Effect Of ATP13A2 Gene Mutation On Endoplasmic Reticulum And Cellular Function In Parkinson’s Disease

Lysosomal transmembrane protein encoded by ATP13A2（PARK9）gene associated with Parkinson’s Disease（PD）belongs to type 5P ATPase subfamily.The wild-type ATP13A2 protein was localized to the lysosome,while all PD-related mutated proteins were localized to the Endoplasmic Reticulum（ER）to cause ER stress.Endoplasmic reticulum stress is an important factor leading to the occurrence of Parkinson’s disease.Mitochondria-associated endoplasmic reticulum（MAMs）,a subdomain of the endoplasmic reticulum,is closely associated and communicates with mitochondria and regulates a number of important cellular functions,including Ca²⁺homeostasis,mitochondrial transport dynamics,bioenergy,ER stress,apoptotic signaling,and the occurrence of inflammatory responses.These cellular dysfunctions are closely associated with the development of Parkinson’s disease.Studies have clearly proved that the deletion of ATP13A2 gene leading to lysosomal dysfunction is an important factor in the pathogenesis of PD.Later studies have also proved that the retention of ATP13A2-related mutated proteins in the ER can lead to the occurrence of ER stress response,but the mechanism is still not clear.In order to further clarify the specific mechanism of endoplasmic reticulum stress caused by ATP13A2mutated proteins’retention in the endoplasmic reticulum,and whether it has an effect on other organelles such as mitochondria by affecting MAMs.We take two ATP13A2 mutants,F182L and G877R,as examples.The overexpression of ATP13A2 wild-type and mutant plasmid in MN9D（mouse dopaminergic neuron）cells was used as positive control group and experimental group,while the overexpression of empty plasmid p IRES was used as negative control group.We investigated the effects of overexpression of ATP13A2 wild-type and mutant plasmids on endoplasmic reticulum,MAMs,and mitochondrial function,providing a strategy for scientific research on the pathogenesis of Parkinson’s disease.Firstly,four recombinant plasmids were amplified and verified.Plasmid DNA electrophoresis experiment and company sequencing results showed that the four recombinant plasmids were amplified correctly.Repeated cell transient pre-experiment showed that the plasmid could be successfully expressed in MN9D cell line by double verification of western blot and immunofluorescence,and cytotoxicity analysis was conducted in the pre-experiment,and it was found that:compared with the control group,the cells overexpressing the mutant plasmid produced irreversible cytotoxicity,which further supports our hypothesis that the retention of ATP13A2 mutant plasmid in the endoplasmic reticulum is related to the pathogenesis of Parkinson’s disease.In the subsequent cell transfection experiment,by immunofluorescence double-marker analysis,we demonstrated that the wild-type ATP13A2 proteins was located in the lysosome,while the F182L and G877R proteins were located in the endoplasmic reticulum and also distributed in the mitochondrial related endoplasmic reticulum subdomain,which communicate with the mitochondrial outer membrane.Endoplasmic reticulum stress caused by ATP13A2 mutation proteins may initiate apoptosis through UPR’s PERK/e IF2α/ATF4/CHOP pathway under overstress,and may also activate other pathways such as ATF6 and XBP1 pathway to regulate homeostasis.Through western blotting analysis,we found that the over-expression of ATP13A2 mutation can put the endoplasmic reticulum in a state of intense stress,which activate the CHOP pathway and inflammasome caspase12 to promote cell apoptosis,and meanwhile activate the phosphorylation of transcription factors ATF6 and IRE1 to regulate homeostasis.Through western blot analysis,we also found that overexpression of ATP13A2 mutant plasmid affected the expression of GRP75-IP3R-VDAC1 protein complex and DISC1,which regulate calcium homeostasis in MAMs,and the expression of PD-related genes localized in MAMs also changed.The up-regulated expression of GRP75 gene leads to the uncontrolled transport of calcium ions,which accumulate in mitochondria in large quantities,leading to mitochondrial dysfunction.In addition,we also found that the expression of MFN2 gene regulating mitochondrial fusion is also significantly down-regulated,both of which disrupt mitochondrial transport and dynamics.The abnormal function of intracellular organelles will stimulate the autophagy protective function in cells,so we analyzed the changes in the expression of autophagy-related proteins ATG5/AKT/m TOR,and found that the over-expression of ATP13A2 mutant proteins damaged the autophagy ability of cells.In addition,the expression of apoptosis-related proteins Bcl2 and Bax was affected,which promoted cell apoptosis.In general,we verified the ATP13A2 wild-type and mutant recombinant plasmids by enzyme digestion,successfully expressed the recombinant plasmid through cell transfection experiments.The toxicity of the transfection experiment to cells was analyzed by CCK8 kit and multifunctional microplate analyzer.We validated the distribution of ATP13A2 wild type and mutant proteins in organelles.It was further found that the overexpression of ATP13A2 mutant proteins caused endoplasmic reticulum stress and affected the occurrence of biological functions related to MAMs.They act on mitochondria through MAMs and cause mitochondrial dysfunction and autophagy.The occurrence of these pathological mechanisms increases cytotoxicity and promotes the death of nerve cells,which is an important factor in aggravating neuronal degeneration in neurodegenerative diseases.These results provide multiple reference directions for exploring the pathogenesis of Parkinson’s disease.