Nur77 exacerbates PC12 cellular injury in vitro by aggravating mitochondrial impairment and endoplasmic reticulum stress

The nuclear orphan receptor, Nur77 plays important roles in neuroimflammation, apoptosis, and dopaminergic neurodegeneration. We conducted a further mechanistic investigation into the association of Nur77 with cell death. Cytosporone B (Csn-B), an agonist for Nur77, and Nur77 knockdown were adopted in the 6-hydroxydopamine (OHDA)-lesioned PC12 cells to investigate the mechanisms underlying Nur77-mediated injury. The 6-OHDA incubation caused Nur77 translocation from the nucleus to cytosol and Endoplasm reticulum (ER) and induced co-localization of Tom20/Nur77 and Protein Disulfide Isomerase (PDI)/Nur77. Nur77 activation further decreased cell viability, aggravated intracellular LDH release, intracellular Ca2+, ROS levels, apoptosis, ER tress and, mitochondrial transmembrane potential (ΔΨm) decline. In addition, Nur77 activation significantly enhanced the efficiency of autophagy as indicated by an up-regulation of Beclin-1/LC-3 and downregulation of p62, and aggravated mitochondrial dysfunctions and ER stress as shown by increased HSP60/Cytochrome C (Cyt C) and CHOP-ATF3 levels respectively. These changes could be partially reversed by Nur77 knockdown. Moreover, Nur77 activation upregulated PINK1 and downregulated Parkin levels. We conclude that Nur77 exacerbates PC12 cell death at least partially by aggravating the mitochondrial impairment and ER stress and enhancing autophagy. We propose that Nur77 is likely a critical target in the PD therapy.


Results
Nur77 exacerbated PC12 cell injury. 6-OHDA significantly increased the expression of Nur77 in a dose-dependent fashion. In the group of 100 μM 6-OHDA for 24 h incubation, Nur77 increased by 4.3-fold compared to the control level (***p < 0.001, n = 5 independent experiments, Fig. 1A). Since the incubation at 100 μM of 6-OHDA for 24 hrs induces valid PD-like changes in in vitro PD model as shown in our previous studies 2 , we use this treatment in the subsequent experiments.
We employed Csn-B, an agonist of Nur77, which specifically binds to the ligand-binding domain of Nur77 and stimulates Nur77-dependent transactivation activity forward 20 . We also carried out an in vitro knockdown experiment by introducing small interfering RNA (siRNA) against Nur77 in the PC12 cells. The results showed that in the present of Csn-B, Nur77 increased by 3 fold (***p < 0.001, n = 5 experiments, Fig. 1B) as compared to the blank groups and siRNA targeting negative control (siCtrl group), while transfection of siRNA targeting Nur77 (siNur77 group) effectively decreased Nur77 to 2.6% of the control level (***p < 0.001, n = 5 experiments, Fig. 1C).
Western blot results showed that 6-OHDA decreased Bcl2 expression, but increased Cytochrome C and cleaved-caspase 3, while co-treatment with Csn-B amplified the alterations in the expression of these proteins (Bcl2: 65.2% of the control group in 6-OHDA-treated group, 41% in the Csn-B + 6-OHDA group; Cytochrome C: 2.3 fold of the control group in 6-OHDA-treated group, 4.2 fold in the Csn-B + 6-OHDA group, n = 5 independent experiments,***p < 0.001; cleaved-caspase 3: 4.9 fold of the control group in 6-OHDA-treated group, 8.4 fold in the Csn-B + 6-OHDA group, n = 5 independent experiments, **p < 0.01, ***p < 0.001, Fig. 2B). Similarly, the densities of Cytochrome C and cleaved-caspase 3 increased and the examination of confocal microscopy showed that they are co-located in cytoplasm (Fig. 2C). Furthermore, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The TUNEL staining slightly increased in the 6-OHDA-treated group as compared to the blank group or Csn-B alone group. However, TUNEL staining was stronger in the group of Csn-B plus 6-OHDA compared to the 6-OHDA group (Fig. 2D).
Nur77 regulated ER Stress via intracellular ROS and Ca 2+ PI3K/AKT, CHOP and ATF3. Recent evidence suggest that Nur77-mediated apoptosis in tumor cells and T cells is associated with Nur77 nucleocytoplasmic translocation. Western blot and immunofluorescent staining with confocal microscopy was performed to determine the expression and localization of Nur77 in 6-OHDA-lesioned PC12 cells with or without Csn-B treatment. We extracted cytosolic fractions without mitochondria to detect Nur77 in endoplasm reticulum with/ without 6-OHDA incubation. Nur77 increased to 11.5% in cytoplasm compared to the control group (n = 5 independent experiments, ***p < 0.001, Fig. 3A). In the 6-OHDA-treated group, PDI increased by 5.55 fold compared to the control (n = 5 independent experiments, ***p < 0.001, Fig. 3A). Moreover, our results showed Scientific RepoRts | 6:34403 | DOI: 10.1038/srep34403 that increased density of cytosolic Nur77 was co-localized with ER protein disulfide isomerase (PDI) in the 6-OHDA-treated group compared with control group (Fig. 3B). Transfection with siRNA targeting Nur77, Nur77 decreases sharply (C). The bar chart shows the relative quantification of Nur77 compared with that of β-actin (A-C). The data are expressed as the relative ratios of the control group, which was set to 1.0, and are expressed as the mean ± SEM of 5 independent experiments, ***p < 0.001. (D,E) Cell viability was measured using MTS assay (D). LDH leakage into the medium was measured using LDH assays (E). For agitated Nur77, PC12 cells were incubated with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. For knockdown of Nur77, PC12 cells were transfected by empty vector or siRNA targeting Nur77 with or without incubation with 100 μM 6-OHDA. The MTS data are expressed as the relative ratios of the control group, which was set to 1.0. The LDH data are expressed as the ratio of LDH release level to the maximal LDH release. Data are expressed as the mean ± SEM of 5 independent experiments, **p < 0.01, ***p < 0.001. (F) Western blots of TH, DAT, Nurr1 expressions in PC12 after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. The bar chart shows the relative quantification of proteins. The data are expressed as the mean ± SEM of 5 independent experiments, **p < 0.01, ***p < 0.001.  (A) Apoptosis was quantified by flow cytometry with double staining annexin V-FITC and PI. For agitated Nur77, PC12 cells were incubated with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. For knockdown Nur77, PC12 cells were transfected by empty vector or siRNA targeting Nur77 with or without incubation with 100 μM 6-OHDA. The bar chart shows the apoptotic rate of PC12 cells. The results are expressed as the mean ± SEM of 5 independent experiments, **p < 0.01, ***p < 0.001. (B) Western blots of Bcl2, Cytochrome C (Cyt C), cleaved caspase 3 expressions in PC12 after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. The bar chart shows the relative quantification of the protein levels compared with that of β-actin. The data are expressed as the relative ratios of the blank group, which was set to 1.0, and are expressed as the mean ± SEM of 5 independent experiments. **p < 0.01, ***p < 0.001. (C) Immunofluorescent staining with confocal microscopy of Cyt C (red fluorescence) and cleaved caspase 3 (green fluorescence) and DAPI (blue fluorescence) after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. Scale bar: 20 μm. (D) Immunofluorescent staining with confocal microscopy of Tunel (green fluorescence) and DAPI (blue fluorescence) after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. Scale bar: 20 μm.  The mRNA expression of ER stress genes CHOP, ATF3 were assessed by quantitative real-time PCR in the PC12 cells after treatment (E). For agitated Nur77, PC12 cells were incubated with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. For knockingdown Nur77, PC12 cells were transfected by empty vector or siRNA targeting Nur77 with or without incubation with 100 μM 6-OHDA. The bar chart shows the ROS levels (C) and intracellular Ca 2+ levels (D) as the average intensity of the recorded fluorescence (NRFU) per cell (U/cell). (F) Western blots of p-AKT, AKT, p-PI3K, PI3K expressions in PC12 after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. The bar charts show the relative quantification of p-AKT/AKT and p-PI3K/PI3K. All the data are expressed as the relative ratios of the blank group, which was set to 1.0, and are expressed as the mean ± SEM of 5 independent experiments, **p < 0.01, ***p < 0.001.
Nur77 induced mitochondrial impairment. We found that Nur77 increased by 15% in the mitochondria of 6-OHDA group compared to the control group in Nurr77 nucleocytoplasmic translocation (n = 5 independent experiments, ***p < 0.001, Fig. 4A). In the 6-OHDA group, HSP60 increased by 4.7 fold compared to the control (n = 5 independent experiments, ***p < 0.001, Fig. 4A). Our results showed that increased density of cytosolic Nur77 was co-localized with the mitochondrial out membrane protein Tom20 in the 6-OHDA-treated group (Fig. 4B) compared with the control group. We also found that Nur77 co-localized with Hsp60 both in the 6-OHDA group and Csn-B with 6-OHDA group, but the co-treatment with Csn-B and 6-OHDA increased the densities of Nur77 and HSP60 (Fig. 4C). This indicates that more Nur77 translocation from nuclear to mitochondria in 6-OHDA-lesioned PC12 cells is involved in mitochondrial impairment. To determine mitochondrial impairment, we used JC-1 staining to examine the mitochondrial membrane potential (ΔΨm). JC-1 aggregate rate represented the ratio of healthy cell with normal membrane potential to the total cells, while JC-1 Monomer rate represented the ratio of unhealthy cell with collapsed membrane potential to the total cells. Incubation with Csn-B showed little effect on the ΔΨm of PC12 cells. However, Csn-B significantly collapsed mitochondrial ΔΨm under 6-OHDA treatment, as JC-1 aggregate rate of Csn-B with 6-OHDA group decreased to 20.5% of the blank control level, while JC-1 aggregate rate in 6-OHDA alone group decreased to 38.9% of the control (n = 5 independent experiments, **p < 0.01, ***p < 0.001, Fig. 4D). In contrast, silencing Nur77 rescued the mitochondrial ΔΨm as the JC-1 aggregate rate was 73.7% in the siNur77 + 6-OHDA group and 67.0% in the siCtrl + 6-OHDA group (n = 5 independent experiments, **p < 0.01, ***p < 0.001, Fig. 4D).
Nur77 induced autophagy through PINK1/Parkin/Beclin-1/p62. Our results showed that mitochondrial out membrane protein Tom 20 of Csn-B plus 6-OHDA group decreased to 7% of the control level, while Tom 20 of 6-OHDA alone group decreased to 38.8% of the control (n = 5 independent experiments, ***p < 0.001, Fig. 5A). Considering that Nur77 is involved in mitochondrial impairment, we proposed that Nur77 is also involved in mitochondrial autophagy.
Under the 6-OHDA condition, Csn-B increased PINK1 expression and decreased Parkin expression (PINK1: 2.2 fold higher in 6-OHDA group and 3.4 fold higher in the Csn-B + 6-OHDA group as compared to the control; Parkin levels as compared to the contrl: 54% in 6-OHDA group and 9% in the Csn-B + 6-OHDA group, n = 5 independent experiments, **p < 0.01, ***p < 0.001, Fig. 5C). In confocal microscopy studies, we observed that Parkin distributed mainly in the cytoplasm but not in the mitochondria. Most of the Parkin did not co-locate with Tom20 in normal condition. However, 6-OHDA induced Parkin accumulation on the mitochondria membrane. The co-location of Tom20 and Parkin in the 6-OHDA group and Csn-B with6-OHDA group, and Parkin density in Csn-B plus 6-OHDA group decreased sharply (Fig. 5B). This indicated that damaged mitochondria were removed via mitochondrial autophagy. In term of the autophagy markers, 6-OHDA treatment slightly increased the Beclin 1 and ratio of LC3 II to LC3 I (Beclin 1: 1.2 fold of the control group; the ratio of LC3 II to LC3 I 1.5 vs. 0.4 in the control group; n = 5 independent experiments, Fig. 5C). In contrast, 6-OHDA decreased p62 to 73.5% of the control level (n = 5 independent experiments, **p < 0.01, ***p < 0.001, Fig. 5C). However, Csn-B promoted the autophagy process as the Western blot results showed that Beclin 1 and the ratio of LC3 II to LC3 I significantly increased by 1.8 fold and by 2.3 fold, respectively, and p62 decreased to17.5% of the control in the Csn-B + 6-OHDA group compared with 6-OHDA group (n = 5, independent experiments, **p < 0.01, ***p < 0.001, Fig. 5C).
We also found that LC3 co-location with Tom20 after treatment with 6-OHDA (Fig. 5D) and a decreased density of Tom 20 and increased density of LC3 under co-treatment of 6-OHDA and Csn-B (Fig. 5D). Interestingly,  in the Can-B with 6-OHDA group, LC3 and Tom20 were fragmented in some cells, indicating the degradation of mitochondria (Fig. 5D(p)).

Discussion
We investigated the roles of Nur77 in PC12 cell injury induced by 6-OHDA and made three novel findings in this study: (1) Nur77 knockdown attenuates 6-OHDA-induced PC12 cell injury, while its activation exacerbates cellular degeneration; (2) Nur77 displays a devastating effects on 6-OHDA-lesioned PC12 cells partially through exacerbating mitochondrial impairment and ER stress; and (3) Nur77 enhances autophagy. To our knowledge, this study is the first to demonstrate that ER stress inducing caspase-dependent apoptosis and autophagic cellular death in 6-OHDA-lesioned PC12 cells is mediated by Nur77 signaling. Even though they are not considered neurons, PC12 cells can easily differentiate into neuron-like cells and share properties similar to neurons, which make PC12 cells useful as a model system for neuronal differentiation 21 . Though our results are not from midbrain dopaminergic neurons, our study using PC12 cells displaying an unspecific catecholaminergic phenotype, still provides a crucial clue that Nur77 may be an important modulator in mediating mitochondrial impairment and cellular death in the pathogenesis of PD.
Several lines of evidence suggest that Nur77 plays important roles in DA neuronal death, differentiation, biochemical activity, dopamine turnover, dopamine-related neuroadaptation processes [22][23][24][25] . Specifically, under the nuclear export signals induced by nerve growth factor (NGF) 25 , Nur77 modulates retinoid signaling through heterodimerization with retinoid-X receptor 24 . One in vivo study by St-Hilaire et al., 25 showed that in Nur77 −/− mice, Nur77 proved to set the threshold level for L-DOPA-induced rotational behavior. L-DOPA-induced rotational response was exacerbated in Nur77 −/− mice, compared to Nur77 +/+ ones, while the up-regulation of encephalin and neurotensin by lesioning the nigrostriatal pathway was significantly reduced in Nur77 −/− mice. In terms of behavioral and molecular adaptations to dopamine denervation and repeated L-DOPA treatment, it does seem to be worsening in Nur77 −/− mice. Therefore, Nur77 may play different roles in different conditions. The total levels of Nur77 increased in a dose-dependent manner under 6-OHDA lesion (Fig. S1). Our findings (Figs 3A and 4A) showed that under 6-OHDA treatment, Nur77 accumulated in cytosol and mitochondria that is consistent with Martinoli' study 26 indicating that 6-OHDA induced the translocation of Nur77. This implies that in 6-OHDA induced cellular death, Nur77 is concurrently contributing to DA neuronal death as a detrimental factor. To investigate the mechanisms of Nur77, we further activated or blocked Nur77 function using its agonist and knockdown respectively in the in vitro model. These findings (Figs 1D-F and 2A) show that Nur77 was positively parallel with the 6-OHDA-mediated injury and aggregated the neurodegeneration in PC12, and further confirming our conclusion that Nur77 may induce deleterious effects on this in vitro model.
An important observation in this study was that in 6-OHDA lesion, Nur77 translocated from nuclear to mitochondria and endoplasm reticulum. Given this translocation to both mitochondria and endoplasm reticulum, we propose that this Nur77 translocation from the nucleus into the cytosol may initiate the cellular death. Deficiencies in mitochondrial dynamics and ER stress under the conditions of Ca 2+ homeostasis, oxidative stress and apoptosis not only result in cellular abnormal energy metabolism, but also accelerate neurodegeneration 26,27 . Indeed, Merkwirth et al., indicated that mitochondrial defects including the perinuclear clustering of mitochondria in hippocampal neurons, mitochondrial fusion and mitochondrial ultrastructure disruption lead to extensive neuro-degeneration associated with behavioral impairments and cognitive deficiencies in mice 28 .
Previous evidence has shown that Nur77 translocation to cytoplasm is necessary for ER-induced stress 29,30 . It has been documented that severe ER stress can induce neuronal death and exacerbate neurodegeneration via different pathways including autophagic induction, cytotoxicity, upregulation of both CHOP and p53, ER mitochondria calcium cycle and apoptosis 3,31-37 . CHOP, a transcription factor, is reported as an ER-stress induced modulator in cellular death and the cascade CHOP-ATF3 is involved in ER-stress 38 . The observation that under oxidative stress the levels of CHOP-ATF3 and p-AKT/p-PI3K were profoundly increased and decreased respectively implies that serious ER stress was activated to produce obvious apoptosis in the in vitro model (Fig. 3E,F). In addition, the increased CHOP together with decreased Bcl 2 following Nur77 activation under oxidative stress suggestes that Nur77 may regulate ER stress and influence the cellular survival, which is consistent with one previous study showing CHOP down-regulated Bcl2 and making cells sensitive to ER stress 39 .
Interestingly, the present study showed that CHOP-ATF3 was upregulated or downregulated in 6-OHDAlesioned PC12 in response to Nur77 activation or knockdown, respectively. It also showed that p-AKT/p-PI3K downregulation following Nur77 activation with 6-OHDA lesion only. This finding further indicates that the translocation of Nur77 from the nuclear may directly exacerbate ER stress and trigger cellular death via of Parkin (red fluorescence) and Tom20 (green fluorescence) and DAPI (blue fluorescence) after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. Scale bar: 20 μm. In the magnifying figures (d Ctrl group and h 6-OHDA group), the scale bar is 10 μm. (C) Western blots of Beclin 1, p62, LC3 expressions in PC12 after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. The bar chart shows the relative quantification of the protein levels compared with that of β-actin. The data are expressed as the relative ratios of the blank group, which was set to 1.0, and are expressed as the mean ± SEM of 5 independent experiments. **p < 0.01, ***p < 0.001. (D) Immunofluorescent staining with confocal microscopy of LC3 (green fluorescence) and Tom20 (red fluorescence) and DAPI (blue fluorescence) after treatment with 100 μM 6-OHDA or 5 μg/ml Csn-B alone, or in combination with both 6-OHDA and Csn-B, and control vehicle was used as control. Scale bar: 20 μm. In the magnifying figures (d Ctrl group and h 6-OHDA group), the scale bar is 10 μm.
Scientific RepoRts | 6:34403 | DOI: 10.1038/srep34403 CHOP-ATF3 and AKT-PI3K signalling pathways. It is noteworhty that our results are different from thoses of previous studies, showing that mild ER stress inhibits neuronal death by promoting autophagy or mitophagy 17,40 . The discrepant results may be due to the fact that 6-OHDA (100 μM) in our study induced severe ER stress that stimulates and aggravates both apoptosis and cellular death. This is while ER stress was relatively mild in previous studies and therefore probably induced autophagy/mitophagy mediated neuroprotection.
In addition to ER stress, mitochondrial impairment was also observed under oxidative stress and aggregated following Nur77 activation, as indicated by HSP60 and Cyt C. We hypothesize that the Nur77 translocation initiates the neuronal death probably through disruption of mitochondrial integrity, excessive mitochondria clearance and further induction of autophagy, subsequently leading to an irreversible cellular death 14,41 . The increased intracellular calcium (Fig. 3D) may influence the sensitivity of mitochondria to oxidative stress, and further aggravates the 6-OHDA-mediated mitochondrial impairment, which may lead to an irreversible cellular death. Previous studies suggest that the subcellular location of NR4A is functionally important and cytoplasmic NR4A acts on the mitochondria and interacts with Bc12 to promote apoptosis 42,43 . For example, Lin et al. 44 and Thompson et al. 43 , showed that translocation of Nur77 from nuclear to mitochondria promoted its interaction with Bcl2 and switched Bcl2 activity from an anti-apoptotic factor into a pro-apoptotic molecule in cancer cells and T cells. In this study, we found that 6-OHDA-induced translocation of Nur77 into the cytosol is enhanced following Nur77 activation and this enhanced translocation of Nur77 is parallel with the downregulation of Bcl2, upregulation of caspase 3, and cellular apoptosis. Therefore, it seems that Nur77 does not only switch Bcl2 activity from an anti-apoptotic factor into a pro-apoptotic molecule, but also reduces the level of Bcl2, leading cell injury. These findings demonstrate that Nur77 positively modulates the mitochondrial impairment and promotes apoptosis via regulating Bcl 2 and caspase 3 under oxidative stress. The fact that the 6-OHDA-induced apoptosis was attenuated after Nur77 knockdown further supports the critical role of Nur77 in cellular death. In addition, we identified the mitochondrial function using JC-1 assay to detect the mitochondrial membrane potential and evaluate its function. Our results showed that the 6-OHDA-induced a sharp loss of ΔΨm and increased JC-1. This was attenuated and aggregated by Nur77 activation and knockdown respectively. Subsequently this verified that Nur77 may be directly involved in the modulation of mitochondrial membrane function. Based on the above observations, the present mechanisms identified in PC12 cells support a role of Nur77 in DA cell loss. However, these findings need to be further validated in other in vitro and in vivo PD models.
As the markers of mitochondria functions, PINK1 and Parkin (encoded by the PARK2 gene) are the key proteins associated with autophagy in the neuro-pathogenesis of PD. They physically associate and functionally label damaged mitochondria for selective degradation via autophagy [45][46][47][48] . Choubey et al., showed that Beclin 1, the autophagy protein, is involved in PARK2 translocation to mitochondria and regulates PINK1 overexpression-induced PARK2 translocation 49 . Wong and Holzbaur indicated that through the LC3-interacting region, another autophagy-related protein, autophagosome assembly around mitochondria was induced after ubiquitinating mitochondria downstream of PARK2 which interacted with OPTN (optineurin) 50 .
To further determine whether Nur77 affects 6-OHDA-lesioned PC12 cells via regulating autophagy and mitochondrial functions and influencing PINK1 and Parkin, we activated Nur77 and then examined the association of autophagy and mitochondrial functions together with PINK1/Parkin levels. Beclin-1 acts as a promoter in autophagy, while p62 is the substance of autophagy and usually behaves as a cargo receptor for ubiquitinated proteins to play a role in selectively delivering various proteins to the autophagosome. The increased Beclin-1 and decreased p62 imply an increased autophagic induction. Our results (Fig. 5A,B) strongly verified that Nur77 has a harmful influence on mitochondrial function. Interestingly, the expressions of Betclin 1 and LC3 are similar to that of PINK1 but contrary to Parkin with Nur77 activation under oxidative stress. On the other hand, the alteration in p62 is different from those of both Beclin-1 and LC3. Our observation on the colocation of Parkin/Tom20 and LC3/Tom20 is consistent with the viewpoint that Parkin binds to depolarized mitochondria and induced autophagy of mitochondria 51 . Both Parkin and PINK1 were involved in cellular response to Nur77 agonist under 6-OHDA condition for up-regulation of PINK1 and down-regulation of Parkin after Nur77 agonist treatment. We propose that PINK1 may act as an upstream factor for Parkin to initiate the autophagic degradation of damaged mitiochondria and is essential for recruiting Parkin from the cytoplasm to depolarized mitochondria to some extent, responding to Nur77 52,53 . PINK1 on the mitochondria is regulated by voltage-dependent proteolysis to maintain low levels of PINK1 for rapidly and constitutively degradation under steady-state conditions, while the loss in mitochondrial membrane potential stabilizes PINK1 mitochondrial accumulation 51,53 . This may be the one of the potential mechanisms of neurotoxicity linked to increase in PINK1. However, there is little evidence showing that the downregulation of PINK1 is directly linked to PD progression. Taken together, this finding strongly suggests that Nur77 may directly regulate cellular autophagy by modulating mitochondrial functions. Mitochondrial potential collapse oppositely influences PINK1 and Parkin. These findings further confirmed that Nur77 may be used as the autophagic modulator by influencing mitochondrial target in the in vitro PD model.

Conclusions
In summary, we have demonstrated an increase in the cytosolic Nur77 and translocation from the nucleus together with mitochondrial impairment as indicated by the upregulation of cytosolic HSP60/Cyt C and increased JC-1 as well as downregulation of Tom20 and sharply loss of ΔΨm following 6-OHDA lesion, while Nur77 activation/knockdown aggravates/attenuates such pathophysiological changes respectively. In addition, Nur77 exacerbates PC12 cellular death by upregulating Beclin-1/LC3, PINK1, CHOP and ATF3 as well as downregulating Parkin in the in vitro model, indicating enhanced autophagic induction and ER stress. Moreover, Nur77 exacerbates Ca 2+ homeostasis, ROS and LDH in the in vitro model. We hypothesize that Nur77 may not only regulate mitochondrial dysfunction, but also modulate endoplasm reticulum stress in the development of neurodegenerative diseases like PD. Importantly, our proposed mechanisms for Nur77 in inducing autophagy and modulating mitochondrial dysfunctions and ER stress in PD pathogenesis increase our understanding of the roles of Nur77 in the disease (Fig. 6). This study provides a clue for the development of an alternative approach to the treatment of neurodegenerative diseases like PD by modulating Nur77-mediated mitochondrial impairment and ER stress as well as Nur77-inducted enhanced autophagy. A better understanding of the roles and relationships between Nur77, mitochondrial dysfunction, ER stress and autophagy may open new perspectives for the treatment of neurodegenerative disorders such as PD by targeting Nur77 signaling.

MTS Assay, LDH and Apoptotic Cell Measurement. Cell viability was measured using CellTiter96
Aqueous Assay (Promega, Madison, WI, Cat: G3582). Briefly, after 6-OHDA incubation for 24 hrs, 20 ul/well of CellTiter96 Aqueous was added and control wells without cells contained culture medium and CellTiter96 Aqueous. One solution was set to correct background 490 nm absorbance, and the absorbance at 490 nm was recorded using a microplate reader (BioTek Instruments Inc., Missouri, USA). Survival of control group was defined as 100%, and other groups were expressed as percentage of the control group. To further investigate the apoptosis, PC12 cells were seeded at a density of 1 × 10 5 cells/well in 6-well plates. After incubation with 6-OHDA for 24 hrs, PC12 cell apoptosis was evaluated by flow cytometry (Bender MedSystems, Burlingame, CA, USA).
The cytotoxicity was evaluated by measuring the LDH leakage into the culture medium using Cytoscan-LDH cytotoxicity assay kit (G-Biosciences Inc., St Louis, MO, USA). After 6-OHDA incubation for 24 hrs, 50 μL supernatant of each wells were transferred to another fresh 96-well plate and mixed with 50 μL reconstituted Substrate Mix, and then these mixtures were incubated at 37 °C and protected from light for 30 mins. Lastly, 50 μL Stop Solution was added to each well to terminate the reaction. The absorbance of the solution was recorded by Microplate Reader with the absorbance wavelength at 490 nm.

Measurement of intracellular ROS and calcium concentrations.
The level of intracellular ROS was measured using a ROS kit. The cells were collected and rinsed twice with PBS then they were incubated with 10 μM of the cell permeable 2′,7′-dichlorofluorescin diacetate (DCFH-DA) for 20 minutes at 37 °C. After that, the cells were rinsed with medium without FBS three times, and then their fluorescence was detected using a flow cytometer. The results were presented as an average of the intensity of the recorded fluorescence (NRFU) (U/cell).
The intracellular Ca 2+ levels in PC12 cells were measured using the fluorescence Ca 2+ indicator Fluo-3 AM. The cells were collected, rinsed twice with PBS, and stained with 5 μg/ml of Fura-3AM for 60 minutes. After staining, the cells were rinsed twice with PBS and incubated for 30 minutes at 37 °C before fluorescence-intensity detection by a flow cytometer. Calcium levels in cells were expressed as NRFU (U/cell).
Measurement of mitochondrial membrane potential (∆Ψm). ∆Ψm of PC12 cells was determined by flow cytometry according to the manufacturer's protocol using JC-1 mitochondrial membrane potential assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). Briefly, cells in 6 well plates were seeded at the density of 5 × 10 5 cells/ml in a CO2 incubator before being treated with 6-OHDA. After 24 hrs, cells from each well were harvested into a plastic tube and rinsed with PBS twice. 50 μL of the JC-1 Staining Solution per 500 μL of culture medium was added to each tube and then incubated in a CO 2 incubator at 37 °C for 25 minutes. Healthy cells with functional mitochondria contained red JC-1 J-aggregates and were detectable in the FL2 channel. Unhealthy cells with collapsed mitochondria contained mainly green JC-1 monomer and were detectable in the FL1 channel.