DJ-1 Activation Raf/ERK Pathways Promotes Autophagy Maturation of PC-12 Cells

Park 7 gene encodes a conserved protein called DJ-1 protein, which involves autophagy stress, but the mechanism is unclear. Therefore, it is necessary to explore the mechanism of DJ-1 regulation PC-12 autophagical stress. Using CRISPR/Cas9 technique to construct DJ-1 knockout PC-12 cell lines, we culture wild-type and DJ-1 knockout PC-12 cell lines, establish oxidative stress cell model by MPP + , and divide them into wild-type control group (WT), wild-type intervention group (WT + MPP + ), DJ-1 knockout control group (KO) and DJ-1 knockout intervention group (KO + MPP + ), and explore the role of DJ-1 in regulating neuronal autophagy stress by cell viability assay, immunofluorescence, confocal, western blotting and electron microscopy. The results show that the growth ability of DJ-1 knockout cells is inferior to that of normal cells, and DJ-1 knockout cells are more sensitive to oxidative stress and more vulnerable to damage than wild-type cells. Exposing to MPP + , DJ-1 proteins undergo oxidative responses at Cys-106 sites, while DJ-1 knockout PC-12 cells do not show similar responses. The wild-type PC-12 cells have the confocal in both anti-oxidant DJ-1 antibody and anti-C-Raf phosphorylation antibody. The activated DJ-1 induces the phosphorylation of C-Raf at Ser338 sites to activate directly C-Raf, and subsequently activates ERK1/2 signaling pathways Ser-338 sites, then activating C-Raf, subsequent activation of the MEK/ERK pathway. DJ-1 promotes autophagy maturation through the C-Raf/ERK pathway, thereby improving cell survival.


Introduction
Park 7 gene encodes a conserved protein called DJ-1, and is found to have an autosomal recessive gene deletion mutation in 2003, which can lead to early onset Parkinson's disease (PD) [1]. DJ-1 consists of 189 amino acids, containing three cysteine residues at sites 46, 53 and 106. Of the three cysteine residues, the 106-site cysteine residues are highly sensitive to oxidative stress [2] [3] [4]. DJ-1 can bind directly to the C-Raf kinase domain rather than RAS, and Cys-106 mutants of DJ-1 show weaker binding capacity than wild-type [5] [6]. The trigger of autophagy is precisely regulated by a series of waterfall events, including MAPK/ ERK and p38 signaling pathways. ERK nuclear translocation is associated with autophagy stress [7] [8] [9], but the mechanism of DJ-1 genes regulating autophagy stress is still not completely clear, especially the effect on autophagy maturation has not been reported, so it is necessary to explore deeply.

Knockout of DJ-1 Genes in PC-12 Cell Lines
Using CRISPR/Cas9 techniques in PC-12 cells, we first designed a sg sequence in DJ-1 functional exon coding regions that could guide Cas9 cleavage, and cloned it into plasmids that express both sgRNA and Cas9 simultaneously. Then the constructed plasmid was transiently transfected into PC12 cells, and the expressed Cas9 was directed to the target region by sgRNA, then the functional exon coding region DJ-1 was cut. The cells will cause frameshift mutation in the process of NHEJ repair, then DJ-1 genes was knock out in the PC12 cells. Finally, the cells will be cloned by finite dilution method to complete the screening of monoclonal cell lines and obtain completely consistent DJ-1 knockout PC12 cell lines, which was called as A12 cell line. A12 genotype was detected to have 25 bp base loss in exon 3, A12: TGCAGTGTAGCCGTG-TCTGGAAGAAGCAA-25 bp, in line with the goal of DJ-1 knockout.

Cell Culture and Treatments
We thaw the preserved wild-type PC12 and DJ-1 knockout PC-12 cells, and incubate them in a incubator for 2 d. Then the cells are collected by trypsin digestion, inoculated in 75 cm 2 culture bottles at a density of 2 × 10 6 /flask. They are incubated with a DMEM containing 10%FBS for 2 days, then digested by trypsin

Immunofluorescence Staining and Confocal
Immunofluorescence staining is based on the literature [10].

Western Blotting
Cell lysates are prepared in RIPA lysis buffer, and western blotting as described in the literature [11]. Nuclear protein is extracted according to nucleoprotein

Electron Microscopy
PC-12 cells ultrastructural changes, especially the changes of autophagy-related organelles, were analyzed by transmission electron microscopy see the literature for details [12]. We used transmission electron microscope (Hitachi, Japan H-7500, Suzhou University) to analyze the characteristics of autophagy vesicles by morphological characteristics. Autophagosomes contained intact cytoplasminto early stage, and partially disintegrating and electron dense in late stage, while autophagolysosomes were the products of autophagy and lysosome binding [8]. We randomly chose 10 micrographs (primary magnification, 10,000)

Statistical Analysis
All data were collected from at least three independent experiments in triplicate.
Through Student t test or single factor ANOVA and Bonferroni multiple comparison test, the significance of the influence between control conditions and experimental conditions was determined. Statistical significance was set to p ≤ 0.05.

Results
The growth ability of DJ-1 knockout cells is significantly lower than that of wild-type PC-12 cells, which is more sensitive to oxidative stress.
Literature studies have shown that DJ-1 plays a regulatory role in cell growth and proliferation, while oxidative stress has a negative effect on them [4] [13] [14] [15], so it is necessary to observe the effect of DJ-1, oxidative stress or both on cell viability. In this study, we observe the effect of various interventions on cell viability by MTT detection (Figure 1). The results show that the OD values of the cells in the intervention groups are significantly lower than that in the control groups, and the cells in the DJ-1 knockout groups are lower than those in the wild type cells, which is statistical differences. DJ-1 knockout cells are is highly sensitive to oxidative stress, and the Cys-106 oxidation state determines the functional level of DJ-1 [13]. Therefore, the oxidation characteristics of different cells under oxidative stress must be observed first. In this study, we eva-  OxDJ-1 binds to C-Raf and causes phosphorylation of Ser-338 sites, leading to C-Raf activation. What is the effect of activated complexes on MAPK/ERK signaling pathways? We culture wild-type and DJ-1 knockout PC-12 cells, divide them into control groups and intervention groups. The intervention groups are exposed to MPP + , and subsequently intervened with sorafenib and PD98059.
The effects of DJ-1 and oxidative stress on MAPK/ERK signaling pathway are observed by immunofluorescence (Figure 4(A)) and western blotting ( Figure   4(B), Figure 4(C)), respectively. In the study, we first observe the changes in the level of intracellular pERK (Figure 4(B)). The results show that knockout DJ-1 can lead to an increase of intracellular pERK, and exposure to MPP + induces a more obvious increase, which reacts to significant differences. The exposure of  DJ-1 knockout cells to MPP + caused further increase in intracellular pERK, but there isn't achieve significant difference. PD98059 can inhibit ERK activation  (Figure 4(C)), and the results show that exposure to MPP + increases the level of wild-type cell nuclear pERK, which reaches a significant difference, while knockout DJ-1 also increases the levels of intranucleus pERK, but there isn't significant difference. PD98059 or sorafenib can inhibit the increase of nuclear pERK, but only sorafenib inhibition to wild-type cell nuclear pERK is more obvious than PD98059, which achieves a significant difference.
DJ-1 can improve cell survival by promoting autophagy maturation. We collect all kinds of experimental cells, process them according to the procedure of electron microscope observation, and observe the morphological characteristics of cells by transmission electron microscope. The results show that DJ-1 knockout cells have more autophagy stress than wild-type PC-12 cells in the base stage, and both wild-type PC-12 cells and DJ-1 knockout cells can induce stronger autophagy stress in the face of oxidative stress ( Figure 5(A)). Wild-type PC-12 cells can produce more autophagolysosomes ( Figure 5(B)), while DJ-1 knockout cells are mainly autophagosomes ( Figure 5(C)), and mitochondria are irregular. PD98059 can reduce autophagy stress in wild-type PC-12 cells under oxidative stress, and the number of autophagolysosomes is also reduced; while sorafenib cannot down-regulate autophagy stress in DJ-1 knockout cells, but the number of autophagolysosomes decrease more significantly in wild-type PC-12 cells (Figure 5(D)). PD98059 can reduce the degree of autophagical stress, and there is no significant change in the proportion of autophagolysosomes/autophagosomes. Sorafenib can also down-regulate the degree

Discussion
Park 7 gene was found to have an autosomal recessive deletion mutation in 2003, leading to early onset Parkinson's disease (PD) [14].  Oxidative stress leads to DJ-1 activation by oxidation at Cys106 sites while stimulating intracellular ERK kinase phosphorylation. The cross-talk between the oxDJ-1 and C-Raf induces phosphorylation of C-Raf at Ser338 sites leads to C-Raf activation, which forms a complex with oxDJ-1. The complex promotes pERK nuclear translocation, subsequently regulates the expression of related genes. The genes regulate the expression of proteins, which promotes autophagosomes to autophagolysosomes transformation and induces autophagy maturation.
can inhibit the activation and nuclear translocation of ERK signaling pathways, and the degree of inhibition is almost similar. In wild-type cells, sorafenib can slightly down-regulate ERK1/2 phosphorylation, but it can significantly inhibit nuclear translocation, which doesn't exist for DJ-1knockout cells, suggesting that C-Raf activation plays a key role in the DJ-1 regulation of signaling pathways.
C-Raf (Ser338) site phosphorylation activates the MEK/ERK signaling pathway, which phosphorylates the ERK1/2 [26]. The activated p-ERK1/2 eventually transfers from the cytoplasm to the nucleus, and pERK1/2 phosphorylates related transcription factors and promotes cell growth or autophagy maturation [25] [27] [28]. Under oxidative stress, the wild-type PC-12 cells show this feature better, but for DJ-1 knockout PC-12 cells, despite some degree of activation of ERK pathways, pERK1/2 nuclear translocation was reduced. Oxidative stress promotes autophagy stress in wild-type PC-12 cells, leading to increased autophagy, as well as for DJ-1 knockout PC-12 cells. Moreover, the inhibition of cells or DJ-1 knockout cells, reduced the extent of autophagical stress, but did not change the proportion of autophagolysosomes/autophagosomes. Therefore, we have reason to speculate that DJ-1 could promote autophagy maturation through C-Raf/ERK signal pathway thus improving cell survival ( Figure 6).