Parkin Protects Mitochondrial Homeostasis By DJ-1 Down-regulation And DJ-1 L166P Mutant SH-SY5Y Cells By Oxidative Stress Pathway

Parkinson’s disease (Parkinson disease, PD), the second most common chronic neurodegenerative diseases to Alzheimer’s disease (AD), is pathological characterized by survival lewy bodies (LB) in neurons intracytoplasmic and selective loss of dopaminergic neurons within the substantia nigra pars compacta. The incidence of PD is grow along with the age. More than90%of the patients are older than55years. The foreign researches revealed that morbidity was about1%in65years old crowd and rose to4%in older than85people. Though the pathogenic mechanisms underlyings PD remains elusive, the majority support a role for combined action of genetic and environmental factors. Mithchondria are dynamic organelles and mitochondria dysfunction is involved in the sporadic or genetic PD.DJ-1is a multi-functional protein that plays roles in anti-oxidative stress, transcriptional regulation and molecular chaperone. It has a variety of ways against the cell damage caused by oxidative stress. As an antioxidant, DJ-1eliminates intracellular excess reactive oxygen species (ROS) by itself cysteines oxidation. In addition, through regulating the transcription of glutathione synthesis, DJ-1could enhance the cell antioxidant ability. It’s mutations, the long deletion of exons1-5or a substitution of a highly conserved leucine into a proline in position of166of DJ-1sequence, can lead to autosomal recessive familial PD. Other mutations are found in in the sporadic PD patients. The point mutant L166P fails to form active homologous or heterologous dimer leading to inaction of the clearance of exogenous hydrogen peroxide even endogenous ROS. DJ-1knockout and its pathological mutation can make the cell to improve oxidative stress sensitivity. Knock out DJ-1gene mouse and fruit flies increase sensitivity to oxidative stress caused by1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), hydrogen peroxide, paraquat, etc., which can lead to cell death of dopaminergic neurons.Loss-of-function mutations of the parkin (PARK2) gene, which encodes a ubiquitin-protein ligase, are a common cause of autosomal recessive juvenile parkinsonism (ARJP). Except as a member of ubiquitin-proteasome system to degradate substrates, Parkin plays an important role in signal transduction, DNA repair, oxidative stress and mitochondrial homeostasis.Both DJ-1and Parkin, associated with autosomal recessive familial PD, involved in regulation mitochondria at multiple levels. However, their specific mechanisms and whether it has interaction between them still not clear. This research pay close attention to the regulation function of DJ-1and Parkin to mitochondrial homeostasis, as well as the mitochondrial protection of Parkin against DJ-1mutation and knock out in SH-SY5Y cell.The main methods and results are as follows:1) Constructe oxidative stress model of SH-SY5Y cells by exogenous H2o2and detecte its mitochondrial function. The experimental results show that intracellular level of ROS and mitochondrial fragment increase.meantime, mitochondrial membrane potential decreases. DJ-1protein increased after incubation with H2O23hours and then decline.2) Construction and identification of DJ-1KD cell model:Design and synthetize DJ-1siRNA interference sequence. The plasmid, confirmed by sequencing, is transfected into SH-SY5Y cells. Both mRNA and protein two levels of DJ-1expression are detected by realtime PCR, Western blot and immunofluorescence. Down-regulated endogenous DJ-1expression, is similar to add exogenous H2o2, also increased the level of ROS, decrease mitochondrial membrane potential, reduce ATP concentration and contribute to release Cyt C into the cytosol.3) Construction and identification of pathological DJ-1L166P point mutation cell model: L166P plasmid, confirmed by sequencing correctly, is transfected into SH-SY5Y cells. Overexpression of pathological DJ-1L166P protein, increase the level of ROS, decrease mitochondrial membrane potential, reduce ATP concentration, contribute to release Cyt C into the cytosol and cause cells apoptosis. Moreover, exogenous H2o2into overexpressed DJ-1L166P cells increase the level of cell oxidative stress.4) The effects of Parkin in oxidative stress on the mitochondria:Immunofluorescence technical detection show mainly distribution of Parkin in the cytoplasm, a few colocalization with mitochondria. The results of Western blot and immunocytochemistry reveal the expression of parkin protein increase after incubation with exogenous H2o2for3h in SH-SY5Y cells, which in line with the expression of DJ-1protein. Exogenous H2o2increase intracellular ROS but no significant effect on ATP concentration in the overexpression of parkin WT cells.5) To investigate the relationship between DJ-1and Parkin, co-Immunoprecipitation (co-IP) experiments were performed. SH-SY5Y cells were transiently co-transfected myc-Parkin with DJ-1WT or not followed by IP with anti-DJ-1antibody or anti-myc antibody. We found that DJ-1could co-immunoprecipitated with WT Parkin. In particular, the treatment of H2O2weakened the specifically interaction between them. Additionally, L166P point mutation decreased the association of DJ-1and Parkin. Immunofluorescence was employed to analysis of Parkin expression and subcellular location under DJ-1changed groups, arkin protein strikingly reduced in DJ-1KD cells but not in over-expressed DJ-1L166P cells.6) The overexpression of Parkin WT could rescue mitochondrial dysfunction caused by DJ-1KD. The experimental results show that overexpressed Parkin can improve abnormal changes of intracellular ROS level, mitochondrial membrane potential and ATP levels due to deficient DJ-1protein.7) The Parkin WT overexpression alleviate mitochondrial dysfunction caused by the overexpressed pathological DJ-1L166P mutational protein in SH-SY5Y cells. The experimental results show that the upexpressed Parkin protected mitochondrial dysfunction of DJ-1L166P overexpression by the way of oxidative stress. However, Parkin have active role in maitaining mitochondrial morphologyOur results provide evidences for the concept that the intracellular level of oxidative stress were increased in SH-SY5Ycells of deficient DJ-1or over-expressed DJ-1L166P, causing dysfunctional mitochondria. Parkin up-regulation rescued the abnormal changes of DJ-1KD cells or partly DJ-1L166P cells by the way of affecting level of oxidative stress.