DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

J. Mol. Biol. (2012) 423, 232–248

doi:10.1016/j.jmb.2012.06.034 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy H. Gao 1, 2 †, W. Yang 1 †, Z. Qi 1 , L. Lu 1 , C. Duan 1 , C. Zhao 1 and H. Yang 1 ⁎ 1

Beijing Institute for Neuroscience, Capital Medical University, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing Center of Neural Regeneration and Repair, Beijing Key Laboratory of Brain Major Disorders—State Key Lab Incubation Base, Beijing Neuroscience Disciplines, Beijing 100069, China 2 Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, China Received 16 May 2011; received in revised form 28 April 2012; accepted 24 June 2012 Available online 14 August 2012 Edited by M. Ostankovitch Keywords: Parkinson's disease; DJ-1; autophagy; rotenone; ERK1/2 pathways

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐ kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD. © 2012 Published by Elsevier Ltd.

*Corresponding author. E-mail address: [email protected] † H.G. and W.Y. contributed equally to this work. Abbreviations used: PD, Parkinson's disease; DA, dopaminergic; SN, substantia nigra; 3-MA, 3-methyladenine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nicked labeling; AAV, adeno-associated virus; TH, tyrosine hydroxylase; TEM, transmission electron microscopy; mTOR, mammalian target of rapamycin; PI3K, phosphoinositol 3-kinase; PS, phosphatidylserine; PFA, paraformaldehyde; GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; PB, phosphate buffer. 0022-2836/$ - see front matter © 2012 Published by Elsevier Ltd.

Neuroprotective Effects of DJ‐1 on DA Neurons

Introduction Many genes have been implicated in familial early- and late-onset idiopathic Parkinson's disease (PD). 1–3 The novel oncogene, DJ-1, originally identified in Ras signaling, 4 has been linked to the PARK7 chromosomal locus associated with autosomal recessive early-onset parkinsonism. 5 Thirteen mutations of the DJ-1 gene, including deletion and point mutations, have been reported in PD patients. 6 The DJ-1 protein is multifunctional, with roles in oncogenesis, 7 male fertility, 8 the control of protein–RNA interactions, 9 and modulation of androgen receptor transcriptional activity. 10 The DJ-1 protein also has protease and chaperone activities. 11–13 Of all the functions ascribed to DJ-1, the most relevant to PD is a putative role in neuroprotection. Overexpression and mRNA knock-down experiments have suggested that DJ-1 promotes cell survival by enhancing Akt phosphorylation with concomitant inhibition of phosphatase and tensin homolog (PTEN) function. 14 There are reports that DJ-1 may affect protein clearance pathways, including the proteosomal pathway. 15,16 Silencing DJ-1 inhibits the cytoplasmic accumulation of autophagic vacuoles and results in enhanced apoptotic cell death after paraquat exposure in SHSY5Y cells. 17 The autophagic pathway is responsible for the degradation of long-lived proteins, protein aggregates, and cytoplasmic organelles. 18 Recent studies have revealed that deregulation of autophagy is evident in the brains of PD patients. 16 One of the first indications for an important role involving autophagy in PD was the demonstration that αsynuclein is degraded by macroautophagy and chaperone-mediated autophagy. 19–21 Aside from the degradation of α-synuclein, the autophagic pathway is involved in the turnover of mitochondria, and mitochondrial dysfunction is a welldocumented pathogenic mechanism in PD. In particular, deficits in mitochondrial complex I have been observed in PD patients. 22 Recently, parkin was found to facilitate the macroautophagy of impaired mitochondria, a process that is also known as mitophagy. 23 Knock-down of PINK1 expression induces mitochondrial fragmentation, followed by activation of autophagy/mitophagy. 24 These observations suggest that the autophagic pathway is essential for the turnover of dysfunctional mitochondria in PD. Failure to activate efficient autophagy may thus serve as a pathogenic mechanism of PD. Recently, many studies have reported that loss of DJ-1 perturbs mitochondrial function, including loss of mitochondrial polarization, fragmentation of mitochondria, and accumulation of markers of autophagy. 25–27 Recent evidence suggests that turnover of damaged mito-

233 chondria by autophagy might be central to the process of recessive parkinsonism; however, it is not fully understood whether or not the autophagic pathway is responsible for the neuroprotective effect of DJ-1 overexpression. This investigation mainly focused on the mechanism of neuroprotection of DJ-1 on dopaminergic (DA) neurons against rotenone-induced apoptosis. Rotenone, an inhibitor of mitochondrial complex I, is known to induce apoptosis. In addition, rotenone exerts neurotoxicity by inhibiting proteasome activity and oxidative stress. 28,29 In the current study, we investigated the neuroprotective effects of DJ-1 on rotenone-induced injury of DA neurons in Sprague– Dawley rats and cultured MN9D cells. We propose that the observed neuroprotective efficacy of DJ-1 was mediated, at least in part, by enhanced autophagy and suppression of mitochondria-dependent apoptosis.

Results DJ-1 attenuated rotenone-induced cell injury in MN9D cells Rotenone induced neurotoxicity in MN9D cells with characteristics of apoptotic cell death. Cultures treated with 100 nM rotenone demonstrated reduced viability that decreased progressively during treatment over 0–48 h (Fig. 1a). Cell viability was reduced 48.2% compared to control cultures after 24 h of rotenone treatment. Rotenone-treated cultures exhibited signs of progressive apoptotic cell death, as the number of annexin-positive cells increased from 0 to 24 h during rotenone treatment (Fig. 1b). Cells overexpressing DJ-1 were much more resistant to rotenone-induced cell death (Fig. 1a) and showed delayed apoptosis, as evidenced by significantly higher cell viability and fewer apoptotic cells at all measured time points (Fig. 1b). To further confirm that rotenone treatment induced apoptosis and that DJ-1 delayed apoptosis, we performed terminal deoxynucleotidyl transferase‐mediated dUTP nicked labeling (TUNEL) assay tests. Cultures treated with 100 nM rotenone had an increased number of apoptotic cells during treatment over 0–6 h (Fig. 1c). Cells overexpressing DJ-1 were much more resistant to rotenone-induced cell death and had delayed apoptosis, as evidenced by significantly fewer apoptotic cells at all measured time points (Fig. 1c). DJ-1 relieves rotenone-induced DA cell death in rat substantia nigra Rotenone caused death of DA neurons in rodents, accompanied by the appearance of eosinophilic

234 Neuroprotective Effects of DJ‐1 on DA Neurons

Fig. 1. DJ-1 attenuated rotenone-induced cell injury in MN9D cells. (a) Cell viability was measured after rotenone exposure using the MTT assay. Rotenone reduced cell viability in DA MN9D cells. Overexpression of WT-DJ-1 significantly enhanced cell viability at all measured time points. (b) Staining with fluorescein‐ isothiocyanate-conjugated annexin-V was used to determine the appearance of PS on the outer lipid bilayer, a cytochemical indicator of apoptosis. Concomitant with enhanced cell viability (a), DJ-1 overexpression significantly reduced annexin-V staining at all time points. The TUNEL assay showed the effect of DJ-1 overexpression on rotenone-induced MN9D cells apoptosis. (c) Representative images of TUNEL results showed MN9D cell apoptosis treated with 100 nM rotenone over 0–6 h in control and DJ-1 transient transfection groups. (d) Cultures were transiently transfected with GFP-DJ-1 plasmid. Images show DJ-1 transfection efficiency. The scale bar represents 50 μm. ⁎Pb0.05 compared to control, **Pb0.001 compared to control, #Pb0.05 compared to the rotenone-treated group. n=3.

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Fig. 2 (legend on next page)

235

236 cytoplasmic inclusions. 10–12 We established a rat model of DJ-1 overexpression in the substantia nigra (SN) using the adeno-associated virus (AAV) containing DJ-1 or green fluorescent protein (GFP)-DJ-1 (AAV-DJ-1 or AAV-GFP-DJ-1) and compared rotenone toxicity to other rat groups injected with virus containing LacZ or saline. Immunofluorescence showed that rats injected with the AAV-DJ-1 overexpressed DJ-1 protein in the ipsilateral SN, but not the contralateral SN (Fig. 2a). Meanwhile, Western blot showed that DJ-1 protein levels increased significantly in the DJ-1‐injected group compared with other groups. Selective and progressive loss of DA neurons is the central pathologic feature of PD. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis and is highly expressed in DA neurons. To determine whether or not DJ-1 expression was increased within THpositive neurons, we found that DJ-1 was expressed in neurons and other cells in the SN of rat brains using AAV-GFP-DJ-1. Exogenous DJ-1 (green) was overexpressed in nearly 60% of the DA neurons in rat SN according to double-labeled immunofluorescent staining with TH (red; Fig. 2b). Several recent studies have reported that DJ-1 overexpression in astrocytes augments the capacity to protect neurons against rotenone and that DJ-1 knockdown impairs astrocyte-mediated neuroprotection against rotenone, 30,31 suggesting that DJ-1 may be involved in the neuroprotective effect of astrocytes on neurons. We quantified the number of THpositive neurons by immunofluorescence in the SN of control rats and rats overexpressing GFP-DJ-1. One week after rotenone injection, the number of TH-positive neurons was reduced significantly compared to the sham group, while overexpressing exogenous GFP-DJ-1 attenuated rotenone-induced DA neuron death (Fig. 2b). At the same time, we also quantified the number of TH-positive neurons by immunochemistry in the SN of control rats and rats overexpressing DJ-1. One week after rotenone injection, the number of TH-positive neurons was

Neuroprotective Effects of DJ‐1 on DA Neurons

reduced to 31.96 ± 8.25% of the contralateral side in the LacZ group and 33.77 ± 6.24% in the NS group, while the number of TH + neurons was reduced to 68.29 ± 12.08% of the contralateral side in rats overexpressing DJ-1 (Fig. 2c). Four weeks postinjection, the number of TH-positive neurons was reduced to 22.75 ± 6.39% and 24.4 ±5.23% in the LacZ and NS groups, respectively, but to 56.36 ± 10.1% in the DJ-1 group, indicating that DJ-1 promoted the survival of DA neurons against this form of metabolic neurotoxicity. Similarly, Western blots from brain lysates showed that TH immunoreactivity in the rats overexpressing DJ-1 was higher than the other treatment groups (Fig. 2d). Overexpression of DJ-1 attenuated rotenone-induced behavioral injuries in rats DJ-1 showed a DA neuroprotective effect in rat SN injected with rotenone. We further investigated whether or not DJ-1 relieved symptoms associated with PD. Specifically, the total distance traveled was reduced in all three rotenone-treated groups (DJ-1, LacZ, and NS) at all tested points from 1 to 12 weeks after rotenone treatment. As speculated, the distance traveled by the rats overexpressing DJ-1 was significantly greater than the LacZ- and NS-treated groups (Pb0.05, n= 6; Fig. 3a). Our results further prove that DJ-1 overexpression can significantly attenuate rotenone-induced depressive-like behaviors in rats (Fig. 3b). However, there were no changes in spontaneous rotational behavior following rotenone (30 μg/kg) injection in the different rat treatment groups (Fig. 3c). In fact, very few behavioral studies are available in the rotenone model. Only one recent report exists regarding the rotational asymmetry following rotenone-induced unilateral damage. 32 One study has reported that rotenone caused 30% and 62% DA depletion following striatal and nigral lesions, respectively, 32 which is not enough to cause spontaneous rotation in the rat and is in agreement with our experiments.

Fig. 2. Overexpression of DJ-1 rescues nigral DA neurons from rotenone-induced cell death in rats. (a) Confocal immunofluorescence microscopy reveals enhanced DJ-1 protein expression following unilateral AAV-DJ-1 injection into the left SN of rats. DJ-1 immunofluorescence is green. Note the lower expression in the non-injected (contralateral) SN. Below, Western blot shows DJ-1 protein expression from lysates prepared from sham-operated, DJ-1-injected, LacZinjected, and normal saline (NS)-injected rat groups. (b) Confocal immunofluorescence microscopy reveals exogenous DJ-1 expression in the TH-positive neurons in sham-operated, GFP-DJ-1-injected, DJ-1+NS, DJ-1+rotenone, and rotenoneinjected rat groups. TH immunofluorescence is red. Exogenous DJ-1 carrying GFP is green. Below, assessment of the total number of TH-positive neurons and double-labeled neurons (representing exogenous DJ-1 within TH positive neurons) in the SN of ipsilateral hemispheres was made according to the optical fractionator principle, using the Olympus Denmark A/S (Albertslund, Denmark) CAST-Grid system. Percentage signal was then calculated as the ratio of the signal in the other four groups versus the sham group and then multiplied by 100 and presented in the graph. (c) Immunochemical staining demonstrated the loss of TH-positive neurons in the SN at 1 and 4 weeks after rotenone injection. The survival of TH-positive neurons in rotenone-treated rats was significantly enhanced by DJ-1 overexpression. (d) Western blots demonstrate that TH protein expression was markedly reduced in the LacZ and NS groups after rotenone treatment and partially rescued by DJ-1 overexpression. ⁎Pb0.05 compared to the sham group, #Pb0.05 compared to the NS group or rotenone treatment group. n=6.

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Fig. 3. Overexpression of DJ-1 attenuated rotenone-induced behavior injuries in rats. (a) Changes in motor behavior following rotenone injection in the different rat treatment groups were measured by the distance traveled (centimeters) over 30 min in the open field. Distance traveled was markedly suppressed by rotenone treatment in the LacZ and NS groups but partially rescued in DJ-1‐overexpressing rats. Mobility was enhanced in rats overexpressing DJ-1 over 12 weeks posttreatment. (b) Depressive-like behaviors elicited by the neurotoxin rotenone injection in the different rat treatment groups are evidenced by the forced swimming test. Swimming times were markedly suppressed by rotenone treatment in the LacZ and NS groups after rotenone treatment, but partially rescued in DJ-1‐overexpressing rats. The values are expressed as the mean±standard error of the mean (n=8–12/group). (c) No changes in spontaneous rotational behavior following rotenone injection in the different rat treatment groups were measured by the number of rotations, which were recorded every 10 min until the rotations became non-apparent and scored for the total number of rotations from the beginning until cessation of rotations in each group. Positive values in the graph denote ipsilateral circling and negative values represent contralateral circling. The data are expressed as rotations/10 min and are depicted as the mean±standard error of the mean. ⁎Pb0.05 compared to the control group, #Pb0.05 compared to the LacZ- or NS-treated groups. ANOVA followed by the Newman–Keuls test.

In addition, rotenone was able to produce depressive-like behaviors assessed through the forced swimming test. 33 These results suggested that overexpression of DJ-1 in the SN of rats caused a long-term reduction in PD symptoms. DJ-1 increases autophagy during rotenone-induced DA cell death The expression of DJ-1 is critical for the activity of Akt and mammalian target of rapamycin (mTOR), 29 two known regulators of autophagy. Transfection of small interfering RNA for DJ-1 inhibits the cytoplasmic accumulation of autophagic vacuoles. 16 We examined whether or not autophagy was altered in neuronal cell cultures overexpressing DJ-1 and found that overexpression

of DJ-1 decreased phospho-mTOR, a negative regulator of autophagy; up-regulated beclin 1, a protein required for the formation of autophagasomes; and increased the ratio of the autophagic marker, LC3II, relative to the inactive precursor, LC3I (Fig. 4A). To clarify whether or not cell vacuolization induced by DJ-1 is involved in autophagy, we performed transmission electron microscopy (TEM) to examine the fine ultrastructure of SN neurons following rotenone treatment (1.0 mg/mL). In the SN of untreated rats, the cytoplasm, organelles, and nuclei of DA neurons exhibited normal morphology (Fig. 4B). The most prominent morphologic change in SN neurons after rotenone injection was the formation of abundant autophagic vacuoles enveloping cytoplasm, mitochondria, and endoplasmic reticulum. Double membranes, giant autophagosomes filled

238 with degraded organelles, and autolysosomes were also observed, suggesting that DJ-1 might exert neuroprotection by enhancing autophagy. DJ-1 cleared aggregated p62, which is often associated with PD As a neuroprotectant for DA neurons, DJ-1 also modulates protein clearance pathways. 11,14 Given the focus on PD, does DJ-1 clear large protein

Neuroprotective Effects of DJ‐1 on DA Neurons

aggregates that are often associated with PD? p62, one of the selective substrates for autophagy, plays a key role in the formation of cytoplasmic proteinaceous inclusion, a hallmark of conformational diseases, such as Alzheimer's disease, PD, and various chronic liver disorders. 34 Importantly, p62 has been identified as a component of inclusion bodies observed in PD. 34 Western blot analysis showed that p62 protein levels decreased significantly in the 6‐h rotenone treatment group

Fig. 4. DJ-1 increases autophagy in rats. (A) Western blots measuring phosphorylated mTOR (a), beclin-1 (b), and the ratio of LC3II-to-LC3I (c) in lysates from the SN of DJ-1, LacZ, and NS rat groups. Overexpression of DJ-1 decreased mTOR phosphorylation, increased beclin-1 expression, and increased the LC3II-to-LC3I ratio, all molecular indices of enhanced autophagy. *Pb0.05 compared to the sham group, **Pb0.001 compared to the sham group, #Pb0.05 compared to the LacZ- or NS-treated groups, ##Pb0.001 compared to the LacZ- or NS-treated groups. n=6. (B) Transmission electron micrographs showing the normal morphology of cytoplasm, organelles, and nuclei of SN neurons in rat brains not treated with rotenone (a and d), and the abnormal ultrastructure of cytoplasm and organelles in SN neurons in rat brains treated with rotenone (b and e). Characteristic ultrastructure of autophagy, including double membranes and autophagic vacuoles (c and f), of SN neurons overexpressing DJ-1 after rotenone treatment (a–c, ×4000; d–f, ×8000). Thick arrows represent autophagosomes.

Neuroprotective Effects of DJ‐1 on DA Neurons Fig. 5. DJ-1 cleared aggregated p62, which are often associated with PD. (a) Western blots show p62 protein levels in the DJ-1 overexpression of MN9D cells after 0, 3, 6, and 12 h of rotenone exposure. (b) Quantitative analysis of p62 protein levels in DJ-1 overexpression of MN9D cells after 0, 3, 6, and 12 h of rotenone exposure. (c) Cells were pretreated with 3-MA (10 mM) for 6 h before transfection. After transfection with GFP-DJ-1 for 48 h and 100 nM rotenone exposure for 0 or 12 h, the cells were fixed and stained with p62 primary antibody, followed by mouse Alexa 594-conjugated secondary antibody. The nucleus was counterstained by DAPI. (d) The staining intensity of p62 was measured and the data are expressed as the mean±SD. (e) The size of p62‐positive puncta was measured and the data are expressed as the mean± SD. Images from 100× objectives were captured with equal exposure times, and the particle size was determined in threshold-matched images using Image J software. DAPI staining was also quantified and used to determine the total cell number/field. Each replicate was assessed with 3–10 fields of view, and three replicates were performed per condition. The scale bar represents 25 μm. ⁎Pb0.05 compared to control, #Pb0.05 compared to the rotenone treatment groups only (MN9D cells after 3, 6, and 12 h of rotenone exposure). ^Pb0.05 compared to the 3‐h rotenone treatment cell group.

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Fig. 6. DJ-1 increases autophagy through the ERK1/2 pathway. ERK plays an important role in modulating autophagy through the PI3K/mTOR pathway. (a) Western blots were used to measure p-ERK levels in the SN of rats injected with DJ-1, LacZ, or NS. Quantitative analysis of p-ERK levels in the SN of rats revealed that DJ-1 rescued the rotenone-induced reduction in p-ERK expression (n=3). (b) Comparison of cell viability with and without rotenone in non-transfected MN9D cells, DJ-1-transfected cultures (DJ-1), DJ-1 cultures pretreated with the ERK inhibitor, U0126 (DJ-1+U0126), and DJ-1 overexpressers pretreated with the autophagy/PIK3 inhibitor, 3-MA (DJ-1+3-MA; n=3) revealed that blockade of ERK or PI3K/autophagy reversed DJ-1-mediated neuroprotection against rotenone. (c) Similarly, 3-MA and U0126 blocked the DJ-1-mediated reduction in apoptosis. (d) Western blots and densitometry were used to measure the ratio of LC3II to LC3I in NM9D cells with rotenone (Ro), starved, DJ-1+rotenone, DJ-1+rotenone+U0126, or DJ-1+rotenone+ 3‐MA. Rotenone induced greater LC3I-to-LC3II conversion (autophagy) in DJ-1‐overexpressing cells, while autophagy was inhibited by the ERK inhibitor, U0126 (n=3). ⁎Pb0.05 compared to the sham or control group, **Pb0.001 compared to control group. n=3.

compared with the 3‐h rotenone group. After 12 h of exposure to rotenone, the p62 protein levels were up-regulated slightly. Interestingly, DJ-1 overexpression promotes p62 protein clearance, which suggests that DJ-1 might be involved in p62 clearance and is associated with PD (Fig. 5a and b). To further demonstrate that DJ-1 clears aggregated p62, MN9D cells were treated with 100 nM rotenone for 12 h and compared to control and GFPDJ-1 transient transfection groups. To examine the effects of autophagy on the clearance of aggregated

p62 by DJ-1, we added 3‐methyladenine (3-MA, 10 mM; Sigma-Aldrich) to DJ-1 overexpression cultures for 6 h before rotenone treatment. Confocal images showed that overexpression of DJ-1 decreased the number of p62-positive puncta in MN9D cells relative to control cultures after 12 h of rotenone treatment (Fig. 5c). Furthermore, the decreased p62 staining intensity and size of p62-positive puncta (79.97 ± 9.75 and 0.43± 0.148, respectively) in rotenone-treated MN9D cells transiently transfected with GFP-DJ-1 were increased to 135± 20.76 and

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Fig. 7. DJ-1 increases autophagic vacuoles aggregation in the presence of rotenone in MN9D cells. Transient transfection of GFP-LC3 resulted in formation of LC3-positive puncta. Overexpression of DJ-1 increased the number of puncta after rotenone exposure. (a) Cells were pretreated with U0126 (5 μM) or 3-MA (10 mM) for 6 h before transfection. After transfection with DJ-1 for 48 h and 100 nM rotenone exposure for 12 h, the cells were fixed and stained with DJ-1 primary antibody followed by mouse Alexa 594-conjugated secondary antibody. The nucleus was counterstained by DAPI. (b) The number of autophagic cells was counted and the data are expressed as the mean±SD. n=20. The scale bar represents 10 μm. **P b0.001 compared to the control group.

1.7900±0.2300, respectively, in cultures treated with 100 nM rotenone for 12 h (Fig. 5d and e) and also significantly increased p62-positive puncta size (1.57± 0.2400) pretreated with 3-MA (Fig. 5c–e), suggesting that enhancing autophagy levels may be involved in the clearance of aggregated p62 by DJ-1. Suppression of autophagy blocked neuroprotection conferred by DJ-1 overexpression The MEK/ERK signaling pathway is known to regulate autophagy through beclin-1. Depletion of ERK partially inhibits autophagy, while specific inhibition of MEK completely inhibits autophagy. 35

We previously reported that the ERK1/2 signaling pathway is involved in DJ-1-induced neuroprotection against oxidative stress. 36 Similarly, ERK phosphorylation protein levels in the SN of rats measured biochemically by Western blotting (Fig. 6a) increased significantly after rotenone treatment in the DJ-1-treated group compared to LacZ- or NStreated groups. To further test whether or not autophagy was up-regulated through the ERK1/2 and phosphoinositol 3‐kinase (PI3K)/mTOR pathways, we treated MN9D cells with the MEK1/2 inhibitor, U0126 (5 μM), or the PI3K inhibitor, 3-MA (10 mM), prior to 12 h of exposure to rotenone. Cell viability measurements revealed that the neuroprotection conferred by DJ-1 was reversed after

242 pretreatment with U0126 or 3-MA (Fig. 6b). 3-MA and U0126 blocked the DJ-1-mediated reduction in apoptosis demonstrated by flow cytometry (Fig. 6c), which also showed that cell death in neurons transiently transfected with DJ-1 was not significantly different from control cultures after pretreatment with U0126 or 3-MA (not shown). Furthermore, Western blotting indicated that U0126 or 3-MA inhibited the autophagy induced by DJ-1. DJ-1‐overexpressing MN9D cells treated with rotenone for 12 h showed a greatly increased LC3II-toLC3 ratio that was reduced to baseline in cultures pretreated with U0126 or 3-MA (Fig. 6d), which suggested that ERK signaling pathway might be involved in DJ-1-induced neuroprotection against rotenone through autophagy. MN9D cells that had been transfected with DJ-1 or the LacZ control vector were co-transfected with GFP-LC3. Activated LC3 protein is a marker of autophagy and, in the activated state, will form punctate structures within the cytoplasm that correspond to autophagic vesicles. Confocal images showed that overexpression of DJ-1 increased the number of LC3-positive puncta in MN9D cells relative to control cultures (Fig. 7). Furthermore, the increased number of LC3-positive puncta in rotenone-treated MN9D cells transiently transfected with DJ-1 was reduced in cultures pretreated with U-0126 or 3-MA (Fig. 7a and b). Enhanced autophagy relieved cell injury after rotenone exposure Macroautophagy, a major degradation pathway of the lysosomal system, plays a unique role in removing cellular organelles and protein aggregates that are too large to be degraded by the ubiquitin– proteasome system. 37 Rotenone treatment also induced several characteristic features of autophagy, including the accumulation of double‐membrane autophagic vacuoles in the cytoplasm, accumulation of the autophagosome marker, LC3II, and negative regulation of protein degradation mediated by mTOR kinase signaling. 16 Because rotenone can also enhance autophagy levels, we need to confirm that enhanced autophagic levels, which exhibited neuroprotective effects, were induced by DJ-1 rather than rotenone. We found that autophagy induced by rotenone alone was time dependent. The ratio of LC3II to LC3I and beclin-1 expression after 6 h of rotenone exposure reached a peak, but both markers returned to baseline by 12 h (Fig. 8a). DJ-1‐overexpressing MN9D cells after 12 h of rotenone treatment showed a greatly increased LC3II-to-LC3I ratio compared with MN9D cells after 12 h of rotenone treatment (Fig. 8b), suggesting that autophagy induced by DJ-1, but not rotenone, showed a neuroprotective effect (Figs. 8a and b and 6b–d). To further verify whether or not changes

Neuroprotective Effects of DJ‐1 on DA Neurons

in the level of DJ-1 expression alter the transient response to rotenone in MN9D cells, Western blot analysis showed a significantly increased LC3II-toLC3I ratio in the DJ-1 overexpression group compared with the control group after 12 h of rotenone treatment; however, the DJ-1 knock-down group showed a minimal LC3II band (Fig. 8b), which further confirmed that after 12 h of rotenone treatment, autophagy was induced by DJ-1, but not rotenone. At the same time, we also found that slightly increased DJ-1 protein induced by 100 nM rotenone was time dependent (data not shown). None of these results showed the effect of rotenoneinduced autophagy levels, further confirming the neuroprotective effect induced by DJ-1-induced autophagy. Autophagy as a cell's defense mechanism to an external stimulus has certain compensatory ability, which occurs early when cells are starved or injured. Thus, using different methods to induce autophagy, an increase in the autophagy level or an extension of the autophagy phase should protect cells from external stimuli. Rapamycin inhibits the activity of mTOR, which normally serves as an agonist of autophagy. We found that pretreatment of cells with rapamycin also salvaged cells from rotenone toxicity, as demonstrated by the increase in cell viability (Fig. 8c), suggesting that autophagy plays a neuroprotective role in rotenone-treated MN9D cells, further confirming our results. Briefly, DJ-1 can increase autophagy levels via ERK pathway activation, which protects MN9D cells against rotenone.

Discussion The majority of PD cases are idiopathic with no clear etiology. A large body of evidence indicates that mitochondrial dysfunction, environmental toxins, oxidative stress, and abnormal protein accumulation can all contribute to disease pathogenesis. The loss of DA neurons in the SN may be partly due to accumulation of aggregated or misfolded proteins or mitochondrial dysfunction. 38 Preventing accumulation of misfolded proteins or degradation of dysfunctional mitochondria might prevent the occurrence of apoptosis and provide potential therapeutic benefit for PD. Mutations in DJ-1 have been implicated in the pathogenesis of PD. Indeed, loss of function is thought to trigger the onset of familial forms of the disease. 9 In the current study, we found that DJ-1 was neuroprotective against rotenone in DA cells in vitro and in vivo. Overexpression of DJ-1 rescued MN9D cells exposed to rotenone, while the number of TH-positive neurons in the SN of rats overexpressing DJ-1 was significantly higher than those of control rats after rotenone injection, indicating that DJ-1 protected nigral DA neurons from rotenone-

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Fig. 8. Enhanced autophagy relieved cell injuries after rotenone exposure. (a) Autophagy induced by rotenone was closely correlated with time. (b) Autophagic levels induced by 100 nM rotenone for 12 h was detected by Western blot in control, DJ-1 interference, and DJ-1 overexpression groups. (c) The MN9D cells were pretreated with 10 nM rapamycin for 12 h followed by addition of 100 nM rotenone for another 0, 6, 12, or 24 h. Pretreatment with rapamycin partly preserved cell viability. ⁎Pb0.05 compared to control, **Pb0.001 compared to control, #Pb0.05 compared to rotenone treatment group. n=3.

induced cell death. Furthermore, rats overexpressing DJ-1 significantly attenuated rotenone-induced behavioral signs of parkinsonism based on the open field and swimming tests. Our results are consistent with previous reports demonstrating protection by recombinant DJ-1 against 6-hydroxydopamine-

induced nigrostriatal neurodegeneration and druginduced rotational asymmetry in hemi-parkinsonian rats. 37 Previous studies have shown that DJ-1 counteracts oxidative stress by acting as a direct free radical scavenger, a redox-sensitive molecular chaperone, or

244 through several interacting signaling pathways. 39–41 Many neurotoxins, including rotenone, paraquat, and MPP+, initiate mitochondrial dysfunction by inhibiting complex I. The present report suggests an alternative mechanism for DJ-1-mediated antioxidant protection; specifically, overexpression of DJ-1 enhanced the autophagic clearance of damaged mitochondria, thus preventing the release of pro-apoptotic molecules, such as cyctochrome c, into the cytosol. A significant body of work indicates that DJ-1 also modulates protein clearance pathways. Knock‐down or deletion of DJ-1 increases susceptibility to proteasome inhibition in cultured cells, 14 while both decreased proteasome activity and increased ubiquitinated protein levels have been reported in DJ-1deficient mice. 15 Recently, one paper have demonstrated that wild-type levels of DJ-1 expression are required for proper turnover of p62, a biomarker of autophagy that is selectively incorporated into autophagosomes and efficiently degraded by autophagy induced by hypoxia. 41 Loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that, under physiologic conditions, would be compensated for by lysosomal clearance. In contrast to DJ-1, mTOR is a major negative regulator of autophagy after phosphorylation by PIK3. The autophagic response involves the cleavage of cytosolic LC3I into a lipidated LC3II, which is then recruited to the autophagosomal membrane. 42,43 The current study demonstrated that overexpression of DJ-1 inhibited the activity of mTOR, promoted the conversion of LC3I to LC3II in the SN and MN9D cells, increased the number of GFPLC3 puncta in MN9D cells following rotenone exposure, and cleared aggregated p62 associated with PD. These results indicate that DJ-1 can upregulate autophagy during oxidative stress and is consistent with previous results showing that silencing DJ-1 inhibits the cytoplasmic accumulation of autophagic vacuoles in SH-SY5Y cells. 16 Interestingly, we found that DJ-1 can up-regulate autophagy by increasing beclin-1 protein levels through suppression of mTOR in DA cells. Beclin-1 expression coincides with autophagic activity and tumor suppression, 44–46 while defective beclin-1 expression or depletion of beclin-1 inhibits autophagy. 45,47,48 Conversely, beclin-1 overexpression triggers autophagy. 45,49 Western blots indicated that DJ-1 overexpression is associated with increased expression of beclin-1. Conversely, autophagy returned to baseline following pretreatment with 3MA, a blocker of PI3K, which normally suppresses mTOR activity. The MEK/ERK signaling cascade regulates autophagy by mediating beclin-1 protein expression through the MEK/ERK–mTOR pathway. 35 We previously reported that DJ-1 may be involved in the increased phosphorylation of ERK1/2 and MEK1/2. 36 The MEK1/2-specific inhibitor, U0126,

Neuroprotective Effects of DJ‐1 on DA Neurons

was used to further explore the role of ERK1/2 in DJ-1-induced autophagy. Indeed, autophagy remained at basal levels in DJ-1‐overexpressing MN9D cells pretreated with U0126 during rotenone treatment. In addition, pretreatment with U0126 or 3-MA reduced the number of autophagic puncta in MN9D cells overexpressing DJ-1. These results indicate that DJ-1 increased the appearance of autophagic puncta through ERK1/2- and mTOR/ PI3K-dependent pathways after rotenone exposure. It was also reported that DJ-1 regulates autophagy through the JNK pathway in cancer cells, but we did not detect activation of JNK pathway in our experimental models (data not shown). As a mitochondrial complex I inhibitor, rotenone most likely damaged DA cells through mitochondrial dysfunction and apoptotic cell death. Interestingly, we also found that rotenone induced the molecular events of autophagy early in treatment, but autophagy appeared to down-regulate with time. The level of autophagy reached a peak 6 h after rotenone exposure and returned to baseline within 12 h. Thus, the reduction in autophagic markers began to decrease concomitant with the onset of cell death, suggesting that as oxidative stress continued, auto-activated autophagy was insufficient to clear damaged mitochondria, resulting in the initiation of apoptosis. That autophagy could be induced in rotenone-treated neuronal cells is also supported by the previous report showing autophagy under enhanced oxidative stress. 50 Rapamycin, an inhibitor of the mTOR pathway, extended both the median and maximum life spans of male and female mice by activating autophagy. 51 In the current study, pretreatment with rapamycin protected DA cells against rotenone, possibly via the inhibition of mTOR and enhanced autophagy. Neurons are one of the few cell types used in the initial identification and characterization of autophagy. Autophagy can be viewed as a protective response that contributes to repair and remodeling after damage to cellular components and that is necessary to sustain normal neuronal function and survival. 52 Our results suggest that DJ-1 is neuroprotective by activating the ERK1/2 pathway and suppressing mTOR in DA cells, leading to enhanced autophagy, more efficient clearance of damaged mitochondria, and reduced apoptosis. These findings provide new insight into the possible pathogenic mechanisms of PD and define molecular targets for the development of therapeutic strategies.

Materials and Methods Animals and cell cultures Male Sprague–Dawley rats (190–210 g) were housed under a 12‐h light/12‐h dark cycle at 20–23 °C with free

Neuroprotective Effects of DJ‐1 on DA Neurons access to food and water. The rats were provided by the Department of Zoology of Capital Medical University. Rats were anesthetized by peritoneal injection with 6 ml/ kg of 6% hydral. An AAV vector encoding DJ-1 or GFPDJ-1 was injected into the SN at the following stereotactic coordinates: −5.2 mm from the bregma, 2.0 mm to the left of the midline, and 7.5 mm below the subdural matter. A 5‐μl AAV-DJ-1 or AAV-GFP-DJ-1 sample (2.5×10 7 TU) was injected into experimental rats, while two control groups received equal volumes of AAV-LacZ or physiologic saline. Four weeks after injection, rats were injected with 30 μg/kg of rotenone at the same site. The MN9D cell line was grown in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% newborn calf serum and transfected with the plasmids (vide infra) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Protein expression was verified by Western blotting. Plasmid constructs The human WT-DJ-1 cDNA was obtained by reverse transcriptase-polymerase chain reaction from human brain RNA with primers (5′-AAGCTTGGGTGCAGGCTTGTAAAC-3′ and 5′-TCTAGAAGTGATCGTCGCAGTTCG3′) designed according to the DJ-1 sequence published in GenBank (NM_007262). The cDNA was directionally cloned into the pcDNA3.1 plasmid (Invitrogen). The orientation of the WT cDNA was verified by DNA sequencing.

Assay of behavior After rotenone injection, motor behavior was assessed at 0, 1, 2, 4, 8, and 12 weeks. Rats were placed in a 40 cm×40 cm open-topped box and free ambulation was recorded for 30 min using a digital video camera (SONY). EthoVision software was used to measure the total distance and mean velocity at 6 frames/s. All trials were performed at 10 a.m. For the spontaneous circling behavior test, different groups of animals were observed for spontaneous behavioral abnormalities during the first 3 days after rotenone injection. One of the striking features following recovery from anesthesia was spontaneous circling behavior (360° in a short axis) in the nigrally lesioned group. The animals were kept in a transparent cage (45 cm diameter and 40 cm height) 24 h following rotenone injection and the spontaneous rotations in the cage were counted by trained/experienced individuals for 30 min. For the modified forced swimming test, the procedure used was a modification of the method proposed by previous studies. 53,54 The test was conducted in two sessions. In the training session, the rats were placed in a tank (20 cm×20 cm×40 cm) containing water at a temperature of 24±1 °C at a depth of 15 cm for 15 min. Twenty-four hours after the training session, the rats were subjected to the forced swimming test for 5 min, which was videotaped for subsequent quantification of the following parameters: immobility (defined as the lack of motion of the whole body, consisting only of the small movements necessary to keep the animal's head above the water), climbing (vigorous movements with the forepaws in and out of the water, usually directed against the wall of

245 the tank), and swimming (coded when large forepaw movements displaced the body around the cylinder more than necessary to merely keep the head above the water). The water was changed after each animal to avoid the influence of substance and temperature. This test was performed 23 days after the rotenone injection to allow complete recovery from the surgical wound. Assessment of cell viability To evaluate the effect of DJ-1 on cell viability, 1×10 4 MN9D cells were transfected with DJ-1 for 24 h and plated in 96-well plates. The day after, a freshly prepared 100‐nM rotenone solution or vehicle was added with or without the MEK1/2 inhibitor, U0126 (5 μM), or the autophagy inhibitor, 3-MA (10 mM). The cells were then incubated for an additional 48 h. Following treatment, cell viability was estimated by the MTT assay that determines relative viable cell count by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to colored formazin. 55 Annexin V&PI detection and TUNEL staining for the apoptosis assay Apoptosis was determined by the Annexin V&PI Apoptosis Detection Kit (Roche Diagnostics). This assay is based on the observation that soon after the initiation of apoptosis, membrane phosphatidylserine (PS) translocates from the inner lipid bilayer to the outer lipid bilayer. When on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of the high-affinity PS binding protein, annexin V. Part of the cultures treated with rotenone (100 nM) were stained for TUNEL during treatment over 0–12 h. The TUNEL staining kit (In Situ Cell Death Detection Kit, POD; Roche) was used to assess cell apoptosis. For apoptosis assessment, MN9D cells were seeded onto poly-L-lysine-coated glass coverslips, grown to 60% confluence, and fixed in 4% paraformaldehyde (PFA) for 20 min. The coverslips were placed in equilibration buffer and incubated with nucleotide mix and rTdT enzyme at 37 °C for 1 h. The reaction was stopped using 2× saline– sodium citrate buffer. Then, the fluorescence signal was converted to a diaminobenzidine signal by the addition of Converter-POD at 37 °C for 1 h. The images were visualized by light microscopy (Leica DM4000B; Wetzlar, Germany). Immunohistochemisty and confocal microscopy Brain tissue sections were prepared as previously described. 56 The brains were cut into 20‐μm-thick sections on a cryostat for immunohistochemisty. Sections were blocked with 5% normal goat serum and then incubated sequentially with anti-TH monoclonal antibody (1:10,000), biotinylated goat anti-mouse IgG (1:500), and horseradish‐ peroxidase-conjugated streptavidin. The bound peroxidase was subsequently revealed using a solution containing diaminobenzidine, hydrogen peroxide, and nickel ammonium sulfate. For immunofluorescent double labeling with TH and DJ-1, sections were incubated with

246 primary mouse monoclonal anti-TH antibody (1:10,000; Sigma-Aldrich) overnight at 4 °C, followed by mouse Alexa 594-conjugated secondary antibody at 1:400 for 30 min at 37 °C. The images were captured by a fluorescent microscope (Leica). Alternatively, MN9D cells were seeded onto poly-Llysine-coated glass coverslips, grown to 60% confluence, and fixed in 4% PFA for 20 min. Coverslips were pretreated with 0.1% Triton X-100 in Tris‐buffered saline for 20 min and then incubated overnight at 4 °C with antibodies against DJ-1 (Sigma-Aldrich) or p62 (Abcam). The signal was detected with the Tyramide Signal Amplification-Direct (Red) system (NEN Life Sciences). Control cultures were transfected with empty vector or GFP vector and immunolabeled in the absence of primary antibodies. Coverslips were mounted with Prolong Gold anti-fading reagent with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Invitrogen). Cells were analyzed with a digital epifluorescence microscope (Olympus BX51) to estimate the percentage of DAPI-stained cells that displayed GFP, DJ-1, or p62 immunoreactivity. On average, 50 cells were imaged per treatment condition, and the individual channel images were merged and analyzed with the ImageJ program. Analysis of autophagy The MN9D cells were grown as described above and then plated onto poly-L-lysine-coated glass coverslips at a density of 5×10 cells per coverslip. Five hours after plating, cells were transfected with DJ-1 and incubated for 48 h. All coverslips were also co-transfected with LC3-GFP (provided by Dr. Spencer Gibson). Most control experiments were performed with cells transfected with empty vector. To examine the effects of ERK on the autophagy pathway, we added U0126 (5 μM) or 3‐MA (10 mM; Sigma-Aldrich) to cultures for 6 h before rotenone treatment. Cells were incubated in rotenone for 12 h, washed twice with serum-free Dulbecco's modified Eagle's medium, and then fed complete or serum-free media for 12 h before fixation with 4% PFA. Briefly, coverslips were treated with Prolong Gold anti-fading reagent with DAPI (Invitrogen) and imaged by laser-scanning confocal microscopy (MRC1024; Bio-Rad) to determine the number of GFP-positive granular structures consistent with autophagosomes. In each case, an average of 50 cells was analyzed using the ImageQuant software. Analysis was performed in duplicate. Samples were coded so that the investigator was blind to treatment history. Additional studies were performed using MN9D cells infected with DJ-1 and analyzed by Western blot for LC3 protein. Analysis was performed with the VersaDoc gel imaging system (Bio-Rad) to determine the ratio of LC3II to LC3I, which are differentiated by a 2‐kDa gel shift. Gel loading values were normalized with an antibody against actin. Preparation of tissue sections and TEM Under anesthesia, rats (n=4) were perfused transcardially with 4% PFA and 2.5% glutaraldehyde in phosphate buffer (PB). The brains were removed and immediately immersed in 2.5% glutaraldehyde in 0.1 M PB (pH 7.4) on ice and fixed for 6 h with shaking. After sufficient washing

Neuroprotective Effects of DJ‐1 on DA Neurons in 0.1 M PB, mesocerebrums were cut into 1‐cm 3 tissue blocks with the SN at the center. Blocks were postfixed in 1% osmium tetroxide for 2 h at 4 °C. The blocks were rinsed several times in distilled water, dehydrated in a graded series of ethanol (20–100%) followed by propylene oxide, infiltrated with Epon 812, and finally polymerized in pure Epon 812 for 48 h at 65 C. Ultrathin sections were cut on an ultramicrotome using a diamond knife, collected on copper grids, and stained with 4% uranyl acetate and Reynold's lead citrate. A minimum of 10 sections from each SN were imaged with a JEM-1230 TEM. SDS‐PAGE and Western blot analyses The SN from the left hemisphere of six animals at 4 weeks posttreatment was isolated and lysed for total cell extracts. In addition, cultured MN9D cells were transfected with plasmid for 48 h; treated with rotenone for 3, 6, or 12 h; and lysed in TNE buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 1 mM ethylenediaminetetraacetic acid; all from Sigma-Aldrich] containing 1% Nonidet P-40 (Calbiochem) with protease and phosphatase inhibitor cocktails (Roche). Total cell extracts were centrifuged at 12,000g for 30 min, and the supernatant protein concentration was determined with a bicinchoninic protein assay kit (Pierce Biotechnology). For Western blot analysis, 20 μg of lysate per lane was loaded onto 4–12% 2‐[bis(2‐ hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol SDS-PAGE gels, separated electrophoretically, and blotted onto polyvinylidene fluoride membranes. Different blots were incubated with antibodies against DJ-1 (SigmaAldrich), beclin-1 (Proteintech Group, Inc.), LC3 (Novus), TH (Sigma-Aldrich), phospho-ERK (Cell Signaling), ERK1/2 (Cell Signaling), phospho-mTOR (Cell Signaling), mTOR (Cell Signaling), p62 (Abcam), and βactin (Sigma-Aldrich), followed by secondary antibodies tagged with horseradish peroxidase (Santa Cruz Biotechnology). Blots were then visualized by enhanced chemiluminescence and densitometry performed with a Versadoc XL imaging apparatus (Bio-Rad). Analysis of β-actin levels was used as a loading control. Statistical analyses Quantitative data were compared by independent sample t tests and ANOVA. Pb0.05 was regarded as significant. Quantified results are presented as the mean± SD, and n indicates the number of independent experiments (n=3–5).

Acknowledgements This work was supported by grants from the National Basic Research Program of China (2011CB504103, 2012CB722407), the National Natural Science Foundation of China (30970940), the Natural Science Foundation of Beijing (5102012, 5102007), the Scientific Research Key Program of Beijing Municipal Commission of Education (KZ201010025022), the open fund of the Key

Neuroprotective Effects of DJ‐1 on DA Neurons

Laboratory for Neurodegenerative Disease of the Ministry of Education (2011SJBX03), the open fund of the Beijing Center of Neural Regeneration and Repair, and the Beijing Key Laboratory of Brain Major Disorders (2011SJZS02).

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