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Protective effects of Ndfip1 on MPP+-induced apoptosis in MES23.5 cells and its underlying mechanisms
Experimental Neurology 273 (2015) 215–224
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Research Paper
Protective effects of Ndfip1 on MPP+-induced apoptosis in MES23.5 cells and its underlying mechanisms Kai Liu a,1, Huamin Xu b,1, Hengwei Xiang a, Peng Sun a,⁎, Junxia Xie b,⁎ a
Department of Neurosurgery, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China b
a r t i c l e
i n f o
Article history: Received 7 May 2015 Received in revised form 22 July 2015 Accepted 18 August 2015 Available online 21 August 2015 Keywords: Parkinson's disease Ndfip1 Dopamine transporter MPP+ Apoptosis
a b s t r a c t Apoptosis has been implicated as one of the important mechanisms involved in the degeneration of dopaminergic neurons in Parkinson's disease (PD). Increasing evidence suggests that Ndfip1 is a neuroprotective protein, and Ndfip1-mediated protein ubiquitination might be a possible survival strategy in neuronal injury. The aim of the present study is to investigate the neuroprotective effect of Ndfip1 on 1-methyl-4-phenylpyridinium (MPP+)-treated MES23.5 cells and the underlying mechanisms. Results showed that overexpression of Ndfip1 could significantly attenuate MPP+-induced cell loss and nuclear condensation. Further experiments demonstrated that Ndfip1 could increase Bcl-2/Bax ratio, suppress cytochrome c release from the mitochondria to cytoplasm and decrease caspase-3 activation induced by MPP+. These results suggested that Ndfip1 protected MES23.5 cells against MPP+ by its anti-apoptotic effect. In addition, we found that Ndfip1 overexpression could decrease the protein level of dopamine transporter (DAT). In parallel, proteasome inhibitor MG132 could markedly reverse Ndfip1-induced degradation of DAT. These data suggest that Ndfip1 exerts its inhibitory effect on DAT by modulating DAT degradation, in which ubiquitin–proteasome system activation might be involved. Collectively, our study indicated that the ability to decrease the DAT of Ndfip1 might be one of the mechanisms underlying its protective effect on MPP+-induced cell damage in MES23.5 cells. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by a preferential loss of dopaminergic neurons in the substantia nigra pars compacta, giving rise to dopamine (DA) depletion in the striatum. Although the etiology of PD remains obscure, a number of factors have been implicated in the pathogenesis of DA neuron loss. For example, oxidative stress, mitochondrial dysfunction (Anantharam et al., 2007; Dauer and Przedborski, 2003; Linazasoro, 2002), apoptosis (Blum et al., 2001) and ubiquitin–proteasome system (UPS) dysfunction (Dauer and Przedborski, 2003; Lim, 2007) are all considered as important mediators of neuronal death in PD. Therefore, new approaches designed to counteract apoptosis and regulate the function of UPS may serve as potential candidates for PD treatment. Protein ubiquitination is a highly ordered post-translational modification of proteins. A major consequence of protein modification by the addition of ubiquitin (Ub) chains is degradation in the 26S proteasome, or sorting and trafficking through various cell machinery (Glickman and Ciechanover, 2002; Lackovic et al., 2012). Ndfip1 (Nedd4 family⁎ Corresponding authors. E-mail addresses: [email protected] (P. Sun), [email protected] (J. Xie). 1 These authors are the co-first authors on this work.
http://dx.doi.org/10.1016/j.expneurol.2015.08.013 0014-4886/© 2015 Elsevier Inc. All rights reserved.
interacting protein 1), also known as Nedd4 WW-domain-binding protein 5 (N4WBP5), is an adaptor and activator protein containing two PPxY motifs, which can interact with several Nedd4 family E3s (such as WWP2 and Nedd4-2) and then recruit E3 ligases to ubiquitinate target proteins (Glickman and Ciechanover, 2002; Shearwin-Whyatt et al., 2004). Recent studies have identified that one of the neuroprotective mechanisms of Ndfip1 is to improve neuron survival following cerebral ischemia and brain injury (Goh et al., 2014; Howitt et al., 2012; Lackovic et al., 2012; Sang et al., 2006). Their results showed that Ndfip1 is up-regulated to combat apoptosis response to stress. Therefore, Ndfip1 is likely to function as an early sensor protein for mobilizing the ubiquitination of the harmful proteins in combination with Nedd4 ligases (Lackovic et al., 2012; Low et al., 2015; Sang et al., 2006). Since apoptosis has been identified as one of the important mechanisms leading to the death of dopaminergic neurons in PD, it is possible that Ndfip1 might have a neuroprotective effect on dopaminergic neurons against PD. Selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to dopaminergic neurons is well-known to be related to an uptake of its active metabolite 1-methyl-4-phenylpyridinium (MPP+) into dopaminergic neurons by dopamine transporter (DAT) (Bezard et al., 1999; Kim et al., 2000; Li et al., 2006; McKinley et al., 2005). Since knockout of DAT gene renders insensitivity of the dopaminergic
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neuron to MPP+ (Bezard et al., 1999; Gainetdinov et al., 1997), DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity (Masoud et al., 2015). It is thus inferred that the blockade of DAT-dependent neurotoxin entrance may protect dopaminergic neurons and may be beneficial to PD. Therefore, in this article, MPP+ is employed to establish a PD cell model in vitro to investigate the possibility that Ndfip1 was involved in the down-regulation of DAT and thus affected MPP+ influx and examine the related regulating mechanism of Ndfip1 in MES23.5 cells. The MES23.5 cells were chosen because they are a dopaminergic cell line hybridized from rat mesencephalic neurons with neuroblastoma–glioma N18TG2 cells, which exhibit several properties similar to the primary neurons that originated in the substantia nigra (Crawford et al., 1992). We demonstrated that Ndfip1-induced DAT degradation maybe one of the underlying mechanisms of the neuroprotective effect of Ndfip1 against MPP+-induced apoptosis. 2. Materials and methods 2.1. Reagents and materials Unless otherwise stated, all chemicals including anti-DAT primary antibody were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary Ndfip1 antibody was from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Anti-cytochrome c and anti-subunit IV of cytochrome c oxidase (Cox IV) antibody were bought from Abcam (Cambridge, MA, USA). Recombinant adenoviral expression vector encoding human NDFIP1 gene and empty vector were provided by GeneChem (Shanghai, China). Primers of human Ndfip1 are: sense primer: 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGGCGTTGGCGTTG GC-3′; antisense primer: 5′-TCACCATGGTGGCGACCGGATAAATAAAGA GAACTCTGGTCC-3′. Dulbecco's modified Eagle's medium Nutrient Mixture-F12 (DMEM/F12) were from Gibco (Grand Island, NY, USA). The ApoAlert® Cell Fractionation Kit was from Clontech Inc. (Hayward, CA, USA). The PE-conjugated monoclonal active caspase-3 antibody apoptosis kit was from BD Bioscience Company (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA). Hoechst 33258 was from Beyotime (Jiangsu, China). Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA). Other chemicals and regents available were from local commercial sources. 2.2. Cell culture MES23.5 cells were offered by Dr. Wei-Dong Le (Baylor College of Medicine, Houston, TX, USA). They were cultured in DMEM/F12 containing Sato's components growth medium supplemented with 5% heat-inactivated fetal bovine serum, 100 mg/mL of streptomycin and 100 units/mL of penicillin at 37 °C, in a humid 5% CO2, 95% air environment. For experiments, cells were seeded at a density of 1 × 105/cm2 in plastic flasks or on glass coverslips. MES23.5 cells were treated with reagents as follows: MES23.5 cells were incubated with 300 μmol/L MPP+ for 24 h for the MPP+-induced apoptosis experiments. For inhibitor treatments, culture medium was supplemented with 2.5 μmol/L MG132 or 10 μmol/L chloroquine. 2.3. Adenovirus-mediated expression of Ndfip1 in MES23.5 cells Recombinant adenovirus containing human NDFIP1 gene was provided by Shanghai GeneChem. The recombinant adenovirus was named Ad.Ndfip1. An empty adenovirus without the gene of interest was also constructed as a control named Ad.GFP. The cells were seeded in 6-well plates and grown to 50–70% confluence prior to viral infection, and then adenovirus at a multiplicity of infection (MOI) of 10 was added to the medium. Green fluorescent protein (GFP) fluorescence was monitored at an indicated time to verify the infective efficiency. Forty-eight
hours after infection, cells were used for further experiments. The cells were divided into the vehicle group, Ad.GFP group, Ad.Ndfip1 prior to MPP+ treatment group, and MPP+ group. To study the protective effects of Ndfip1, cells were pretreated with vehicle (saline), Ad.GFP or Ad.Ndfip1 for 48 h, and then incubated with MPP+ (300 μmol/L) for 24 h. 2.4. MTT assay MES23.5 cells were seeded in a 96-well plate at a density of 2 × 104 cells per well. Cell viability was measured by spectrophotometry using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After 24 h treatment, the cells were incubated in MTT (5 mg/mL) at 37 °C for 4 h, and then cell viability was measured at 494 nm by a spectrophotometer (Tecan, Grodig, Austria). 2.5. Total RNA extraction and real-time PCR MES23.5 cells were seeded in 6-well plates and pretreated with vehicle, Ad.GFP or Ad.Ndfip1 for 48 h, followed by incubation with MPP+ for 24 h. Total RNA was isolated by Trizol Reagent (Invitrogen) according to the manufacturer's instructions. Then 5 μg of total RNA was reverse-transcribed in a 20 μL reaction using the AMV reversetranscription system (Promega Corporation, Madison, WI, USA). Realtime PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in the Applied Biosystems 7500 real-time PCR System. PCR primer sequences were as follows: GAPDH (forward: 5′-TTC ACC ACC ATG GAG AAG GC-3′; reverse: 5′GGC ATG GAC TGT GGT CAT GA-3′); Bcl-2 (forward: 5′-GTC CCG CCT CTT CAC CTT-3′; reverse: 5′-CCC ACT CGT AGC CCC TCT-3′); and Bax (forward: 5′-GGC GAA TTG GAG ATG AAC-3′; reverse: 5′-CCG AAG TAG GAG AGG AGG-3′). The expression of house-keeping gene, GAPDH mRNA, was served as the standardized control. Amplification and detection were performed with the following conditions: an initial hold at 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. Relative mRNA expression level was calculated by the 2− ΔΔCt method. 2.6. Mitochondria and cytoplasm protein extraction The 2 × 104 cells/mL of the MES23.5 cell suspension were seeded in polylysine-precoated T75 cell culture flasks, 20 mL per flask. Then, the cells were treated as described above. Mitochondria were isolated from MES23.5 cells using an ApoAlert® Cell Fractionation Kit. The isolation procedure followed the manufacturer's recommended procedures. Protein concentration was measured with a Bradford assay kit (Bio-Rad). Samples for separation on a 12% sodium dodecyl sulfate polyacrylamide gel were prepared as described in the Western blot section. 2.7. Measurement of caspase-3 activation An active caspase-3 assay was measured by a PE-conjugated monoclonal active caspase-3 antibody apoptosis kit. Briefly, cells were seeded in 6-well plates and treated as described above. After washing twice with cold phosphate-buffered saline (PBS), cells were resuspended in a Citofix/Cytoperm™ solution at a density of 1 × 106 cells/0.5 mL. After incubating on ice for 20 min, cells were washed twice with Perm/Washing buffer, and then incubated with an antibody (100 μL Perm/Washing buffer plus 20 μL antibody per sample) for 30 min. After washing once with Perm/Washing buffer, cells were resuspended with 0.5 mL Perm/Washing buffer and analyzed by flow cytometry at 523 nm excitation and 658 nm emission. Caspase-3 activation was determined by counting the number of active caspase-3 immunoreactive cells as a percentage of total MES23.5 cells using Cellquest Software (BD Bioscience).
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2.8. Hoechst 33258 staining
3. Results
Nuclear morphology was detected using the method previously described in our lab (Xu et al., 2008; Zhang et al., 2011). MES23.5 cells grown on coverslips in 12-well plates were treated as described above. Cells were fixed in 4% paraformaldehyde for 10 min, washed twice with PBS, and stained with Hoechst 33258 dye according to the manufacturer's instructions (Beyotime, Jiangsu, China). After washing 3 times to remove the excessive dye, the cells were examined and photographed under a fluorescence microscope (Zeiss, Oberkochen, Germany). Apoptotic cells were defined on the basis of nuclear morphological changes including chromatin condensation and fragmentation. The total number of condensed cells was counted manually by researchers blinded to the treatment schedule using unbiased stereology (Feng and Zhang, 2004). For each well, we delineated a 400 μm2 frame and counted all condensed and normal nuclei in at least 10 different fields. Average sum of condensed and normal nuclei was calculated per well. The data were presented as the percentage of condensed nuclear number to the total number.
3.1. Ndfip1 attenuated MPP+-induced cytotoxicity and morphological damage in MES23.5 cells After 48 h infection, increased Ndfip1 expression was confirmed by Western blots. Data showed that Ndfip1 protein levels (Fig. 1A) increased significantly in Ad.Ndfip1 infected cells compared with Ad.GFP infected cells and the normal controls. As shown in Fig. 1B, after exposure to 300 μmol/L MPP+ for 24 h, cell viability reduced significantly by 25.45%. However, its cytotoxic effects were ameliorated when cells were infected with Ad.Ndfip1 in advance. Ndfip1 overexpression could significantly increase the cell viability compared to MPP+ treatment. The morphological change was visualized by phase-contrast imaging. The results showed that the cell bodies were evenly attached with regular shape in the control group while shrinkage and detachment were observed in the cells treated with MPP+ for 24 h. Overexpression of Ndfip1 significantly inhibited the morphological damage caused by MPP+ (Fig. 1C). In addition, treatment of Ad.GFP alone did not have a significant effect on cell viability and morphology compared with the control group.
2.9. Western blot analysis Cells were lysed in lysis solution (Ambion, Grand Island, NY, USA) and incubated at 95 °C for 10 min. Protein concentration was determined by the Bradford assay kit (Bio-Rad). Twenty micrograms of total proteins was separated by 10–12% sodium dodecylsulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. Blots were probed with rabbit monoclonal anti-DAT (1:1000), mouse monoclonal anti-cytochrome c (1:5000) and rabbit monoclonal anti-Ndfip1 antibody (1:500). Blots were also probed with rabbit monoclonal anti-β-actin antibody (Sigma, 1:10,000) as a loading control. For mitochondria samples, the blots were performed with mouse monoclonal anti-Cox IV antibody (1:5000) as a mitochondrial loading control. Anti-rabbit and anti-mouse secondary antibodies conjugated to horseradish peroxidase were used at 1:10,000 (Santa Cruz Biotechnology). UVP BioSpectrum® CCD imaging system was used for imaging and analysis. Camera settings were manipulated in preview mode to optimize the exposure and determine the appropriate final exposure settings. Exposures of 30 s up to 5 min were used for data collection. Results were analyzed through scanning densitometry by UVP VisionWorks LS Software (UVP, Cambridge, UK).
2.10. Immunocytochemistry MES23.5 cells were seeded on sterile cover glasses in 12-well plates and incubated with vehicle or Ad.Ndfip1 for 48 h respectively, then, cells were fixed in 4% paraformaldehyde in PBS for 10 min, and then permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum for 1 h at room temperature. Subsequently, the cells were incubated with rabbit monoclonal anti-DAT antibody (1:500) at 4 °C overnight. After washing with PBS, Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibodies (1:500) were used for fluorescence labeling. Nuclei were detected by co-staining with Hoechst 33258 for 15 min. Samples were examined under a fluorescence microscope (Zeiss, Oberkochen, Germany).
2.11. Statistical analysis Results were presented as means ± standard error of the mean. Differences between means in two groups were compared using the unpaired-sample t test. One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test were used to compare the differences between means in more than two groups. A probability value of P b 0.05 was considered to be statistically significant.
3.2. Ndfip1 increased the ratio of Bcl-2/Bax in the MPP+-treated MES23.5 cells In this study, the Bcl-2/Bax ratio was used to determine whether cells have undergone apoptosis. Overexpression of Ndfip1 could inhibit the decrease of Bcl-2 mRNA and the increase of Bax mRNA caused by MPP+. Therefore, the decrease of the Bcl-2/Bax ratio in the MPP+-treated cells were significantly reduced by Ndfip1 (Fig. 2). Results also showed that the ratio of bcl-2/bax protein levels decreased in the MPP+/Ad.GFP group. This could be reversed by Ndfip1 overexpression. These results suggested a notion that Ndfip1 pretreatment shifted the balance between positive and negative regulators of apoptosis towards cell survival. In addition, treatment with Ad.GFP did not affect the ratio of Bcl-2/Bax.
3.3. Ndfip1 antagonized cytochrome c release from the mitochondria to cytoplasm induced by MPP+ Results showed that levels of cytoplasmic cytochrome c were increased in the MPP+ treatment group after normalization to β-actin level (Fig. 3, A and B), and the levels of cytochrome c in mitochondria were decreased after normalization to Cox IV level (Fig. 3, C and D). However, Ndfip1 overexpression could significantly inhibit cytochrome c release from the mitochondria.
3.4. Ndfip1 inhibited caspase-3 activity and cell apoptosis in the MPP+treated MES23.5 cells We further observed the caspase-3 activity in different treatment groups. Consistent with the above observation, increased caspase-3 activity was observed in MPP+-treated MES23.5 cells. Overexpression of Ndfip1 could inhibit MPP+-induced increase in caspase-3 activity. The same as before, Ad.GFP alone did not have a significant effect on the activity of caspase-3 (Fig. 4A). Nuclear condensation and DNA fragmentation are hallmarks of cell apoptosis. Morphological changes of the cells were further observed by Hoechst 33258 staining. In uninfected and Ad.GFP infected cells, nuclei appeared with regular contours exhibiting a round form and large size. However, 300 μmol/L MPP+ treatment caused nuclear condensation in MES23.5 cells, Ndfip1 overexpression significantly attenuated this effect (Fig. 4B).
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Fig. 1. Ndfip1 attenuated MPP+-induced cell death and morphological damage. (A) MES23.5 cells were infected with Ad.Ndfip1 for 48 h, then Ndfip1 protein levels were detected by Western blots. The protein levels of Ndfip1 were higher in Ad.Ndfip1 infected cells than in the other two groups. β-Actin was used as a loading control. Data were presented as mean ± S.E.M. of 6 independent experiments ( ### P b 0.001, compared with Ad.GFP group and the control). (B) Cells were pretreated with Ad.Ndfip1 for 48 h and then MPP+ (300 mmol/L) was added for an additional 24 h and cell viability was analyzed with MTT assay (###P b 0.001, compared with the control; ***P b 0.001, compared with the MPP+/Ad.GFP group; n = 6). (C) The morphological change was visualized by phase-contract imaging.
3.5. Overexpressed Ndfip1 dramatically decreased DAT protein levels in MES23.5 cells
simultaneously for 48 h (Fig. 6). Therefore, it is plausible that the degradation of DAT may be regulated by proteasomal pathways.
To verify whether overexpression of Ndfip1 is involved in DAT degradation in MES23.5 cells, the DAT protein levels were detected by Western blots and immunocytochemistry. Results showed that the protein levels of DAT were obviously decreased with Ad.Ndfip1 infection compared to the Ad.GFP infected cells and uninfected cells. Additionally, Ad.GFP treatment alone had no apparent effect on the protein levels of DAT when compared with the control, as shown in Fig. 5A. In parallel, DAT fluorescence (red fluorescence) decreased in the cells with Ndfip1 overexpression compared to Ad.GFP infected cells and uninfected cells (Fig. 5B). As shown above, it is reasonable to speculate that the dopaminergic neuroprotective property of Ndfip1 may be associated with reduced MPP+ entry resulting from functional degradation of DAT.
4. Discussion
3.6. Proteasomal pathway was involved in the degradation of DAT To determine the factors responsible for the regulation of DAT, MES23.5 cells were incubated with the proteasome inhibitor MG132 or the lysosomal inhibitor chloroquine, and infected with Ad.Ndfip1 at the same time. After incubation with the chemicals for 48 h respectively, the cells were then harvested for Western blots. As we expected, results showed that the expression of DAT was decreased in the Ad.Ndfip1 group. Similarly, decreased expression of DAT was observed in the Ad.Ndfip1 and chloroquine co-treatment group. However, MG132 could markedly reverse the Ndfip1-induced down-regulation in DAT expression, after cells were incubated with MG132 and Ad.Ndfip1
In this study, we have showed that Ndfip1 could potently inhibit the uptake of MPP+ through the induction of DAT degradation in the cell membrane, which was largely mediated via the ubiquitin–proteasome system activation pathway. Though Ndfip1 has been reported to possess multiple bioactivities, this article documented that Ndfip1-induced DAT degradation maybe one of the underlying mechanisms of the neuroprotective effects of Ndfip1 against MPP+-induced apoptosis. Ndfip1 is a protein which is critical in protecting against neuronal apoptosis in vitro and associated with neuronal survival in vivo following traumatic brain injury and cerebral ischemia (Howitt et al., 2012; Lackovic et al., 2012; Sang et al., 2006). Binding of Ndfip1 to target proteins leads to Nedd4-mediated ubiquitination, resulting in degradation (Jia et al., 2015) or trafficking (Howitt et al., 2012; Low et al., 2015) of Ndfip1-bound substrates. Previous studies indicated a number of mechanisms through which Ndfip1 can combat apoptosis, including Ndfip1mediated ubiquitination and nuclear trafficking of BRAT1 (breast cancer type 1-associated ataxia telangiectasia mutated activator 1) to inhibit apoptosis during the DNA damage response (Low et al., 2015), degradation of DMT1 (divalent metal transporter 1) to prevent metal toxicity of neurons (Jia et al., 2015) as well as Ndfip1-dependent trafficking of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) into the nucleus to promote pAkt-mediated cell survival (Howitt et al., 2012). Thus, ubiquitin modification and signaling of diverse protein targets are multifaceted, suggesting that Ndfip1-
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Fig. 2. Changes of Bcl-2 and Bax mRNA and protein levels in MES23.5 cells. Ndfip1 overexpression inhibited MPP+-induced down-regulation of Bcl-2 mRNA and up-regulation of Bax mRNA (A) and down-regulation of Bcl-2/Bax ratio (B). GAPDH mRNA was used as an internal standard (##P b 0.01, ###P b 0.001, compared with the control; *P b 0.05, **P b 0.01, compared with the MPP+/Ad.GFP group). (C) Bcl-2 and Bax protein levels in MES23.5 cells. Ndfip1 overexpression inhibited MPP+-induced down-regulation of Bcl-2/Bax ratio (#P b 0.05, compared with the control; *P b 0.05, compared with the MPP+/Ad.GFP group). Each value represented the mean ± S.E.M. of 6 independent experiments.
mediated ubiquitination might have diverse physiological consequences. The diversity of function is reflected in the varied nature of Ndfip1 substrates, which, to date, included the transcription factor Jun
B (Oliver et al., 2006), divalent metal transporter DMT1 (Jia et al., 2015), BRAT1 (Low et al., 2015) and tumor suppressor PTEN (Howitt et al., 2012), and it is likely that future studies will increase this tally.
Fig. 3. The cytochrome c protein levels in MES23.5 cells. 300 μmol/L MPP+ increased the release of cytochrome c from the mitochondria to the cytoplasm, and this effect was inhibited by overexpression of Ndfip1 in MES23.5 cells. (A) Western blots showed cytochrome c released to cytoplasmic fractions. β-Actin was used as a loading control. (C) Cytochrome c was detected in the mitochondrial fraction using COX IV as the mitochondrial loading control. (B, D) Statistical analysis. Data were expressed as mean ± S.E.M. of 6 independent experiments (###P b 0.001, compared with the control; **P b 0.01, compared with the MPP+ group).
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Fig. 4. Measurement of caspase-3 activity and morphological changes in MES23.5 cells. 300 μmol/L MPP+ treatment increased the activity of caspase-3 compared with that of the control, and Ndfip1 overexpression protected against MPP+-induced increase of caspase-3 activity. (A) Representatives of the fluorometric assay on the activity of caspase-3 in different groups. Data were presented as mean ± S.E.M. of 6 independent experiments and analyzed via Newman–Keuls post one-way ANOVA; fluorescence values of the control were set to 100% (###P b 0.001, compared with the control; ***P b 0.001, compared with the MPP+ group). (B) Representative photographs of Hoechst 33258 staining from vehicle, Ad.GFP infected cells, Ad.Ndfip1 infected cells incubated with MPP+ for 24 h, and MPP+ treatment cells. The nuclei of MPP+-treated cells appeared hypercondensed (brightly stained) and showed fragmentation of chromatin (indicated by arrows); however, Ndfip1 pretreatment significantly attenuated MPP+-induced nuclear condensation. Data were presented as mean ± S.E.M. of 6 independent experiments (###P b 0.001, compared with the control; **P b 0.01, compared with the MPP+ group).
In the present study, we demonstrated that Ndfip1 effectively protected MES23.5 cells from MPP+-induced neurotoxicity. MPTP, a common neurotoxin for inducing PD models after being transported
into the brain, is oxidized by monoamine oxidase-B (MAO-B) into MPP+ (Arora et al., 1990). This compound is actively transported into presynaptic dopaminergic nerve terminals by DAT, inhibiting complex
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I of the mitochondrial respiratory chain, and may cause the mitochondrial dysfunction and oxidative stress (Bezard et al., 1999; Cassarino et al., 1999; Kim et al., 2000; Zhang et al., 2011). Increasing evidence has proved that mitochondrial dysfunction and oxidative stress played an important role in the etiology of PD (Anantharam et al., 2007; Andersen, 2004; Dauer and Przedborski, 2003; Linazasoro, 2002). Mitochondrion is not only the main target of reactive oxygen species
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(ROS), but also the major site of ROS production as well as the primary target of oxidative molecular damage (Dauer and Przedborski, 2003). So the damaged mitochondrial membrane is implicated as key events in the pathogenic cascades. It breaks the balance of the Bcl/Bax system and leads to the release of proapoptotic proteins from the mitochondria (Oka et al., 2008; Zhang et al., 2011). As an anti-apoptotic member of the Bcl-2 family, Bcl-2 can bind Bax to form Bcl-2/Bax heterodimers, thereby
Fig. 5. Ndfip1 overexpression induced a decrease in DAT protein levels in MES23.5 cells. (A) Decreased protein levels of DAT were observed in the Ad.Ndfip1 group compared with the Ad.GFP group and the control. β-Actin was used as a loading control. Data were presented as mean ± S.E.M. of 6 independent experiments (#P b 0.05, compared with the Ad.GFP group and the control). (B) Immunocytochemistry with anti-DAT antibodies indicate basal levels of DAT expression (red fluorescence) in cultured MES23.5 cells and Ad.GFP infected cells, but down-regulation (arrow heads) when exposed to Ad.Ndfip1 for 48 h.
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Fig. 6. Degradation of DAT may be regulated by proteasomal pathways. The down-regulation of DAT induced by Ad.Ndfip1 was reversed by treatment with the proteasome inhibitor MG132 in MES23.5 cells. β-Actin was used as a loading control. (A) Western blots of DAT expression in MES23.5 cells of each indicated experimental group. (B) Statistical analysis. Data were presented as mean ± S.E.M. of 6 independent experiments (#P b 0.05, *P b 0.05, compared with the control, MG132 + Ad.Ndfip1 and MG132 + chlor + Ad.Ndfip1 groups).
attenuating the pro-apoptotic effect of Bax (Oltvai et al., 1993). Among proapoptotic proteins, the efflux of cytochrome c is thought to be a key event in the subsequent apoptotic processes. A subclass of the Bcl-2 family proteins that includes Bak and Bax regulates the efflux of cytochrome c by redistributing to the mitochondria, resulting in the permeabilization of the outer mitochondrial membrane and cytochrome c escaping to the cytoplasm (Bouchier-Hayes et al., 2005; Er et al., 2006; Scorrano and Korsmeyer, 2003). Our study showed that overexpression of Ndfip1 could partially inhibit MPP+-induced dysfunction of the Bcl/Bax system and then inhibited cytochrome c release during apoptosis. The release of cytochrome c from mitochondria which promotes massive activation of executioner caspase-3 is a crucial checkpoint in cell commitment to apoptosis (Danial and Korsmeyer, 2004; Enari et al., 1998; Galluzzi et al., 2012; Thornberry and Lazebnik, 1998). In this study, we confirmed that Ndfip1 could significantly decrease caspase-3 activation and nuclear morphological changes induced by MPP+. Evidence showed that MPP+-induced cell death could be through a caspase-3 dependent or independent pathway (Kaul et al., 2003; Chu et al., 2005). Kaul et al. proved that caspase-3 activation was involved in the apoptosis in rat mesencephalic dopaminergic N27 cells. Their results showed MPP+ (300 μM) caused a 115% increase in DNA fragmentation. Caspase-3 specific inhibitor Z-DEVD-fmk and broad-spectrum caspase inhibitor Z-VAD-fmk could inhibit an MPP+induced increase in DNA fragmentation (Kaul et al., 2003). Another study showed that AIF nuclear translocation was involved in MPP+induced apoptosis, and that this process is independent of caspase-3. In their study, 5 μM MPP+ was used (Chu et al., 2005). Although the precise mechanisms underlying this discrepancy is not clear, one possibility is that different concentrations of MPP+ might lead to different types of apoptosis. In our study, 300 μM MPP+ was used, and we also observed that 300 μM MPP+ induced a significant increase in caspase-3 activity and cytochrome c release which is consistent with the results of Kaul et al. This made the possibility that in our experiment, the caspase-3 dependent
pathway was involved in MPP+-induced apoptosis in MES23.5 dopaminergic cells. However, the mechanisms underlying the neuroprotective effects of Ndfip1 have not been fully elucidated. DAT is located on the cell membrane of dopaminergic neurons, and exerts its function by rapidly taking up dopamine from the extracellular space into the presynaptic neuron (Masoud et al., 2015). DAT provides not only an integral component of dopaminergic neurotransmission but also a molecular gateway for the uptake of some neurotoxins such as MPP+ (Li et al., 2006). The importance of DAT for the selectivity of MPP+ toxicity in dopaminergic neurons has been demonstrated by several in vivo and in vitro studies showing that DAT-blocking agents can reduce MPP+-mediated neurotoxicity (Bezard et al., 1999; Jaber et al., 1997; Ricaurte et al., 1985). DAT is a critical regulator of dopamine disposition in the brain and also a crucial determiner to the neurotoxicity of 6-OHDA (Van Kampen et al., 2000) and MPP+ (Bezard et al., 1999; Storch et al., 2004), which are two wellknown experimental neurotoxins in PD. Proper inhibition of DAT activity, therefore, is proposed to increase synaptic dopamine level and reduce DAT-dependent neurotoxicity, which may be beneficial to PD (Li et al., 2006). Therefore, we hypothesized that the neuroprotective property of Ndfip1 might be associated with the regulation of DAT. To further identify this hypothesis, we detected the protein levels of DAT in different experiment groups. Results showed that DAT expression in the Ad.Ndfip1 group decreased significantly compared with control. Therefore, it is possible that the decrease of DAT is associated with the ubiquitination and degradation of DAT in Ad.Ndfip1 infected MES23.5 cells. This was confirmed by our further results, which showed that the decrease of DAT induced by Ndfip1 was reversed in the presence of MG132, indicating that degradation of DAT by Ndfip1 is via the proteasome pathway. In conclusion, the results presented here give support to a sequence of events whereby Ndfip1 plays a key role in MPP+-induced apoptosis in MES23.5 dopaminergic cells. The simplified depiction of effects of Ndfip1 on MPP+-induced cytotoxicity was summarized in Fig. 7. In our present study, we demonstrated that overexpression of Ndfip1 caused DAT degradation, resulting in the decreased MPP+ influx and MPP+-induced neurotoxicity. Furthermore, this study provides further insight into the underlying mechanisms of Ndfip1, including inhibiting MPP+ uptake and ameliorating DAT-dependent neurotoxicity, which may be beneficial to PD.
Conflict of interest There is no conflict of interest for all authors.
Acknowledgments We thank Dr. Wei-dong Le who gave us the MES23.5 cell line. This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of the People's Republic of China (2011CB504102), the National Natural Science Foundation of China (81430024, 81100955, 31371081, 31271131, 81470059, and 81171208), the Department of Science and Technology of Shandong Province (ZR2015JL011), the Excellent Innovative Team of Shandong Province, the Taishan Scholars Construction Project, the Ministry of Education of China (20123706110002), the Shandong Provincial Education Department (J11LC05), and the Science and Technology Development Plan of Shandong Province (2011gsf11831).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2015.08.013.
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Fig. 7. A schematic diagram showing how Ndfip1 attenuated MPP+-induced cytotoxicity in dopaminergic neurons. The neuroprotective effect of Ndfip1 on MPP+-treated MES23.5 cells is mediated via reducing DAT levels, which resulted in decreased MPP+ uptake and the protection of cells from MPP+-induced neurotoxicity.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Research Paper
Protective effects of Ndfip1 on MPP+-induced apoptosis in MES23.5 cells and its underlying mechanisms Kai Liu a,1, Huamin Xu b,1, Hengwei Xiang a, Peng Sun a,⁎, Junxia Xie b,⁎ a
Department of Neurosurgery, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China b
a r t i c l e
i n f o
Article history: Received 7 May 2015 Received in revised form 22 July 2015 Accepted 18 August 2015 Available online 21 August 2015 Keywords: Parkinson's disease Ndfip1 Dopamine transporter MPP+ Apoptosis
a b s t r a c t Apoptosis has been implicated as one of the important mechanisms involved in the degeneration of dopaminergic neurons in Parkinson's disease (PD). Increasing evidence suggests that Ndfip1 is a neuroprotective protein, and Ndfip1-mediated protein ubiquitination might be a possible survival strategy in neuronal injury. The aim of the present study is to investigate the neuroprotective effect of Ndfip1 on 1-methyl-4-phenylpyridinium (MPP+)-treated MES23.5 cells and the underlying mechanisms. Results showed that overexpression of Ndfip1 could significantly attenuate MPP+-induced cell loss and nuclear condensation. Further experiments demonstrated that Ndfip1 could increase Bcl-2/Bax ratio, suppress cytochrome c release from the mitochondria to cytoplasm and decrease caspase-3 activation induced by MPP+. These results suggested that Ndfip1 protected MES23.5 cells against MPP+ by its anti-apoptotic effect. In addition, we found that Ndfip1 overexpression could decrease the protein level of dopamine transporter (DAT). In parallel, proteasome inhibitor MG132 could markedly reverse Ndfip1-induced degradation of DAT. These data suggest that Ndfip1 exerts its inhibitory effect on DAT by modulating DAT degradation, in which ubiquitin–proteasome system activation might be involved. Collectively, our study indicated that the ability to decrease the DAT of Ndfip1 might be one of the mechanisms underlying its protective effect on MPP+-induced cell damage in MES23.5 cells. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by a preferential loss of dopaminergic neurons in the substantia nigra pars compacta, giving rise to dopamine (DA) depletion in the striatum. Although the etiology of PD remains obscure, a number of factors have been implicated in the pathogenesis of DA neuron loss. For example, oxidative stress, mitochondrial dysfunction (Anantharam et al., 2007; Dauer and Przedborski, 2003; Linazasoro, 2002), apoptosis (Blum et al., 2001) and ubiquitin–proteasome system (UPS) dysfunction (Dauer and Przedborski, 2003; Lim, 2007) are all considered as important mediators of neuronal death in PD. Therefore, new approaches designed to counteract apoptosis and regulate the function of UPS may serve as potential candidates for PD treatment. Protein ubiquitination is a highly ordered post-translational modification of proteins. A major consequence of protein modification by the addition of ubiquitin (Ub) chains is degradation in the 26S proteasome, or sorting and trafficking through various cell machinery (Glickman and Ciechanover, 2002; Lackovic et al., 2012). Ndfip1 (Nedd4 family⁎ Corresponding authors. E-mail addresses: [email protected] (P. Sun), [email protected] (J. Xie). 1 These authors are the co-first authors on this work.
http://dx.doi.org/10.1016/j.expneurol.2015.08.013 0014-4886/© 2015 Elsevier Inc. All rights reserved.
interacting protein 1), also known as Nedd4 WW-domain-binding protein 5 (N4WBP5), is an adaptor and activator protein containing two PPxY motifs, which can interact with several Nedd4 family E3s (such as WWP2 and Nedd4-2) and then recruit E3 ligases to ubiquitinate target proteins (Glickman and Ciechanover, 2002; Shearwin-Whyatt et al., 2004). Recent studies have identified that one of the neuroprotective mechanisms of Ndfip1 is to improve neuron survival following cerebral ischemia and brain injury (Goh et al., 2014; Howitt et al., 2012; Lackovic et al., 2012; Sang et al., 2006). Their results showed that Ndfip1 is up-regulated to combat apoptosis response to stress. Therefore, Ndfip1 is likely to function as an early sensor protein for mobilizing the ubiquitination of the harmful proteins in combination with Nedd4 ligases (Lackovic et al., 2012; Low et al., 2015; Sang et al., 2006). Since apoptosis has been identified as one of the important mechanisms leading to the death of dopaminergic neurons in PD, it is possible that Ndfip1 might have a neuroprotective effect on dopaminergic neurons against PD. Selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to dopaminergic neurons is well-known to be related to an uptake of its active metabolite 1-methyl-4-phenylpyridinium (MPP+) into dopaminergic neurons by dopamine transporter (DAT) (Bezard et al., 1999; Kim et al., 2000; Li et al., 2006; McKinley et al., 2005). Since knockout of DAT gene renders insensitivity of the dopaminergic
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neuron to MPP+ (Bezard et al., 1999; Gainetdinov et al., 1997), DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity (Masoud et al., 2015). It is thus inferred that the blockade of DAT-dependent neurotoxin entrance may protect dopaminergic neurons and may be beneficial to PD. Therefore, in this article, MPP+ is employed to establish a PD cell model in vitro to investigate the possibility that Ndfip1 was involved in the down-regulation of DAT and thus affected MPP+ influx and examine the related regulating mechanism of Ndfip1 in MES23.5 cells. The MES23.5 cells were chosen because they are a dopaminergic cell line hybridized from rat mesencephalic neurons with neuroblastoma–glioma N18TG2 cells, which exhibit several properties similar to the primary neurons that originated in the substantia nigra (Crawford et al., 1992). We demonstrated that Ndfip1-induced DAT degradation maybe one of the underlying mechanisms of the neuroprotective effect of Ndfip1 against MPP+-induced apoptosis. 2. Materials and methods 2.1. Reagents and materials Unless otherwise stated, all chemicals including anti-DAT primary antibody were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary Ndfip1 antibody was from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Anti-cytochrome c and anti-subunit IV of cytochrome c oxidase (Cox IV) antibody were bought from Abcam (Cambridge, MA, USA). Recombinant adenoviral expression vector encoding human NDFIP1 gene and empty vector were provided by GeneChem (Shanghai, China). Primers of human Ndfip1 are: sense primer: 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGGCGTTGGCGTTG GC-3′; antisense primer: 5′-TCACCATGGTGGCGACCGGATAAATAAAGA GAACTCTGGTCC-3′. Dulbecco's modified Eagle's medium Nutrient Mixture-F12 (DMEM/F12) were from Gibco (Grand Island, NY, USA). The ApoAlert® Cell Fractionation Kit was from Clontech Inc. (Hayward, CA, USA). The PE-conjugated monoclonal active caspase-3 antibody apoptosis kit was from BD Bioscience Company (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA). Hoechst 33258 was from Beyotime (Jiangsu, China). Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA). Other chemicals and regents available were from local commercial sources. 2.2. Cell culture MES23.5 cells were offered by Dr. Wei-Dong Le (Baylor College of Medicine, Houston, TX, USA). They were cultured in DMEM/F12 containing Sato's components growth medium supplemented with 5% heat-inactivated fetal bovine serum, 100 mg/mL of streptomycin and 100 units/mL of penicillin at 37 °C, in a humid 5% CO2, 95% air environment. For experiments, cells were seeded at a density of 1 × 105/cm2 in plastic flasks or on glass coverslips. MES23.5 cells were treated with reagents as follows: MES23.5 cells were incubated with 300 μmol/L MPP+ for 24 h for the MPP+-induced apoptosis experiments. For inhibitor treatments, culture medium was supplemented with 2.5 μmol/L MG132 or 10 μmol/L chloroquine. 2.3. Adenovirus-mediated expression of Ndfip1 in MES23.5 cells Recombinant adenovirus containing human NDFIP1 gene was provided by Shanghai GeneChem. The recombinant adenovirus was named Ad.Ndfip1. An empty adenovirus without the gene of interest was also constructed as a control named Ad.GFP. The cells were seeded in 6-well plates and grown to 50–70% confluence prior to viral infection, and then adenovirus at a multiplicity of infection (MOI) of 10 was added to the medium. Green fluorescent protein (GFP) fluorescence was monitored at an indicated time to verify the infective efficiency. Forty-eight
hours after infection, cells were used for further experiments. The cells were divided into the vehicle group, Ad.GFP group, Ad.Ndfip1 prior to MPP+ treatment group, and MPP+ group. To study the protective effects of Ndfip1, cells were pretreated with vehicle (saline), Ad.GFP or Ad.Ndfip1 for 48 h, and then incubated with MPP+ (300 μmol/L) for 24 h. 2.4. MTT assay MES23.5 cells were seeded in a 96-well plate at a density of 2 × 104 cells per well. Cell viability was measured by spectrophotometry using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After 24 h treatment, the cells were incubated in MTT (5 mg/mL) at 37 °C for 4 h, and then cell viability was measured at 494 nm by a spectrophotometer (Tecan, Grodig, Austria). 2.5. Total RNA extraction and real-time PCR MES23.5 cells were seeded in 6-well plates and pretreated with vehicle, Ad.GFP or Ad.Ndfip1 for 48 h, followed by incubation with MPP+ for 24 h. Total RNA was isolated by Trizol Reagent (Invitrogen) according to the manufacturer's instructions. Then 5 μg of total RNA was reverse-transcribed in a 20 μL reaction using the AMV reversetranscription system (Promega Corporation, Madison, WI, USA). Realtime PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in the Applied Biosystems 7500 real-time PCR System. PCR primer sequences were as follows: GAPDH (forward: 5′-TTC ACC ACC ATG GAG AAG GC-3′; reverse: 5′GGC ATG GAC TGT GGT CAT GA-3′); Bcl-2 (forward: 5′-GTC CCG CCT CTT CAC CTT-3′; reverse: 5′-CCC ACT CGT AGC CCC TCT-3′); and Bax (forward: 5′-GGC GAA TTG GAG ATG AAC-3′; reverse: 5′-CCG AAG TAG GAG AGG AGG-3′). The expression of house-keeping gene, GAPDH mRNA, was served as the standardized control. Amplification and detection were performed with the following conditions: an initial hold at 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. Relative mRNA expression level was calculated by the 2− ΔΔCt method. 2.6. Mitochondria and cytoplasm protein extraction The 2 × 104 cells/mL of the MES23.5 cell suspension were seeded in polylysine-precoated T75 cell culture flasks, 20 mL per flask. Then, the cells were treated as described above. Mitochondria were isolated from MES23.5 cells using an ApoAlert® Cell Fractionation Kit. The isolation procedure followed the manufacturer's recommended procedures. Protein concentration was measured with a Bradford assay kit (Bio-Rad). Samples for separation on a 12% sodium dodecyl sulfate polyacrylamide gel were prepared as described in the Western blot section. 2.7. Measurement of caspase-3 activation An active caspase-3 assay was measured by a PE-conjugated monoclonal active caspase-3 antibody apoptosis kit. Briefly, cells were seeded in 6-well plates and treated as described above. After washing twice with cold phosphate-buffered saline (PBS), cells were resuspended in a Citofix/Cytoperm™ solution at a density of 1 × 106 cells/0.5 mL. After incubating on ice for 20 min, cells were washed twice with Perm/Washing buffer, and then incubated with an antibody (100 μL Perm/Washing buffer plus 20 μL antibody per sample) for 30 min. After washing once with Perm/Washing buffer, cells were resuspended with 0.5 mL Perm/Washing buffer and analyzed by flow cytometry at 523 nm excitation and 658 nm emission. Caspase-3 activation was determined by counting the number of active caspase-3 immunoreactive cells as a percentage of total MES23.5 cells using Cellquest Software (BD Bioscience).
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2.8. Hoechst 33258 staining
3. Results
Nuclear morphology was detected using the method previously described in our lab (Xu et al., 2008; Zhang et al., 2011). MES23.5 cells grown on coverslips in 12-well plates were treated as described above. Cells were fixed in 4% paraformaldehyde for 10 min, washed twice with PBS, and stained with Hoechst 33258 dye according to the manufacturer's instructions (Beyotime, Jiangsu, China). After washing 3 times to remove the excessive dye, the cells were examined and photographed under a fluorescence microscope (Zeiss, Oberkochen, Germany). Apoptotic cells were defined on the basis of nuclear morphological changes including chromatin condensation and fragmentation. The total number of condensed cells was counted manually by researchers blinded to the treatment schedule using unbiased stereology (Feng and Zhang, 2004). For each well, we delineated a 400 μm2 frame and counted all condensed and normal nuclei in at least 10 different fields. Average sum of condensed and normal nuclei was calculated per well. The data were presented as the percentage of condensed nuclear number to the total number.
3.1. Ndfip1 attenuated MPP+-induced cytotoxicity and morphological damage in MES23.5 cells After 48 h infection, increased Ndfip1 expression was confirmed by Western blots. Data showed that Ndfip1 protein levels (Fig. 1A) increased significantly in Ad.Ndfip1 infected cells compared with Ad.GFP infected cells and the normal controls. As shown in Fig. 1B, after exposure to 300 μmol/L MPP+ for 24 h, cell viability reduced significantly by 25.45%. However, its cytotoxic effects were ameliorated when cells were infected with Ad.Ndfip1 in advance. Ndfip1 overexpression could significantly increase the cell viability compared to MPP+ treatment. The morphological change was visualized by phase-contrast imaging. The results showed that the cell bodies were evenly attached with regular shape in the control group while shrinkage and detachment were observed in the cells treated with MPP+ for 24 h. Overexpression of Ndfip1 significantly inhibited the morphological damage caused by MPP+ (Fig. 1C). In addition, treatment of Ad.GFP alone did not have a significant effect on cell viability and morphology compared with the control group.
2.9. Western blot analysis Cells were lysed in lysis solution (Ambion, Grand Island, NY, USA) and incubated at 95 °C for 10 min. Protein concentration was determined by the Bradford assay kit (Bio-Rad). Twenty micrograms of total proteins was separated by 10–12% sodium dodecylsulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. Blots were probed with rabbit monoclonal anti-DAT (1:1000), mouse monoclonal anti-cytochrome c (1:5000) and rabbit monoclonal anti-Ndfip1 antibody (1:500). Blots were also probed with rabbit monoclonal anti-β-actin antibody (Sigma, 1:10,000) as a loading control. For mitochondria samples, the blots were performed with mouse monoclonal anti-Cox IV antibody (1:5000) as a mitochondrial loading control. Anti-rabbit and anti-mouse secondary antibodies conjugated to horseradish peroxidase were used at 1:10,000 (Santa Cruz Biotechnology). UVP BioSpectrum® CCD imaging system was used for imaging and analysis. Camera settings were manipulated in preview mode to optimize the exposure and determine the appropriate final exposure settings. Exposures of 30 s up to 5 min were used for data collection. Results were analyzed through scanning densitometry by UVP VisionWorks LS Software (UVP, Cambridge, UK).
2.10. Immunocytochemistry MES23.5 cells were seeded on sterile cover glasses in 12-well plates and incubated with vehicle or Ad.Ndfip1 for 48 h respectively, then, cells were fixed in 4% paraformaldehyde in PBS for 10 min, and then permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum for 1 h at room temperature. Subsequently, the cells were incubated with rabbit monoclonal anti-DAT antibody (1:500) at 4 °C overnight. After washing with PBS, Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibodies (1:500) were used for fluorescence labeling. Nuclei were detected by co-staining with Hoechst 33258 for 15 min. Samples were examined under a fluorescence microscope (Zeiss, Oberkochen, Germany).
2.11. Statistical analysis Results were presented as means ± standard error of the mean. Differences between means in two groups were compared using the unpaired-sample t test. One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test were used to compare the differences between means in more than two groups. A probability value of P b 0.05 was considered to be statistically significant.
3.2. Ndfip1 increased the ratio of Bcl-2/Bax in the MPP+-treated MES23.5 cells In this study, the Bcl-2/Bax ratio was used to determine whether cells have undergone apoptosis. Overexpression of Ndfip1 could inhibit the decrease of Bcl-2 mRNA and the increase of Bax mRNA caused by MPP+. Therefore, the decrease of the Bcl-2/Bax ratio in the MPP+-treated cells were significantly reduced by Ndfip1 (Fig. 2). Results also showed that the ratio of bcl-2/bax protein levels decreased in the MPP+/Ad.GFP group. This could be reversed by Ndfip1 overexpression. These results suggested a notion that Ndfip1 pretreatment shifted the balance between positive and negative regulators of apoptosis towards cell survival. In addition, treatment with Ad.GFP did not affect the ratio of Bcl-2/Bax.
3.3. Ndfip1 antagonized cytochrome c release from the mitochondria to cytoplasm induced by MPP+ Results showed that levels of cytoplasmic cytochrome c were increased in the MPP+ treatment group after normalization to β-actin level (Fig. 3, A and B), and the levels of cytochrome c in mitochondria were decreased after normalization to Cox IV level (Fig. 3, C and D). However, Ndfip1 overexpression could significantly inhibit cytochrome c release from the mitochondria.
3.4. Ndfip1 inhibited caspase-3 activity and cell apoptosis in the MPP+treated MES23.5 cells We further observed the caspase-3 activity in different treatment groups. Consistent with the above observation, increased caspase-3 activity was observed in MPP+-treated MES23.5 cells. Overexpression of Ndfip1 could inhibit MPP+-induced increase in caspase-3 activity. The same as before, Ad.GFP alone did not have a significant effect on the activity of caspase-3 (Fig. 4A). Nuclear condensation and DNA fragmentation are hallmarks of cell apoptosis. Morphological changes of the cells were further observed by Hoechst 33258 staining. In uninfected and Ad.GFP infected cells, nuclei appeared with regular contours exhibiting a round form and large size. However, 300 μmol/L MPP+ treatment caused nuclear condensation in MES23.5 cells, Ndfip1 overexpression significantly attenuated this effect (Fig. 4B).
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Fig. 1. Ndfip1 attenuated MPP+-induced cell death and morphological damage. (A) MES23.5 cells were infected with Ad.Ndfip1 for 48 h, then Ndfip1 protein levels were detected by Western blots. The protein levels of Ndfip1 were higher in Ad.Ndfip1 infected cells than in the other two groups. β-Actin was used as a loading control. Data were presented as mean ± S.E.M. of 6 independent experiments ( ### P b 0.001, compared with Ad.GFP group and the control). (B) Cells were pretreated with Ad.Ndfip1 for 48 h and then MPP+ (300 mmol/L) was added for an additional 24 h and cell viability was analyzed with MTT assay (###P b 0.001, compared with the control; ***P b 0.001, compared with the MPP+/Ad.GFP group; n = 6). (C) The morphological change was visualized by phase-contract imaging.
3.5. Overexpressed Ndfip1 dramatically decreased DAT protein levels in MES23.5 cells
simultaneously for 48 h (Fig. 6). Therefore, it is plausible that the degradation of DAT may be regulated by proteasomal pathways.
To verify whether overexpression of Ndfip1 is involved in DAT degradation in MES23.5 cells, the DAT protein levels were detected by Western blots and immunocytochemistry. Results showed that the protein levels of DAT were obviously decreased with Ad.Ndfip1 infection compared to the Ad.GFP infected cells and uninfected cells. Additionally, Ad.GFP treatment alone had no apparent effect on the protein levels of DAT when compared with the control, as shown in Fig. 5A. In parallel, DAT fluorescence (red fluorescence) decreased in the cells with Ndfip1 overexpression compared to Ad.GFP infected cells and uninfected cells (Fig. 5B). As shown above, it is reasonable to speculate that the dopaminergic neuroprotective property of Ndfip1 may be associated with reduced MPP+ entry resulting from functional degradation of DAT.
4. Discussion
3.6. Proteasomal pathway was involved in the degradation of DAT To determine the factors responsible for the regulation of DAT, MES23.5 cells were incubated with the proteasome inhibitor MG132 or the lysosomal inhibitor chloroquine, and infected with Ad.Ndfip1 at the same time. After incubation with the chemicals for 48 h respectively, the cells were then harvested for Western blots. As we expected, results showed that the expression of DAT was decreased in the Ad.Ndfip1 group. Similarly, decreased expression of DAT was observed in the Ad.Ndfip1 and chloroquine co-treatment group. However, MG132 could markedly reverse the Ndfip1-induced down-regulation in DAT expression, after cells were incubated with MG132 and Ad.Ndfip1
In this study, we have showed that Ndfip1 could potently inhibit the uptake of MPP+ through the induction of DAT degradation in the cell membrane, which was largely mediated via the ubiquitin–proteasome system activation pathway. Though Ndfip1 has been reported to possess multiple bioactivities, this article documented that Ndfip1-induced DAT degradation maybe one of the underlying mechanisms of the neuroprotective effects of Ndfip1 against MPP+-induced apoptosis. Ndfip1 is a protein which is critical in protecting against neuronal apoptosis in vitro and associated with neuronal survival in vivo following traumatic brain injury and cerebral ischemia (Howitt et al., 2012; Lackovic et al., 2012; Sang et al., 2006). Binding of Ndfip1 to target proteins leads to Nedd4-mediated ubiquitination, resulting in degradation (Jia et al., 2015) or trafficking (Howitt et al., 2012; Low et al., 2015) of Ndfip1-bound substrates. Previous studies indicated a number of mechanisms through which Ndfip1 can combat apoptosis, including Ndfip1mediated ubiquitination and nuclear trafficking of BRAT1 (breast cancer type 1-associated ataxia telangiectasia mutated activator 1) to inhibit apoptosis during the DNA damage response (Low et al., 2015), degradation of DMT1 (divalent metal transporter 1) to prevent metal toxicity of neurons (Jia et al., 2015) as well as Ndfip1-dependent trafficking of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) into the nucleus to promote pAkt-mediated cell survival (Howitt et al., 2012). Thus, ubiquitin modification and signaling of diverse protein targets are multifaceted, suggesting that Ndfip1-
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Fig. 2. Changes of Bcl-2 and Bax mRNA and protein levels in MES23.5 cells. Ndfip1 overexpression inhibited MPP+-induced down-regulation of Bcl-2 mRNA and up-regulation of Bax mRNA (A) and down-regulation of Bcl-2/Bax ratio (B). GAPDH mRNA was used as an internal standard (##P b 0.01, ###P b 0.001, compared with the control; *P b 0.05, **P b 0.01, compared with the MPP+/Ad.GFP group). (C) Bcl-2 and Bax protein levels in MES23.5 cells. Ndfip1 overexpression inhibited MPP+-induced down-regulation of Bcl-2/Bax ratio (#P b 0.05, compared with the control; *P b 0.05, compared with the MPP+/Ad.GFP group). Each value represented the mean ± S.E.M. of 6 independent experiments.
mediated ubiquitination might have diverse physiological consequences. The diversity of function is reflected in the varied nature of Ndfip1 substrates, which, to date, included the transcription factor Jun
B (Oliver et al., 2006), divalent metal transporter DMT1 (Jia et al., 2015), BRAT1 (Low et al., 2015) and tumor suppressor PTEN (Howitt et al., 2012), and it is likely that future studies will increase this tally.
Fig. 3. The cytochrome c protein levels in MES23.5 cells. 300 μmol/L MPP+ increased the release of cytochrome c from the mitochondria to the cytoplasm, and this effect was inhibited by overexpression of Ndfip1 in MES23.5 cells. (A) Western blots showed cytochrome c released to cytoplasmic fractions. β-Actin was used as a loading control. (C) Cytochrome c was detected in the mitochondrial fraction using COX IV as the mitochondrial loading control. (B, D) Statistical analysis. Data were expressed as mean ± S.E.M. of 6 independent experiments (###P b 0.001, compared with the control; **P b 0.01, compared with the MPP+ group).
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Fig. 4. Measurement of caspase-3 activity and morphological changes in MES23.5 cells. 300 μmol/L MPP+ treatment increased the activity of caspase-3 compared with that of the control, and Ndfip1 overexpression protected against MPP+-induced increase of caspase-3 activity. (A) Representatives of the fluorometric assay on the activity of caspase-3 in different groups. Data were presented as mean ± S.E.M. of 6 independent experiments and analyzed via Newman–Keuls post one-way ANOVA; fluorescence values of the control were set to 100% (###P b 0.001, compared with the control; ***P b 0.001, compared with the MPP+ group). (B) Representative photographs of Hoechst 33258 staining from vehicle, Ad.GFP infected cells, Ad.Ndfip1 infected cells incubated with MPP+ for 24 h, and MPP+ treatment cells. The nuclei of MPP+-treated cells appeared hypercondensed (brightly stained) and showed fragmentation of chromatin (indicated by arrows); however, Ndfip1 pretreatment significantly attenuated MPP+-induced nuclear condensation. Data were presented as mean ± S.E.M. of 6 independent experiments (###P b 0.001, compared with the control; **P b 0.01, compared with the MPP+ group).
In the present study, we demonstrated that Ndfip1 effectively protected MES23.5 cells from MPP+-induced neurotoxicity. MPTP, a common neurotoxin for inducing PD models after being transported
into the brain, is oxidized by monoamine oxidase-B (MAO-B) into MPP+ (Arora et al., 1990). This compound is actively transported into presynaptic dopaminergic nerve terminals by DAT, inhibiting complex
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I of the mitochondrial respiratory chain, and may cause the mitochondrial dysfunction and oxidative stress (Bezard et al., 1999; Cassarino et al., 1999; Kim et al., 2000; Zhang et al., 2011). Increasing evidence has proved that mitochondrial dysfunction and oxidative stress played an important role in the etiology of PD (Anantharam et al., 2007; Andersen, 2004; Dauer and Przedborski, 2003; Linazasoro, 2002). Mitochondrion is not only the main target of reactive oxygen species
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(ROS), but also the major site of ROS production as well as the primary target of oxidative molecular damage (Dauer and Przedborski, 2003). So the damaged mitochondrial membrane is implicated as key events in the pathogenic cascades. It breaks the balance of the Bcl/Bax system and leads to the release of proapoptotic proteins from the mitochondria (Oka et al., 2008; Zhang et al., 2011). As an anti-apoptotic member of the Bcl-2 family, Bcl-2 can bind Bax to form Bcl-2/Bax heterodimers, thereby
Fig. 5. Ndfip1 overexpression induced a decrease in DAT protein levels in MES23.5 cells. (A) Decreased protein levels of DAT were observed in the Ad.Ndfip1 group compared with the Ad.GFP group and the control. β-Actin was used as a loading control. Data were presented as mean ± S.E.M. of 6 independent experiments (#P b 0.05, compared with the Ad.GFP group and the control). (B) Immunocytochemistry with anti-DAT antibodies indicate basal levels of DAT expression (red fluorescence) in cultured MES23.5 cells and Ad.GFP infected cells, but down-regulation (arrow heads) when exposed to Ad.Ndfip1 for 48 h.
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Fig. 6. Degradation of DAT may be regulated by proteasomal pathways. The down-regulation of DAT induced by Ad.Ndfip1 was reversed by treatment with the proteasome inhibitor MG132 in MES23.5 cells. β-Actin was used as a loading control. (A) Western blots of DAT expression in MES23.5 cells of each indicated experimental group. (B) Statistical analysis. Data were presented as mean ± S.E.M. of 6 independent experiments (#P b 0.05, *P b 0.05, compared with the control, MG132 + Ad.Ndfip1 and MG132 + chlor + Ad.Ndfip1 groups).
attenuating the pro-apoptotic effect of Bax (Oltvai et al., 1993). Among proapoptotic proteins, the efflux of cytochrome c is thought to be a key event in the subsequent apoptotic processes. A subclass of the Bcl-2 family proteins that includes Bak and Bax regulates the efflux of cytochrome c by redistributing to the mitochondria, resulting in the permeabilization of the outer mitochondrial membrane and cytochrome c escaping to the cytoplasm (Bouchier-Hayes et al., 2005; Er et al., 2006; Scorrano and Korsmeyer, 2003). Our study showed that overexpression of Ndfip1 could partially inhibit MPP+-induced dysfunction of the Bcl/Bax system and then inhibited cytochrome c release during apoptosis. The release of cytochrome c from mitochondria which promotes massive activation of executioner caspase-3 is a crucial checkpoint in cell commitment to apoptosis (Danial and Korsmeyer, 2004; Enari et al., 1998; Galluzzi et al., 2012; Thornberry and Lazebnik, 1998). In this study, we confirmed that Ndfip1 could significantly decrease caspase-3 activation and nuclear morphological changes induced by MPP+. Evidence showed that MPP+-induced cell death could be through a caspase-3 dependent or independent pathway (Kaul et al., 2003; Chu et al., 2005). Kaul et al. proved that caspase-3 activation was involved in the apoptosis in rat mesencephalic dopaminergic N27 cells. Their results showed MPP+ (300 μM) caused a 115% increase in DNA fragmentation. Caspase-3 specific inhibitor Z-DEVD-fmk and broad-spectrum caspase inhibitor Z-VAD-fmk could inhibit an MPP+induced increase in DNA fragmentation (Kaul et al., 2003). Another study showed that AIF nuclear translocation was involved in MPP+induced apoptosis, and that this process is independent of caspase-3. In their study, 5 μM MPP+ was used (Chu et al., 2005). Although the precise mechanisms underlying this discrepancy is not clear, one possibility is that different concentrations of MPP+ might lead to different types of apoptosis. In our study, 300 μM MPP+ was used, and we also observed that 300 μM MPP+ induced a significant increase in caspase-3 activity and cytochrome c release which is consistent with the results of Kaul et al. This made the possibility that in our experiment, the caspase-3 dependent
pathway was involved in MPP+-induced apoptosis in MES23.5 dopaminergic cells. However, the mechanisms underlying the neuroprotective effects of Ndfip1 have not been fully elucidated. DAT is located on the cell membrane of dopaminergic neurons, and exerts its function by rapidly taking up dopamine from the extracellular space into the presynaptic neuron (Masoud et al., 2015). DAT provides not only an integral component of dopaminergic neurotransmission but also a molecular gateway for the uptake of some neurotoxins such as MPP+ (Li et al., 2006). The importance of DAT for the selectivity of MPP+ toxicity in dopaminergic neurons has been demonstrated by several in vivo and in vitro studies showing that DAT-blocking agents can reduce MPP+-mediated neurotoxicity (Bezard et al., 1999; Jaber et al., 1997; Ricaurte et al., 1985). DAT is a critical regulator of dopamine disposition in the brain and also a crucial determiner to the neurotoxicity of 6-OHDA (Van Kampen et al., 2000) and MPP+ (Bezard et al., 1999; Storch et al., 2004), which are two wellknown experimental neurotoxins in PD. Proper inhibition of DAT activity, therefore, is proposed to increase synaptic dopamine level and reduce DAT-dependent neurotoxicity, which may be beneficial to PD (Li et al., 2006). Therefore, we hypothesized that the neuroprotective property of Ndfip1 might be associated with the regulation of DAT. To further identify this hypothesis, we detected the protein levels of DAT in different experiment groups. Results showed that DAT expression in the Ad.Ndfip1 group decreased significantly compared with control. Therefore, it is possible that the decrease of DAT is associated with the ubiquitination and degradation of DAT in Ad.Ndfip1 infected MES23.5 cells. This was confirmed by our further results, which showed that the decrease of DAT induced by Ndfip1 was reversed in the presence of MG132, indicating that degradation of DAT by Ndfip1 is via the proteasome pathway. In conclusion, the results presented here give support to a sequence of events whereby Ndfip1 plays a key role in MPP+-induced apoptosis in MES23.5 dopaminergic cells. The simplified depiction of effects of Ndfip1 on MPP+-induced cytotoxicity was summarized in Fig. 7. In our present study, we demonstrated that overexpression of Ndfip1 caused DAT degradation, resulting in the decreased MPP+ influx and MPP+-induced neurotoxicity. Furthermore, this study provides further insight into the underlying mechanisms of Ndfip1, including inhibiting MPP+ uptake and ameliorating DAT-dependent neurotoxicity, which may be beneficial to PD.
Conflict of interest There is no conflict of interest for all authors.
Acknowledgments We thank Dr. Wei-dong Le who gave us the MES23.5 cell line. This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of the People's Republic of China (2011CB504102), the National Natural Science Foundation of China (81430024, 81100955, 31371081, 31271131, 81470059, and 81171208), the Department of Science and Technology of Shandong Province (ZR2015JL011), the Excellent Innovative Team of Shandong Province, the Taishan Scholars Construction Project, the Ministry of Education of China (20123706110002), the Shandong Provincial Education Department (J11LC05), and the Science and Technology Development Plan of Shandong Province (2011gsf11831).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2015.08.013.
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Fig. 7. A schematic diagram showing how Ndfip1 attenuated MPP+-induced cytotoxicity in dopaminergic neurons. The neuroprotective effect of Ndfip1 on MPP+-treated MES23.5 cells is mediated via reducing DAT levels, which resulted in decreased MPP+ uptake and the protection of cells from MPP+-induced neurotoxicity.
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