The Function And Mechanism Of ATP13A2 Regulating Astrocytic Mitochondiral Fission In The Pathological Process Of Parkinson’s Disease

objective:Kufor-Rakeb syndrome is a juvenile onset Parkinson’s disease(PD)syndrome caused by mutations in ATP13A2.The main pathological feature of PD is progressive loss of dopaminergic neurons(DA)in the substantia nigra pars compacta(SNc).Presentiy,the treatment of PD still relies on drugs to relieve symptoms or delay the progression,which cannot be fundamentally curative.Therefore,it is urgent to find new drug targets for PD treatment.Astrocytes are the most abundant cell type in the brain and play an important role in the nervous system,which continuously providing neurotrophic factor to neurons,regulating the formation of neuronal synapses and transmission of synaptic signals,maintaining neuronal homeostasis and providing support for neuronal metabolism.Mitochondrial homeostasis is particularly important for astrocytes and for maintaining normal physiological functions in the brain,as astrocytes exert all the above functions dependent on mitochondrial energy supply.Here in,we investigated the cause and mechanism of ATP13A2 loss induced mitochondrial dysfunction in astrocytes in vivo by constructing PD model mouse and ATP13A2 knockout mouse,and elucidating the role of ATP13A2 regulating astrocytic mitochondrial dynamics in the neurodegrneration of PD.Furthermore,ATP13A2 interfering lentivirus was applied to clarify the underlying molecular mechanism of ATP13A2 regulating mitochondrial fission in astrocytes in vitro.In conciusion,this article have elucidated the new mechanism by which ATP13A2regulates astrocytic mitochondrial fission-mediated neuroinflammation in the pathological development of PD,and providing novel molecular targets for improving therapeutic strategies in PD.Methods:(1)Acute MPTP PD model mouse was constructed and the relevance of ATP13A2 gene deletion to the development of PD was elucidated by behaviorally testing whether ATP13A2 knockout mouse showed motor dysfunction similarity to that of PD model mouse;(2)Correlation of neuronal loss in the midbrain of ATP13A2 knockout mouse with PD model mouse was detected by immunofluorescence,immunohistochemistry,and Nissl staining;(3)Expression levels of proteins related to brain mitochondrial dynamics in MPTP PD model mouse and ATP13A2 knockout mouse were measured by Western Blot;(4)Correlation between the proliferation activation of brain astrocytes and the expression level of Dynamin-related protein 1(Drp1)in ATP13A2 knockout mouse;(5)Astrocytes were cultured in isolation and divided into LV-NC group,LV-NC+MPP~+group,LV-ATP13A2-si RNA group,LV-ATP13A2-si RNA+Mdivi-1 group and LV-ATP13A2-si RNA+LV-ATP13A2-sh RNA group by lentiviral transfection,and the changes in mitochondrial function and mitochondrial dynamics-related protein expression levels in different treatment groups of astrocytes were detected by flow cytometry and Western Blot;(6)The levels of the exocytotic inflammatory cytokines Interleukin-6(IL-6)and Tumour Necrosis Factor-alpha(TNF-α)were measured by Elisa assay in the astrocytes of the different treatment groups.Results:(1)Compared to controls,ATP13A2 knockout mouse showed similar motor dysfunction as MPTP PD model mouse,including PD-like motor slowing,reduced voluntary movement and reduced motor coordination,loss of DA neurons in the SNc and proliferation activation of astrocytes;(2)The expression of Drp1 and Mitochondrial fission 1 protein(Fis1)was upregulated,and the expression of Mitochondrial fusion protein 1(Mfn1)and Mitochondrial fusion protein 2(Mfn2)was downregulated in the midbrain of ATP13A2 knockout mouse;(3)Enhanced fluorescent expression of GFAP and Drp1 in the SNc region of the midbrain in ATP13A2knockout mouse,and upregulation of Drp1 expression was found in proliferation-activated astrocytes;(4)Astrocytes overexpressing LV-ATP13A2-sh RNA or adding the drug Mitochondrial division inhibitor 1(Mdivi-1)significantly inhibited ATP13A2 knockdown induced mitochondrial dysfunction and upregulation of mitochondrial fission-related protein Drp1 and Fis1 expression;(5)Overexpression of LV-ATP13A2-sh RNA in astrocytes or addition of the drug Mdivi-1significantly inhibited the upregulation of IL-6 and TNF-αexpression levels in astrocyte supernatants induced by ATP13A2 knockdown.Conclusions:(1)ATP13A2 knockout induces motor dysfunction such as PD-like bradykinesia,reduced voluntary movement and reduced motor coordination,neuronal cell loss,astrocyte activation and abnormal mitochondrial fission in the midbrain of mouse;(2)Astrocyte ATP13A2 knockdown induced mitochondrial dysfunction,enhanced mitochondrial fission and upregulation of pro-inflammatory factors IL-6 and TNF-αexpression induced neuroinflammatory responses;(3)The mitochondrial fission inhibitor Mdivi-1 or astrocyte overexpression of ATP13A2 inhibits the abnormal mitochondrial fission and its mediated neuroinflammatory response in astrocytes induced by ATP13A2 deficiency,exerting a neuroprotective effect.