J Mol Neurosci (2015) 55:798–802 DOI 10.1007/s12031-014-0481-6

Bcl-2-Associated Athanogene 2 Prevents the Neurotoxicity of MPP+ via Interaction with DJ-1 Zhenhai Song & Shuo Xu & Bin Song & Qinghua Zhang

Received: 23 August 2014 / Accepted: 21 September 2014 / Published online: 20 January 2015 # Springer Science+Business Media New York 2015

Abstract Bcl-2-associated athanogene 2 (BAG2) is an important member in the BAG family which is characterized by their property of interaction with a variety of partners involved in modulating the proliferation/death balance. The role of BAG family proteins in Parkinson’s disease (PD) has not been elucidated. In this study, we demonstrated that overexpressing BAG2 ameliorates the effects of 1-methyl-4phenylpyridinium (MPP+) in mitochondrial membrane potential (MMP) collapse, reactive oxygen species (ROS) generation, and mitochondrial release of cytochrome C. However, knockdown of DJ-1 abolished the neuroprotective effects of BAG2 against MPP+-induced neuronal toxicity. With coimmunoprecipitation and pulldown experiments, we present a direct physical interaction between BAG2 and DJ-1. Coexpression of BAG-2 together with DJ-1 increased the ratio of dimer/monomer staining intensity at the basal level. Our results indicated that MPP+ treatment leads to the disassociation of the homodimer of DJ-1. When BAG2 was overexpressed in cells, the homodimer of DJ-1 was increased. Importantly, BAG2 prevents MPP+-induced monomerization of DJ-1. Thus, our data showed that the neuroprotective effects of BAG2 are mediated by its interaction with DJ-1, strengthening the link between the BAG proteins and PD-related neurodegeneration. Z. Song : Q. Zhang (*) Department of Neurology, Shandong Provincial Hospital, No 324 Jingwuweiqi road, Ji’nan City, Shandong Province 250021, China e-mail: [email protected] S. Xu Department of Radiology, Shandong Provincial Hospital, Ji’nan City, China B. Song Emergency Department, Shandong Provincial Hospital, Ji’nan City, China

Keywords Parkinson’s disease . Bcl-2-associated athanogene 2 . DJ-1 . MPP+ . Mitochondrial dysfunction

Introduction Parkinson’s disease (PD) is a chronic and progressive neurodegenerative movement disorder. The majority of PD cases are sporadic with obscure etiology. However, sequence or copy number variants in several genes that result in familial forms of PD have been identified (Bekris et al. 2010). Among them, the PARK7 locus corresponds to the gene which encodes DJ-1 protein (Bonifati et al. 2003). DJ-1 is a small 20kD protein described as highly conserved across diverse species. DJ-1 is an oxidative stress response protein, which can eliminate hydrogen peroxide and hence protect cells from cytotoxicity caused by oxidative stress and apoptosis. It has been reported that DJ-1 deficiency led to altered sensitivity to oxidative stressors (Meulener et al. 2005). In vivo studies indicated that mice lacking DJ-1 displayed nigrostriatal dopaminergic deficits and hypokinesia (Goldberg et al. 2005). Crystal structure analysis has shown that homodimer formation is critical for DJ-1 activities (Honbou et al. 2003). Familial PD-associated DJ-1 L166P mutation leads to the impairment of DJ-1 homodimerization and increased degradation rate, which induces the loss of function of DJ-1 in antioxidative stress, maintaining mitochondrial function, and antiapoptosis (Maita et al. 2013). 1-Methyl-4-phenylpyridinium (MPP+), a major product of the oxidation of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been widely used to model PD in various studies. DJ-1 has a role in protecting cell death against MPP+ treatment (Taira et al. 2004). The Bcl-2 associated athanogene (BAG) family is a multifunctional group that can function as the chaperone modulators. The BAG family members contain the BAG domain

J Mol Neurosci (2015) 55:798–802

(BD), which mediates direct interaction with the adenosine triphosphatase (ATPase) domain of Hsp70/Hsc70 molecular chaperones (Kabbage and Dickman 2008). Recently, BAG2 has been shown to inhibit ubiquitylation of misfolded proteins by CHIP by a co-chaperone-dependent regulatory mechanism (Arndt et al. 2005), suggesting that BAG2 plays an endogenous role in regulating proteasome-mediated degradation of chaperone substrates. Most of the BAG family proteins play important roles in the regulation of apoptosis, cell survival, and stress response. However, the effects of BAG2 in regulating the neurotoxicity of MPP+ in PD are still unknown. In this study, we reported that BAG2 is able to protect neuronal cells from MPP+-induced oxidative stress, mitochondrial dysfunction, and apoptosis. Mechanistically, the neuroprotective effects of BAG2 depend on its interaction with DJ-1. In addition, BAG2 promotes the dimerization of DJ-1.

Materials and Methods Cell Culture and Transfection Human neuroblastoma SH-SY5Y cells were cultured in Eagle’s minimum essential medium (EMEM) and Ham’s F12 medium containing fetal bovine serum (FBS) (10 %) and penicillin and streptomycin (1 %; Invitrogen, USA). Human DJ-1 and a nonspecific control small interfering RNA (siRNA) were acquired from the Thermo Fisher Scientific, USA. siRNA was transiently transfected into cells using Lipofectamine RNAiMAX (Invitrogen).

Preparation of Protein Extracts and Western Blot Analysis Cell lysates were prepared using RIPA buffer supplemented with complete protease inhibitor cocktail (Roche) for 15 min at 4 °C, followed by a 10-min centrifugation at 13,000 rpm at 4 °C. Subcellular fractionation was performed using ProteoExtract Subcellular Proteome Extraction kit (EMD). Lysates were subjected to the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10 %) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), as previously described (Sheng et al. 2012). Membranes were blocked for 1 h at room temperature (RT) in 5 % fat-free milk powder in tris-buffered saline with tween (TBST), followed by sequentially incubated with primary antibodies for 3 h and horseradish peroxidaseconjugated secondary antibodies for 2 h at room temperature (RT). Bands were visualized using enhanced chemiluminescence (ECL) reagent (Santa Cruz, USA) according to the manufacturer’s protocol.

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Co-immunoprecipitation (Co-IP) Cells were cultured and transfected in 6-well plates. Then, cells were lysed by cell lysis buffer containing 50 mM TrisHCl (pH 7.5), 150 mM NaCl, 0.1 % Nonidet P-40, 1 mM dithiothreitol, protease inhibitor mixture, sodium orthovanadate, and sodium fluoride. A total of 5 μg of antiBAG2 antibody (Abcam, USA) was incubated with 1 mg of whole cell lysates to form the antigen-antibody complex overnight at 4 °C. The formed antigen-antibody complex was immunoprecipitated with protein A/G-agarose beads (Santa Cruz) for 2 h at 4 °C. Then, the beads were eluted in SDS sample buffer and the protein solution was subjected to Western blot analysis. Determination of Intracellular Reactive Oxygen Species (ROS) The generation of intracellular ROS in SY-SH5Y cells was investigated using the 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). After being incubated with the indicated concentrations of MPP+, SY-SH5Y cells were rinsed with Krebs’ ringer solution (100 mM NaCl, 2.6 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 11 mM glucose). Then, cells were loaded with 10 μM DCFH-DA and incubated at 37 °C for 1 h. Fluorescence signals were recorded by using a fluorescence microscope (Sheng et al. 2009). The average fluorescent density of intracellular areas was calculated by Image-Pro Plus to index the ROS level. Measurement of Mitochondrial Membrane Potential (MMP) MMP in SY-SH5Y cells was examined by using the fluorescence dye tetramethylrhodamine methyl ester (TMRM) (Invitrogen, USA) according to the manual instructions. Briefly, after indicated incubation and transfection, SYSH5Y cells were loaded with 20 nmol/L of TMRM and incubated for 1 h at 37 °C. Fluorescence signals were recorded by using a fluorescence microscope. The average fluorescent density of intracellular areas was calculated by Image-Pro Plus to index MMP. Cytochrome C Assay After indicated treatment and transfection, cells were washed with cold PBS three times. Then, cells were resuspended and incubated on ice for 30 min in ice-cold cytosolic extraction buffer (250 mM sucrose, 20 mM Hepes pH 7.4, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM pepstatin A, 10 mg/mL leupeptin, and 2 mg/mL aprotinin) followed by gently homogenized with a glass Dounce homogenizer and a tight-fitting pestle. After

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centrifugation at 2500 rpm at 4 °C for 10 min, the supernatant was collected and then centrifuged at 13,000 rpm at 4 °C for 30 min to yield a cytosolic extract and the supernatant. Cytochrome C levels in the cytosolic extract were determined by Western blot analysis. Statistical Analysis All quantitative results are shown as mean±standard error of the mean (SEM) from at least three experiments. One-way analysis of variance (ANOVA) was used to assess statistical significance of differences among treatment groups. P

Bcl-2-associated athanogene 2 prevents the neurotoxicity of MPP+ via interaction with DJ-1.

Bcl-2-associated athanogene 2 (BAG2) is an important member in the BAG family which is characterized by their property of interaction with a variety o...
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