochondrial SIRT4 In Retinal Müller Glial Cells

Sir2 homologous family proteins(Sirtuins)are NAD+-dependent histone deacetylases belonging to the third group of histone deacetylases.The silent information regulator protein 4(SIRT4)is a member of this family,mainly in mitochondria.Compared with the silent information regulator protein 1(SIRT1),silent information regulator protein 6(SIRT6),and silent information regulator protein 7(SIRT7),which exist in the nucleus,SIRT4 does not have the effect of deacetylation.Instead,it has the activities of ADP-ribosyl transferase,lipoamidase,and deacetylase,which can inhibit the activity of glutamate dehydrogenase(GDH)and limit the metabolism of glutamate and glutamine.SIRT4 also hydrolyzes the lipoamide cofactors in the DLAT E2 component of the DLAT dehydrogenase(PDH)complex and inhibits PDH activity.SIRT4 is highly expressed in retinal Müller glial cells.However,the role of SIRT4 in retinal Müller glial cells has not been clear until now.Therefore,this study intended to explore further the function of SIRT4 in retinal Müller glial cells.Part Ⅰ: The effects of SIRT4 on the cell activity,oxidative stress,and mitochondrial function of retinal Müller glial cellsPurpose: To investigate the effects of SIRT4 on the cell activity,oxidative stress,and mitochondrial function of retinal Müller glial cells under normal and injured conditions.Methods: 1.The localization of SIRT4 in rat retina and mouse-derived Müller glial cells(r MC-1)was verified by tissue and cell immunofluorescence staining.2.After overexpression and downregulation of SIRT4 in r MC-1 and human-derived Müller glial cells(MIO-M1),we detected cell apoptosis and intracellular reactive oxygen species(ROS)levels by flow cytometry.Apoptotic and pro-apoptotic proteins,as well as mitochondrial dynamic-related proteins,were detected by western blotting.We observed the mitochondrial morphology and number after Ds Red2-Mito plasmid transfection.3.After overexpression and downregulation of SIRT4 in r MC-1 cells,we treated r MC-1 cells with serum deprivation for 24 hours(cultured cells in a serum-free medium for 24 hours).Finally,we observed the state of r MC-1 cells under the microscope and detected apoptosis,the level of ROS,and cleaved caspase-3 by flow cytometry and western blot.Results: 1.SIRT4 was mainly located in the mitochondria of retinal Müller glial cells.2.The upregulation or downregulation of SIRT4 alone could not affect apoptosis.Overexpression of SIRT4 could reduce intracellular ROS,reduce the BAX/BCL2 protein ratio,up-regulate the L-OPA/S-OPA1 ratio,up-regulate the level of mitochondrial fusion protein MFN2 and mitochondrial cleavage protein FIS1,and make mitochondria tend to be fused.Knockdown of SIRT4 could increase intracellular ROS,increase the BAX/BCL2 protein ratio,decrease the ratio of L-OPA/S-OPA1,down-regulate the levels of mitochondrial fusion protein MFN2,total OPA1 protein,and mitochondrial cleavage protein DRP1,and make mitochondria tend to divide.3.Down-regulation of SIRT4 could increase intracellular ROS,cell death,and the expression of cleaved caspase-3 caused by serum deprivation.Overexpression of SIRT4 could reduce intracellular ROS,cell death,and the expression of cleaved caspase-3 caused by serum deprivation.Conclusions: SIRT4 can reduce oxidative stress in Müller glial cells,increase intracellular vitality,affect the level of mitochondrial dynamic-related protein,and make mitochondria tend to fuse.SIRT4 can also reduce the oxidative stress damage and cell death of Müller glial cells induced by serum deprivation.Part Ⅱ: SIRT4 can promote the proliferation of retinal Müller glial cells and the expression of stem-related genes of retinal progenitor cellsPurpose: To investigate whether SIRT4 can affect the proliferation of retinal Müller glial cells and maintain the characteristics of progenitor cells.Methods: 1.We overexpressed SIRT4 in MIO-M1 and rat primary Müller glial cells and detected cell proliferation by counting Brd U-positive cells.By western blot assay,we also detected the expression of SIRT4,glutamine synthetase(GS),Vimentin,glial fibrillary acidic protein(GFAP),proliferating cell nuclear antigen(PCNA),Notch homolog 1(NOTCH1),and SRY-related high-mobility-group(HMG)-box protein-2(SOX2).2.we knocked down SIRT4 in MIO-M1 cells and observed the cell status under the fluorescence microscope.We also detected the protein expressions of SIRT4,GS,Vimentin,PCNA,and GFAP by western blotting.3.we treated MIO-M1 cells and rat primary Müller glial cells with resveratrol at different concentrations(0 μM,1 μM,5 μM,10 μM)and detected cell proliferation by Brd U assay.Expression of SIRT4,GS,PCNA,hairy and enhancer of split 1(HES1),NOTCH1,and SOX2 were detected by Western blotting,too.We intraperitoneally injected rats with resveratrol(20 mg/kg)for a long time in vivo.After the injection,we observed the changes in the distribution and quantity of GS and SIRT4 proteins in Müller glial cells by immunofluorescence staining.We detected the proliferation of Müller glial cells in the retina by Brd U assay.The expressions of SIRT4,GS,Vimentin,PCNA,GFAP,and stem-related proteins of retinal progenitor cells were detected by western blotting.4.Under long-term intraperitoneal injection of resveratrol in rats,the proliferation of Müller glial cells treated with NMDA at different times was detected by Brd U staining.The expressions of SIRT4,GS,Vimentin,PCNA,GFAP,and stem-related proteins of retinal progenitor cells were detected by western blotting.Results: 1.SIRT4 up-regulated the expression of glutamine synthetase and stem-related protein of retinal progenitor cells in MIO-M1 and primary Müller glial cells.Moreover,SIRT4 promoted the proliferation of Müller glial cells.2.Low expression of SIRT4 could reduce the expression of PCNA and GS in MIO-M1 cells.3.Resveratrol could up-regulate SIRT4,GS,PCNA,and stem-related proteins of retinal progenitor cells in MIO-M1,rat primary Müller glial cells,and in vivo retinal Müller glial cells.Moreover,resveratrol promoted the proliferation of Müller glial cells,too.4.Resveratrol promoted the proliferation and dedifferentiation potential of retinal Müller glial cells induced by NMDA.Conclusions: SIRT4 can promote the proliferation of retinal Müller glial cells and the expression of the stem-related protein of retinal progenitor cells.As an antagonist of the Sirtuins family,resveratrol may promote the proliferation of Müller glial cells and the expression of the stem-related protein of retinal progenitor cells by up-regulating the expression of SIRT4 with the normal condition or damage by NMDA.