Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Analytical Biochemistry 407 (2010) 205–210

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Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death Il Sang Yoon, Qingyan Au, Jack R. Barber, Shi Chung Ng, Bin Zhang ⇑ Department of Biology, CytRx Corporation, 3030 Bunker Hill Street, Suite 101, San Diego, CA 92109, USA

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Article history: Received 3 June 2010 Received in revised form 26 July 2010 Accepted 4 August 2010 Available online 10 August 2010 Keywords: Rotenone High-throughput screening High-content analysis Small molecule Parkinson’s disease Carnosic acid

a b s t r a c t Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by a-synucleinpositive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a highthroughput screening format with Z0 factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD. Ó 2010 Elsevier Inc. All rights reserved.

Parkinson’s disease (PD)1 is the second most common age-related neurodegenerative disorder that affects approximately 1 million persons in the United States [1]. PD is clinically characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta coupled with juxtanuclear ubiquitinated proteinaceous inclusions known as Lewy bodies [2,3]. Although research efforts led to the hypothesis that misfolding of proteins and dysfunction of the ubiquitin-proteasome pathway are critical to PD pathogenesis [3], we still have limited understanding of the key molecular targets or pathways that provoke neurodegeneration. Mitochondrial dysfunction, protein phosphorylation, and oxidative stress may also contribute to the progressive accumulation of misfolded proteins [2,4,5], which eventually cause deleterious effects in vulnerable dopaminergic neurons. The current treatment is to use the dopamine precursor levodopa primarily as a replacement strategy, with serious side effects occurring in a majority of patients, such as dyskinesia (involuntary movements) and fluctuations in motor response [3]. Therefore, discovery of small molecules for a more effective therapy is valuable for clinical therapeutics in PD patients. Several experimental systems have been previously reported to study the molecular mechanisms in PD pathogenesis, including gene-based models (also referred to as ‘‘etiologic models”) and tox-

⇑ Corresponding author. Fax: +1 858 273 2697. E-mail address: [email protected] (B. Zhang). Abbreviations used: FBS, fetal bovine serum; MEM, minimum essential medium; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PGA1, prostaglandins A1; SDS, sodium dodecyl sulfate. 1

0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.08.011

in-based models (also referred to as ‘‘pathologic models”) [3,6]. Much progress has been made in recent years in toxin-induced models, based on the proposition that dopaminergic neurons have a common death cascade which can be mimicked by insults of small-molecule toxins [3]. Several neurotoxins are widely used to create in vitro and in vivo models for dopaminergic neurodegeneration, including 6-hydroxydopamine, MPTP, paraquat, and rotenone [3,6,7]. Rotenone is a classic mitochondrial complex I inhibitor, which activates the apoptosis pathway through release of reactive oxygen species [7]. Greenamyre and colleagues reported selective degeneration of nigrostriatal dopaminergic neurons accompanied by a-synuclein-positive inclusions after administration to rats with low-dose intravenous rotenone [8]. Reports from other studies also demonstrated that rotenone inhibited complex I uniformly throughout the brain in rats with selective cell loss in the nigrostriatal dopaminergic system [9,10]. There were also several reports in cell-based assays that link a-synuclein aggregation to neuronal cell death [11–13]. Thus, rotenoneinduced cell death provides an excellent model system for small-molecule drug discovery research for PD. Although there is a clear demand to develop small molecules that protect neuronal cells from rotenone or other toxin-induced death, few reports are available on screening for cytoprotective agents, particularly in a high-throughput format. Previously, cyclopentenone prostaglandins A1 (PGA1) was reported to exert neuroprotective effects in rotenone-treated SH-SY5Y cells, evidently through the induction of HSP70 and HSP27 and attenuation of caspase-3 activation [14]. The effective dose of PGA1 was relatively

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HTS assay for cytoprotective agents in rotenone-induced cell death / I.S. Yoon et al. / Anal. Biochem. 407 (2010) 205–210

high (20–80 lM) with cytotoxicity observed at a concentration of 160 lM [14]. In an effort to identify more potent compounds, we developed a cell-based system to screen for additional small-molecule cytoprotective agents that can reverse rotenone-induced cytotoxicity. Our results suggested that this cell-based assay is sensitive, robust, and amenable for large-scale compound screening. The initial screen led to the identification of carnosic acid, a catechol ring-containing compound known to be neuroprotecive [15]. Further analysis of the molecular mechanisms confirmed that carnosic acid significantly induced the expression levels of HSP70 in a dose-dependent manner. In addition, carnosic acid also exhibited an inhibitory function to caspase-3 activation.

Invitrogen. RIPA cell lysis buffer was purchased form Sigma. Complete protease inhibitor cocktail tablets were from Roche (Palo Alto, CA).

Materials and methods

MTS assay in rotenone-treated SK-N-SH cells

Reagents and compound library

SK-N-SH neuroblastoma cells (passage number 3–6) were seeded in 96-well plates at a density of 8000 cells/well in 120 ll of rotenone assay media (MEM/0.1% FBS), and cultured overnight (Day 1). Using the Sciclone liquid handling system, 30 ll of fresh assay media containing various concentrations of CytRx compounds and rotenone (final concentration: 200 nM) was added. The cells were cultured for 48 h (Day 3). On Day 4, cell viability was measured using an MTS assay kit (Promega, Madison, WI). Cellular protection from rotenone stress is defined as follows:

A chemical library composed of 1120 bioactive compounds was purchased from MicroSource Discovery Systems Inc. (Gaylordsville, CT). Rotenone was purchased from Sigma (St. Louis, MO) and dissolved in DMSO to make a 1 mM stock solution. Tetrazolium salt powder (MTS) was purchased from Promega (Madison, WI). Phenazine methosulfate was ordered from Fluka (St. Louis, MO). Sodium dodecyl sulfate (SDS) was purchased from Bio-Rad (Hercules, CA). DMSO was from Sigma (St. Louis, MO). DAPI, Hoechst 33342, propidium iodide, and Yopro were purchased from Invitrogen (Carlsbad, CA). Paraformaldehyde was purchased from EMS (Hatfield, PA). Antibodies against HSP70 (Catalog No. SPA901) and b-actin (Catalog No. 4967) were purchased from Assay Design, Inc. (Ann Arbor, MI) and Cell Signaling Technology (Danvers, MA), respectively. FITC-labeled anti-mouse antibodies (Catalog No. 115-096-146) were obtained from Jackson ImmunoResearch (West Grove, PA). Caspase-3 colorimetric assay kit was purchased from R&D Systems, Inc. (Minneapolis, MN). Minimum essential medium (MEM) was purchased from Mediatech Inc. (Manassas, VA), and fetal bovine serum (FBS) was ordered from

SK-N-SH cells were purchased from ATCC (Manassas, VA). For growth and maintenance of the cell line, cells were cultured in MEM containing 10% FBS and 4 mM glutamine at 37 °C. For rotenone treatment experiments, 0.1% FBS was used instead of 10% FBS since it has been reported previously that FBS quenches rotenone in the culture media [16].

Increase of cell viability % ¼ ðMTScompound  MTSrotenone Þ=MTSrotenone  100 ð%Þ:

Sciclone-based high-throughput screening SK-N-SH cells were seeded on 96-well plates 18 h before compound treatment as described above. The 1120 compounds from the Microsource library were added with a final concentration of 2 lM along with 200 nM rotenone in the assay media. Samples treated with 1 lM CYT600 [17] in column 1 were used as positive

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Fig.1. Optimization of rotenone treatment conditions in SK-N-SH cells for compound screening. (A) Time-dependent cell killing up to 48 h by rotenone treatment at 37 °C. SKN-SH cells were prepared in a 96-well plate in assay media containing 200 nM rotenone. At each time point, cells were collected for MTS assay to measure cell viability as described under Materials and methods. Rotenone treatment for 24 and 48 h significantly lowered cell viability. Error bar represents mean ± standard deviation (n = 24). (B) Dose-dependent cell killing of rotenone treatment and protection by CYT600. SK-N-SH cells were treated with increasing doses of rotenone (R) for 48 h along with DMSOtreated controls. CYT600 at two different doses (0.6 and 1.2 lM) showed significant cytoprotective effects compared with rotenone-alone treated groups. Error bar represents mean ± standard deviation (n = 12).

HTS assay for cytoprotective agents in rotenone-induced cell death / I.S. Yoon et al. / Anal. Biochem. 407 (2010) 205–210

controls. Samples treated with 0.33% DMSO (v/v) in column 12 were used as negative controls. Cells were further cultured for 48 h followed by MTS assay for evaluation of cell viability. Z0 value for CYT600 was calculated with the following formula:

Z 0 ¼ 1  3  ½ðSDCYT600 þ SDDMSO Þ= j averageCYT600  averageDMSO j:

Western blot analysis SK-N-SH cells were lysed with RIPA buffer containing protease inhibitor cocktail after rotenone treatment. After centrifugation at 14,000 rpm for 10 min, the supernatants were mixed with LDS sample buffer (Invitrogen) followed by heat denaturation at 75 °C for 10 min. The prepared samples were subjected to the SDS–PAGE using Novex 4–12% Bis–Tris gel (Invitrogen). Western blot images were obtained with HSP70 primary antibody, IRDye 680-conjugated secondary antibody and the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Fluorescence intensities of HSP70 band and b-actin band were analyzed using Odyssey infrared imaging software. Percentage of fluorescence intensity ratio was calculated using the following formula:

Fluorescence intensity ratio % ¼ intensity of HSP70 band= intensity of b-actin band  100 ð%Þ

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High-content image-based method For the fixed cell imaging assay, SK-N-SH cells were seeded in Costar 96-well assay plates (Costar 3904, Corning Inc., NY) at a density of 12,000 cells/well approximately 16–24 h before compound treatment. Forty-five microliters of 16% paraformaldehyde was mixed with the culture medium (total volume at 150 ll) with a final concentration of 4%. The plates were incubated at room temperature for 30 min before washing with PBS. Permeabilization of cellular membrane was achieved using 0.2% Triton X-100 in PBS for 30 min. After washing three times with PBS, 80 ll of 5% FBS/ PBS was applied to the plates at room temperature for 1 h. For antibody staining, a 1:500 dilution of anti-HSP70 antibody in 1% FBS/ PBS was added to the plates. The plates were incubated at room temperature for 2 h or at 4 °C overnight. Finally, a mixture of DAPIand FITC-labeled secondary antibodies was added into the plates at a final concentration of 1:5000 (for DAPI at 5 mg/ml), 1:500 (for FITC labeled anti-rabbit secondary antibodies). After 1 h at room temperature, the plates were washed with PBS and stored at 4 °C. Image acquisition and analysis were performed using an INcell 1000 with a 10 objective (GE Healthcare, Piscataway, NJ). The setting for image acquisition was three images captured per well at 500 ms for DAPI and 100 ms for FITC. The threshold for HSP70 positively stained cells was determined with gating values

SK-N-SH cells were seeded at 8,000 cell per well in a 96-well plate overnight

Addition of 200 nM rotenone & 2 μM Microsource compounds in duplicate. 1 μM CYT600 & DMSO were used as positive & negative control, respectively. All concentrations are final.

Cell culture was allowed to incubate for 48 hours followed by MTS assay

Compounds with cell viability>20% were selected for EC50 evaluation

Confirmed hits were further evaluated with western blot and HCS assays Compound number 0

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Fig.2. Sciclone-based high-throughput screening and confirmation of two screening hits. (A) Schematic flow chart of our screen. (B) High-throughput screening of Microsource compound library () at a final concentration of 2 lM. A 1 lM CYT600 (j) was used as a positive control. Four compounds (labeled with 1–4) were identified with more than 20% increased cell viability as compared to 0.33% DMSO-treated controls. Compounds 1 and 2 are carnosic acid and isoliquiritigenin, respectively. (C) Chemical structures of carnosic acid and isoliquiritigenin.

HTS assay for cytoprotective agents in rotenone-induced cell death / I.S. Yoon et al. / Anal. Biochem. 407 (2010) 205–210

equivalent to the average of DMSO-treated samples plus 2 or more standard deviations. For the live cell imaging assay, SK-N-SH cells were labeled with 0.5 lg/ml Hoechst 33342, 1 lg/ml propidium iodide (PI), and 0.5 lg/ml Yopro for 15 min at 37 °C. Image acquisition was performed with INcell 1000 with a setting of 3 images per well at 100 ms for Hoechst and 50 ms for PI or Yopro. Yopro intensity and Hoechst nuclear intensity CV (coefficient of variation) were chosen for determination of apoptotic cells. The thresholds for apoptotic cells were determined with gating values similar to the previous description. Caspase-3 activity assay Caspase-3 colorimetric assay was performed according to the manufacturer’s manual (R&D Systems, Minneapolis, MN). Briefly, SK-N-SH cells were plated in a 60-mm dish (4  106 cells per dish) in 10% FBS/MEM culture media. On the following day, cell culture media were replaced with rotenone assay media (0.1% FBS/MEM) containing CytRx compound, and further cultured for 24 or 48 h. The cells were collected and lysed by lysis buffer followed by centrifugation. The supernatant (50 ll) was mixed with the same volume of 2 reaction buffer 3 and 5 ll of caspase-3 colorimetric substrate. The reaction mixture was incubated for 2 h at 37 °C. The absorbance at 405 nm was measured using Envision Excite (Perkin Elmer, Wellesley, MA).

Assay development in SK-N-SH We treated SK-N-SH cell, a human neuroblastoma cell line that possesses many features of dopaminergic neurons, with different concentrations of rotenone in a time frame up to 48 h. Our intention was to optimize the cell-based assay with a relative long-term rotenone treatment, mimicking the chronic onset of PD. It was previously reported that rotenone treatment can induce Lewy body-like aggregates in the cytoplasm [12]. Comparing to DMSO controls, we also observed more than 2-fold increase of ubiquitinated proteins when SK-N-SH cells were exposed to rotenone treatment (data not shown), suggesting a biologically relevant cell model system. As shown in Fig. 1A, 200 nM rotenone treatment for 48 h caused 50% cell loss as evaluated by MTS assay. Although more cell loss was observed when longer rotenone treatment times (>48 h) or higher concentrations (>1 lM) of rotenone were applied, we experienced poor reproducibility of assay with significantly larger CV. CYT600, an internal compound known to protect cells from rotenone-induced cytotoxicity [17], rescued more than 50% dead cells at 1.2 lM reproducibly (Fig. 1B). Therefore, the optimized assay condition was set at 200 nM rotenone treatment for 48 h with CYT600 as a positive control. Rotenone has a reactive ketone group. To rule out the possibility of potential intermolecular interaction

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Fig.3. Cytoprotective effects of carnosic acid were confirmed by a high-content image-based assay (A and B) and a caspase-3 biochemical assay (C). SK-N-SH cells were treated with 4 lM carnosic acid (CA) or 0.33% DMSO control in rotenone (R)-induced stress for 24 or 48 h. SK-N-SH cells with a value of Yopro intensity (A) or nuclear intensity CV (B) higher than the preset threshold were considered as positively stained apoptotic cells. The percentage of positively stained cells was determined using Multi Target Analysis module (GE Healthcare). (C) Carnosic acid also lowered caspase-3 activity in rotenone-treated cells. # Statistical analysis revealed a significant increase of parameters measured in (A–C) after treatment of 200 nM rotenone. *Statistical analysis revealed a significant decrease (cytoprotective effects) of parameters measured in A–C when carnosic acid was applied. Error bar represents mean ± standard deviation (n = 3).

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HTS assay for cytoprotective agents in rotenone-induced cell death / I.S. Yoon et al. / Anal. Biochem. 407 (2010) 205–210

Fig. 2A shows the screening flow chart. The primary screening results are shown in Fig. 2B with 1120 bioactive compounds from Microsource at 2 lM. CYT600 at 1 lM showed an average value of 20.56 ± 2.78% increased viable cells with Z0 factor of 0.56, suggesting a reliable assay for high-throughput screening. From four initial hits identified with this assay, carnosic acid and isoliquiritigenin were further confirmed in a dose-dependent study (Fig. 2C). Among them, carnosic acid, a naturally occurring catechol-type electrophilic compound, demonstrated the most potent cytoprotective effect with an EC50 value of 2.4 lM in rescuing rotenoneinduced cytotoxicity. Isoliquiritigenin showed significantly weaker activity in this assay. In the meantime, LC-MS data confirmed that carnosic acid does not interact with rotenone (data not shown). Treatment of carnosic acid alone did not cause cytotoxicity in SK-N-SH cells at concentrations up to 25 lM (data not shown). Therefore, a series of follow-up experiments were performed with carnosic acid for the potential underlying mechanisms of cytoprotection in rotenone-treated cells.

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Confirmation of cytoprotective effectives by carnosic acid To further validate the cytoprotective effects of carnosic acid, we performed a live-cell image-based high-content analysis. A multiplexing image-based approach allows sensitive segmentation and quantification of apoptotic cells featured as positively labeled with Yopro dye, which is only fluorescent (green) in cells that undergo apoptosis [18]. In addition, apoptotic fragmented nuclei can be also monitored by evaluation of coefficient of variation of Hoechst nuclear staining [19,20]. As shown in Fig. 3A and B, a significant reduction of apoptotic cells, as measured by both Yopro intensity and Hoechst nuclear intensity CV, was recorded 24 and 48 h after 4 lM carnosic acid treatment. Since caspase-3, a final executioner of apoptosis, was reported to be involved in rotenone-induced cell death [21], we measured caspase-3 activity in rotenone-treated SK-N-SH cells in the presence or absence of carnosic acid. More than 2-fold elevation of caspase-3 activity was observed in rotenone-treated samples, demonstrating activation of the apoptosis pathway (Fig. 3C). Consistent with our morphology study results, carnosic acid treatment significantly inhibits activation of caspase-3 at both 24- and 48-h treatment points (Fig. 3C). Therefore, carnosic acid is a genuine and potent cytoprotective agent against rotenone-induced cellular stress.

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between rotenone and any tested compounds that may compromise cytotoxicity of rotenone, we routinely examined the mixture of rotenone and selected compounds with LC-MS and excluded compounds that interact with rotenone.

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** p <0.01 Fig.4. Effects of carnosic acid (CA) treatment on HSP70 expression in rotenone (R)induced stress. (A) High-content analysis of expression levels of HSP70 in SK-N-SH cells. The percentage of positively stained HSP70 cells was determined by MTA with a preset threshold. (B) Western blot confirmation of increased HSP70 levels in 4 lM carnosic acid-treated samples. Quantification of Western blot results was performed using Odyssey infrared imaging software according to the manufacturer’s instructions. Percentage of fluorescence intensity ratio was calculated as described under Materials and methods. Each data point is the mean of four determinations. Student’s t test revealed significant increase in HSP70 level after carnosic acid treatment compared with rotenone-only treatment group.

can induce HSP70 chaperone protein for rescuing cytotoxicity in the rotenone model system.

Discussion Carnosic acid induces expression of HSP70 in rotenone-treated cells PGA1, a proven inducer of HSP70, can reverse the cytotoxicity effects induced by rotenone [14]. Many reports have shown that the induction of HSP70 protects cells from apoptotic death and may even clear intracellular inclusions in neuronal cells [6]. To investigate the underlying cytoprotection mechanisms of carnosic acid, we performed multiplexing high-content analysis of carnosic acid-treated samples for measurements of HSP70. As shown in Fig. 4A, carnosic acid significantly induced the percentage of HSP70 positively stained cells compared to DMSO-treated controls. Conventional Western blot assay was also performed with more than 3-fold increase of expression levels of HSP70 recorded in carnosic acid-treated samples in comparison to the samples treated with rotenone only (Fig. 4B). Thus, similar to PGA1, carnosic acid

Discovery of small-molecule neuroprotective agents presents a great challenge for neuroscience. Although reports of agents offering neuroprotection have begun to appear in the literature [22], there are few available biological relevant assays that can systematically screen for neuroprotective agents. We have developed and validated a high-throughput assay for screening compounds rescuing rotenone-induced neurotoxicity. Carnosic acid was identified with single-digit micromolar potency in the cytoprotection of rotenone-treated SK-N-SH cells. Therefore, a large-scale screening with this assay platform is warranted for small-molecule lead compounds for PD therapeutics. With some optimizations, this assay platform can be generally applied to other complex I inhibitors (MPTP and paraquat) or other neurotoxins for screening of cytoprotective agents.

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HTS assay for cytoprotective agents in rotenone-induced cell death / I.S. Yoon et al. / Anal. Biochem. 407 (2010) 205–210

Molecular chaperones such as HSP70 are the major defense molecules against environmental insults such as high temperature, oxidative exposure, and chemical toxins that result in protein misfolding and denaturation [23,24]. It was previously reported that HSP70 and HSP27 are highly inducible in glial cells and neurons following stress [25]. An intra- or extracellular accumulation of misfolded, aggregated, and ubiquitinated proteins was found in PD (Lewy bodies) and other neurodegenerative diseases, suggesting an underlying incapacity of molecular chaperone machinery in preventing the accumulation of misfolded proteins [24]. Auluck et al. [26] reported convincing results that overexpressed HSP70 suppresses the loss of dopaminergic neurons in a Drosophila model of PD. The authors also demonstrated that a small percentage of Lewy bodies in PD brain tissue react with HSP70 and HSP40 antibodies, suggesting that increased levels of chaperones may provide an effective therapeutic. In addition, overexpression of HSP70 in mice that are transgenic for a-synuclein significantly reduces the formation of detergent-insoluble material by a-synuclein [27]. Our results from high-content image-based assay and Western blot confirmed that carnosic acid can profoundly induce the expression of HSP70 (Fig. 4). The resulting high levels of HSP70 expression may reduce the toxicity caused by protein inclusions and more importantly, soluble misfolded proteins. The induced HSP70 by carnosic acid prevents or inhibits apoptosis and rescues rotenone-based cytotoxicity (Fig. 3). It was recently reported that carnosic acid may have multiple cellular targets in neuroprotection [28]. For example, carnosic acid activates the Keap1/Nrf2 transcription pathway by binding to specific Keap1 cysteine residues, thus protecting neurons from oxidative stress and excitotoxicity [15]. Our initial results indicated that heat shock factor 1 (HSF1), the master transcription factor controlling expression of HSPs, may be responsible for induction of HSP70 expression in the rotenone stress system [29]. On the other hand, the biological profiles of carnosic acid are similar to PGA1, which was previously reported to inhibit nuclear translocation of NF-jB and subsequently block programmed cell death [14]. Therefore, further study on cytoprotective mechanisms of carnosic acid is necessary to facilitate structure activity relationship studies for target-based therapy. Acknowledgments The authors thank everyone in Biology Department and Chemistry Department of CytRx Corporation for their strong support during the project. References [1] C.W. Olanow, W.G. Tatton, Etiology and pathogenesis of Parkinson’s disease, Annu. Rev. Neurosci. 22 (1999) 123–144. [2] A. Wood-Kaczmar, S. Gandhi, N.W. Wood, Understanding the molecular causes of Parkinson’s disease, Trends Mol. Med. 12 (2006) 521–528. [3] W. Dauer, S. Przedborski, Parkinson’s disease: mechanisms and models, Neuron 39 (2003) 889–909. [4] P.M. Abou-Sleiman, M.M. Muqit, N.W. Wood, Expanding insights of mitochondrial dysfunction in Parkinson’s disease, Nat. Rev. Neurosci. 7 (2006) 207–219. [5] A. Reynolds, C. Laurie, R.L. Mosley, H.E. Gendelman, Oxidative stress and the pathogenesis of neurodegenerative disorders, Int. Rev. Neurobiol. 82 (2007) 297–325. [6] D.W. Ethell, Q. Fei, Parkinsons-linked genes and toxins that impact neuronal cell death through the Bcl-2 family, Antioxid. Redox Signal. 11 (2008) 529–540.

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