JOURNAL OF NEUROCHEMISTRY

| 2014 | 130 | 280–290

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doi: 10.1111/jnc.12629

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*Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA †Kobe Creative Center, Senju Pharmaceutical Corporation Limited, Kobe, Japan

Abstract Complex pathophysiology of Parkinson’s disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinson’s disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. To unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes, respectively, and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP+ and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP+ and rotenone dose-dependently elevated the levels of intracellular free Ca2+ and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP+ and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species more profoundly in SH-

SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators cyclooxygenase-2 (Cox-2 and cleaved p10 fragment of caspase-1) were up-regulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP+ or rotenone-induced reactive oxygen species generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1–3 h after exposure to MPP+ or rotenone. Taken together, these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. Keywords: calpain, cell viability, experimental parkinsonism, inflammation, neuroprotection, oxidative stress. J. Neurochem. (2014) 130, 280–290.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by impaired motor functions, which are predominantly associated with degeneration of nigral dopaminergic neurons tyrosine hydroxylase (TH positive) and reduced striatal dopamine (DA) neurotransmission (Hornykiewicz 2008). Nevertheless, the complex pathophysiology of PD is extended much beyond the selective nigrostriatal degeneration to several extranigral and extrastriatal regions (Olanow et al. 2011; Giza et al. 2012). The spinal cord is one such site. Its involvement in PD pathology is implicated based on the findings of significant degeneration of spinal neurons in human PD, postmortem PD spinal cord and animal models of experimental PD (Braak et al. 2007; Knaryan et al. 2011; Vivacqua et al. 2011, 2012; Del Tredici and Braak 2012; Samantaray et al. 2013a). We previously reported degeneration of cholinergic choline acetyltransferase positive (ChAT) spinal motoneurons in

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- and rotenoneinduced experimental parkinsonism in mice and rats, respectively (Ray et al. 2000; Chera et al. 2002, 2004; Samantaray et al. 2007, 2008a), and in post-mortem spinal cord specimens of human PD (Samantaray et al. 2013a).

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Received September 18, 2013; revised manuscript received November 11, 2013; accepted November 26, 2013. Address correspondence and reprint requests to Naren L. Banik, Department of Neurosciences, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 309 CSB, MSC 606, Charleston, SC 29425, USA. E-mail: [email protected] 1 These authors contributed equally to this study. Abbreviations used: BDNF, brain-derived neurotrophic factor; ChAT, choline acetyltransferase; Cox-2, cyclooxygenase-2; DA, dopamine; DAT, DA transporter; DBH, DA b-hydroxylase; IR, immunoreactivity; PD, Parkinson’s disease; PMA, phorbol 12-myristate 13-acetate; RA, retinoic acid; ROS, reactive oxygen species; SBDP, spectrin breakdown products; TH, tyrosine hydroxylase.

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However, the selective mechanisms of such degeneration are not well understood. In vitro studies conducted in hybrid VSC 4.1 cells differentiated into cholinergic spinal motoneurons and exposed to MPP+ or rotenone showed that mitochondrial toxins cause specific intracellular damage in spinal motoneurons (Samantaray et al. 2011). The common underlying mechanisms of spinal cord motoneuron degeneration found in vivo and in vitro involve aberrant Ca2+ homeostasis, up-regulation and activation of Ca2+-dependent cysteine proteases calpain and caspase-3, and limited proteolysis of their intracellular substrates, including cytoskeletal protein such as a-spectrin (Samantaray et al. 2007, 2011). A key role for calpain up-regulation and activation in neuronal death in substantia nigra and locus coeruleus has been previously reported in PD (Mouatt-Prigent et al. 2000; Crocker et al. 2003). Dysregulation of calpain and the sole endogenous inhibitor calpastatin was found associated with degeneration of spinal motoneurons in post-mortem spinal cord of PD patients (Samantaray et al. 2013a) much like the findings in PD brain (Mouatt-Prigent et al. 2000; Crocker et al. 2003). To this end, calpain inhibitors MDL-28170 and calpeptin tested in animal models of parkinsonism showed beneficial effects (Crocker et al. 2003; Samantaray et al. 2013b). Progression of PD also involves associated inflammatory responses, activation of astrocytes and microglia, generation of reactive oxygen species (ROS), which are known to be involved in degeneration of the dopaminergic neurons in PD (Teismann et al. 2003; Vijitruth et al. 2006; Roy et al. 2012). Involvement of calpain in inflammatory processes has been shown in neurodegenerative diseases, multiple sclerosis and studied in its animal model (Shields and Banik 1998; Shields et al. 1999). It is likely that calpain could be involved in inflammatory processes associated with PD pathology as well thus, validating calpain inhibition as an interventional target. Currently, there is no cure for PD; the widely accepted L-3,4-dihydroxyphenylalanine treatment has many side effects and it does not block the disease progression. Therefore, there is an urgent need to develop new therapeutic strategies, which can help to protect discrete cell types involved in PD, including nigral dopaminergic and spinal cholinergic motoneurons. Although inhibition of calpain by calpeptin, a cell permeable peptide aldehyde inhibitor, substantially attenuated MPP+- and rotenone-induced toxicity in vitro in spinal motoneurons (Samantaray et al. 2011) yet, calpeptin is limited by its lack of water solubility. To this end, a new water-soluble calpain inhibitor SNJ-1945 (amphipathic ketoamide) developed by Senju Pharmaceutical Co. Ltd. (Kobe, Japan) may serve as a better alternative. SNJ-1945 has been suggested as a novel potential drug for the treatment of diseases that share common etiology and are associated with overt calpain activation and proteolysis of its intracellular substrates; such as neuroprotection against retinal

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degeneration (Ma et al. 2009; Shimazawa et al. 2010), prevention of retinal ganglionic cell death (Shanab et al. 2012), as a neuroprotective agent in animal models of stroke (Koumura et al. 2008) and traumatic brain injury (Bains et al. 2013) and also as additive cardioprotective agent (Yoshikawa et al. 2010; Takeshita et al. 2013). The present in vitro study is designed to address the damaging effects of MPP+ and rotenone in SH-SY5Y human neuroblastoma cells; SH-SY5Y cells were chosen as they can be differentiated into diverse phenotypes as dopaminergic or cholinergic (Presgraves et al. 2004a,b; Cheng et al. 2009; Mastroeni et al. 2009; Xie et al. 2010). Distinct responses were seen in cholinergic versus dopaminergic phenotypes thus, providing better understanding of toxic mechanisms induced by MPP+ or rotenone depending upon the neuronal subtype. Examination of SNJ-1945 showed its neuroprotective efficacy against multiple factors that are involved in MPP+ and rotenone-induced toxicity, including calpain activation, inflammatory mediators and ROS generation, which may culminate into the demise of the respective cell types.

Material and methods Cell culture, differentiation, and treatments The human neuroblastoma cell line SH-SY5Y (ATCC, Manassas, VA, USA) was cultured and differentiated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were maintained in complete medium comprising of Dulbecco’s modified Earle’s medium/Ham’s F12 50/50 mix with L-glutamine and 15 mM HEPES (Cellgro, Mediatech, Manassas, VA, USA) which was supplemented with penicillin (100 IU/mL), streptomycin (100 lg/ mL), and 10% of heat-inactivated fetal bovine serum. At 60–70% confluence, cells were sub-cultured and differentiated into either cholinergic (ChAT-positive) phenotype with 10 lM retinoic acid over 6 days following (Cheng et al. 2009; Xie et al. 2010) or dopaminergic TH-positive phenotype with 10 lM retinoic acid (RA) for the first 3 days followed with 80 nM phorbol 12-myristate 13-acetate (PMA) for the next 3 days (Presgraves et al. 2004a,b; Xie et al. 2010) or 50 ng/mL brain derived neurotrophic factor (BDNF) for the next 3 days (Mastroeni et al. 2009; Xie et al. 2010; Table 1). Upon differentiation TH and ChAT immunoreactivity (IR) were up-regulated in the respective groups of cells, which are henceforth designated as SH-SY5Y-DA or SH-SH5Y-ChAT cells, respectively. Differentiation medium contained reduced serum (3–5%). Different concentrations of MPP+ (50, 100 or 500 lM) or rotenone (10, 50, or 100 nM) were used to expose the cells for a period of 24 h. To test cytoprotection, cells were pre-treated with three concentrations (50, 100, or 250 lM) of the calpain inhibitor SNJ-1945 (Senju Pharmaceutical Co. Ltd.) 30 min prior to or 1–3 h post-neurotoxicant exposure. Intracellular free Ca2+ assay Fura-2 was used to assess intracellular free Ca2+ in cells exposed to MPP+ or rotenone following previously published method (Grynkiewicz et al. 1985; Samantaray et al. 2011). After 24 h of

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Table 1 Differentiation of SH-SY5Y cells Day 1

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RA, 10 lM

PMA, 80 nM BDNF, 50 ng/mL RA, 10 lM

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Day 7

References

Dopaminergic (TH, DAT and DBH positive)

Presgraves et al. (2004a,b), Xie et al. (2010) Mastroeni et al. (2009), Xie et al. (2010) Cheng et al. (2009), Xie et al. (2010)

Cholinergic (ChAT positive)

neurotoxicant exposure, cells were washed, re-suspended in modified Locke’s buffer (NaCl: 154 mM, KCl: 5.6 mM, NaHCO3: 3.4 mM, MgCl2: 1.2 mM, glucose: 5.6 mM, Hepes: 5 mM [pH 7.4], and CaCl2: 2.3 mM), and counted on a hemocytometer. In each experimental group, equal number of cells (1 9 106 cells/mL) were loaded with the fluoroprobe Fura-2 AM (5 lM) (Molecular Probes, Carlsbad, CA, USA) at 37 °C for 30 min. Cells were spun and washed twice in ice-cold Locke’s buffer. Concentration of Rmin)/ [Ca2+]i was calculated using the equation [Ca2+]i = Kd(R R). Spectrophotometric analysis of the fluorescence ratio (Rmax (R) was done using SLM 8000 fluorometer at 340 nm and 380 nm wavelengths (Thermospectronic, Thermoscientific, Pittsburgh, PA, USA). Maximal (Rmax) and minimal (Rmin) ratios were determined using 25 lM digitonin and 5 mM EGTA, respectively. Percent of [Ca2+]i increase in exposed cells compared to control was plotted. Immunocytofluorescent staining Cells were cultured and differentiated in six-well plates with cover slips inserted inside the wells. To test the differentiation protocol, TH (1 : 100, overnight at 4 °C; Novus Biologicals, Littelton, CO, USA) staining was performed in undifferentiated cells, and SH-SY5Y cells differentiated with RA/PMA or RA/RA. Cells were also exposed to respective concentrations of neurotoxicants with or without SNJ-1945 in each plate for 24 h. Plates were centrifuged to sediment the nonadherent cells. Cells were fixed with 95% ethanol for 10 min followed by 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min; in between steps, cells were washed with phosphate-buffered saline (3 9 5 min). Cover slips containing the cells were removed from wells, placed on glass microscope slides, and blocked with goat serum in PBS for 1 h followed by incubation with active l-calpain antibody (1 : 100; Banik et al. 1983) overnight at 4 °C. Immunostaining was visualized with DyLight 488 or 594 conjugated anti-rabbit secondary IgG for active calpain and TH, respectively (Thermo Scientific, Rockford, IL, USA), aided with antifade VectashieldTM (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were viewed and captured in Olympus BH-2 microscope (Melville, NY, USA) at 2009 magnification. Cell viability assay and in situ Wright staining Procedures were performed as described previously (Samantaray et al. 2011). 3-(4, 5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma, St. Louis, MO, USA) was used to assess cell viability. Following neurotoxicant exposure, cells were incubated with 3-(4, 5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide reagent (0.1 mg/mL) in 0.5% serum containing medium at 37 °C for 1 h. Formazan crystals were precipitated by centrifugation at 1900 g (Eppendorf Centrifuge 5804R; Eppendorf, Hamburg, Germany), medium was aspirated, and crystals were dissolved in dimethylsulfoxide. Plates were read in Emax. Precision Microplate reader at

570 nm with reference wavelength set at 630 nm using SoftMax Pro software (Molecular Devices, Sunnyvale, CA, USA). Optical density was compared setting the control at 100%. In situ Wright staining was performed as described previously (Samantaray et al. 2011) and the images were captured at 2009 magnification. Intracellular ROS assay ROS were detected using cell-permeable CM-H2DCFDA (Life Technologies, Grand Island, NY, USA) reagent following manufacturer’s protocol. Following respective treatments, cells were gently harvested from flasks with warm Hank’s Balanced Salt Solution (HBSS, 1X, Cellgro) into tubes and spun. Pellets were re-suspended in HBSS and loaded with 10 lM of CM-H2DCFDA for 30 min at 37 °C. After short centrifugation, the excess dye was aspirated; cells were resuspended with warm HBSS and transferred into 24-well plates; the end-point arbitrary fluorescent units were recorded setting the excitation and emission wavelengths at 485 nm and 538 nm, respectively. For in situ measurements, cells were grown in 6-well plates with coverslips inserted in them and processed for ROS assay. Fluorescent images representing the total intracellular ROS in cells were captured in Olympus BH-2 microscope at 2009 magnification. Western blot Immunoblotting was performed as described previously (Samantaray et al. 2011). Control and neurotoxicant-exposed cells were harvested; pellets were sonicated in homogenizing buffer [50 mM Tris–HCl, (pH 7.4) with 5 mM EGTA, and freshly added 1 mM phenylmethylsulfonyl fluoride]. Samples were diluted 1 : 1 in sample buffer [62.5 mM Tris–HCl, pH 6.8, 2 % sodium dodecyl sulfate, 5 mM b-mercaptoethanol, 10 % glycerol] and boiled. Protein concentration was adjusted to a concentration of 1.5 mg/ mL with 1 : 1 v/v mix of homogenizing buffer and sample buffer containing 0.01 % bromophenol blue. Samples were resolved in 4–20 or 7.5 % [for spectrin breakdown products (SBDP)] precast sodium dodecyl sulfate–polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA) at 100 V for 1 h or 1 and 1/2 h, respectively; transferred to the ImmobilonTM-polyvinylidene fluoride microporous membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5 % non-fat milk in Tris–HCl buffer (0.1 % Tween-20 in 20 mM Tris–HCl, pH 7.6). Following overnight incubation at 4 °C with appropriate primary IgG antibodies, blots were incubated with horseradish peroxidaseconjugated corresponding secondary IgG antibodies at 20–23°C. Between incubations, membranes were washed 3 9 5 min in Tris–HCl buffer. Immunoreactive protein bands were detected with chemiluminescent reagent (ECL or ECL prime, Amersham, UK); images were acquired using Alpha Innotech FluorChem FC2 Imager (Cell Biosciences Inc., Santa Clara, CA, USA).

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Antibodies used in the study included rabbit polyclonal anticaspase-1, cleaved caspases-1 p10 fragment, anti-caspase-3, anticaspase-8, anti-calpastatin and mouse monoclonal anti-Cox-2, (all diluted 1 : 250; Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti a-Fodrin (aII-spectrin, 1 : 10 000; Enzo Life Sciences, Farmingdale, NY, USA); mouse monoclonal anti-bactin (1 : 10 000, Sigma), and rabbit polyclonal anti-calpain (1 : 500; Banik et al. 1983). The bound antibodies were visualized by corresponding peroxidase-conjugated IgG antibodies (1 : 2000; MP Biomedicals, Solon, OH, USA).

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Statistical analyses Each assay was performed in duplicate and the experiment was repeated thrice. Optical density (OD) of protein IR bands obtained from western blotting was analyzed with NIH ImageJ 1.45 software. Results were assessed in Stat View software (Abacus Concepts, Piscataway, NJ, USA) and compared by using one-way ANOVA with Fisher’s protected least significant difference post hoc test at 95% confidence interval. Data were expressed as mean  SEM (n ≥ 3). The difference was considered significant at p ≤ 0.05. Neurotoxicant-induced changes in levels of protein (%) were considered significant at *p ≤ 0.05, compared with control, and @p ≤ 0.05, compared to SNJ-1945 pre-treatment or post-treatment. ARRIVE experimental guidelines were followed along with institutional approval during the course of this study.

Results MPP+ and rotenone-induced rise in [Ca2+]i and calpain up-regulation Aberrant intracellular Ca2+ homeostasis is one of the mechanisms involved in PD. Whether MPP+ or rotenone induced rise in [Ca2+]i in SH-SY5Y cells was tested with the ratiometric dye Fura-2 AM. A significant dose-dependent elevation in levels of [Ca2+]i ranging from 30 to 60% (p ≤ 0.05) were observed in SH-SY5Y-DA cells exposed to MPP+ (50, 100, or 500 lM) or rotenone (10, 50, or 100 nM; Fig. 1a). We had previously reported a similar dose-dependent rise in [Ca2+]i in ChAT-positive VSC 4.1 cells exposed to MPP+ or rotenone (Samantaray et al. 2011). Next, we investigated whether MPP+ or rotenone-induced rise in [Ca2+]i was accompanied with activation of calpain in these cells. Compared with control, active l-calpain IR was significantly elevated in SH-SY5Y-DA cells by exposure to MPP+ (100 lM) or rotenone (50 nM; Fig. 1b). Up-regulation of active calpain was also observed in the cells that survived after exposure to higher concentrations of neurotoxicants; the similar trend was observed in SH-SY5Y-ChAT cells (data not presented); hence, efficacy of the calpain inhibitor SNJ-1945 was tested in SH-SY5Y-DA and –ChAT cells. SNJ-1945-mediated protection of cell viability and morphology Effects of calpain inhibitor SNJ-1945 on the survival of differentiated SH-SY5Y cells following exposure to MPP+ or rotenone was tested next. Cell viability assay showed that

Fig. 1 (a) Elevation of free [Ca2+]i levels in SH-SY5Y-DA cells. Following exposure to different concentrations of MPP+ (50, 100, or 500 lM) and rotenone (10, 50, or 100 nM) for 24 h, cells were loaded with ratiometric dye Fura-2AM. A dose-dependent rise in [Ca2+]i was recorded by monitoring the emission at 510 nm and dual excitement at 340 and 380 nm, respectively. Bar graphs represent the per cent increase of [Ca2+]i compared to the control cells; data are expressed as mean  SEM (n ≥ 3); *p ≤ 0.05, significantly different from control. (b) Up-regulation of calpain in SH-SY5Y-DA cells. Representative images from three independent experiments (n = 3) illustrate that active l-calpain IR (green) was significantly increased in SH-SY5Y-DA cells following MPP+ (100 lM) and rotenone (50 nM) exposure, compared with the control cells.

both SH-SY5Y-DA and SH-SY5Y-ChAT cells responded to both neurotoxicants in a dose-dependent manner (data presented in SH-SY5Y-DA cells, Fig. 2a and b). MPP+ was found effective at micromolar range (50–500 lM), whereas rotenone was found to be effective at nanomolar range (10–100 nM); such log scale differences in the effective concentration of these neurotoxicants were previously reported in ChAT-positive VSC 4.1 cells (Samantaray et al. 2011). We used similar concentrations of MPP+ and rotenone in SH-SY5Y-DA and SH-SY5Y-ChAT cells in subsequent experiments. Three doses of the calpain inhibitor SNJ-1945 (10, 100 or 250 lM) were tested for protective capacity against MPP+ or rotenone (Fig. 2a and b, respectively). SNJ-1945 alone at its highest concentration (250 lM) had no overt on these cells. SNJ-1945 (100 and 250 lM) was found significantly protective against MPP+ and rotenone. Loss in cell viability following neurotoxicant exposure was associated with distinct alterations in morphology of SHSY5Y cells, which were assessed with in situ Wright staining. Microscopic observation of stained cells showed morphological alterations in cells exposed to MPP+ or rotenone compared to control cells; the apoptotic cell nuclei were deeply stained and shrunken. MPP+ or rotenone-

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MPP+ (Fig. 4b). Pre-treatment with SNJ-1945 (250 lM) could significantly attenuate the elevated levels of ROS in SH-SY5Y-DA cells (Fig. 4a, lower panel; Fig. 4b). Importantly, such elevations in ROS were not found in SH-SY5YChAT cells exposed to MPP+ or rotenone for 24 h. MPP+ or rotenone-induced elevation of ROS was selectively associated with the DA phenotype and absent in ChAT phenotype, so we verified expression of TH IR with immunofluorescent staining in undifferentiated cells, and SH-SY5Y cells differentiated with RA/PMA or RA/RA as shown in Fig. 5.

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Fig. 2 (a and b) SNJ-1945-mediated protection of viability in SHSY5Y-DA cells. 3-(4, 5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay performed following 24 h exposure to MPP+ (50– 500 lM) or rotenone (10–100 nM) showed reduction in cell viability in a dose-dependent manner. Pre-treatment with SNJ-1945 (100 or 250 lM) was found protective, whereas the lowest dose used (10 lM) was ineffective. Bar graphs represent data expressed as mean  SEM of cell viability (%), obtained from four to six independent assays; *p ≤ 0.05, significantly different from viability of control cells; @ p ≤ 0.05 significantly different from viability of neurotoxicant-exposed cells, respectively.

induced morphological alterations were observed in SHSY5Y-DA cells (Fig. 3), SH-SY5Y-ChAT cells (data not shown) and ChAT-positive VSC 4.1, as reported previously (Samantaray et al. 2011). Importantly, these alterations could be ameliorated by pre-treatment with SNJ-1945 dosedependently. Differential induction of ROS, and SNJ-1945-mediated protection Mitochondrial dysfunction and aberrant Ca2+ homeostasis subsequently lead to the induction of ROS. Elevated levels of ROS as imaged with fluorescent dye CM-H2DCFDA was observed when SH-SY5Y-DA cells were exposed to MPP+ (100 lM) or rotenone (50 nM) for 24 h (Fig. 4a); this effect was still evident following prolonged incubation for 72 h with

Differential induction of inflammatory mediators, and SNJ-1945-mediated protection Next, the generation of inflammatory mediators, Cox-2, caspase-1 and the cleaved p10 fragment of caspase-1 were examined in both SH-SY5Y-DA and SH-SY5Y-ChAT cells following exposure to MPP+ or rotenone. Interestingly, the neurotoxicants did not induce any significant changes in the profiles of any inflammatory mediator tested in SH-SY5YDA cells; importantly, the differentiation protocol to induce dopaminergic phenotype vide RA/PMA or RA/BDNF did not alter the outcomes as shown in the left and right panels of Figure S1. However, significantly high levels of Cox-2 (35% and 32%), caspase-1 (20% and 23%), and p10 (45% and 35%) were induced by MPP+ (Fig. 6a and b) and rotenone (Fig. 6c and d), respectively, in SH-SY5Y-ChAT cells compared with control. Pre-treatment with SNJ-1945 (50 or 100 or 250 lM) dose-dependently attenuated the neurotoxicant-induced levels of inflammatory mediators in SH-SY5YChAT cells (Fig. 6). SNJ-1945-mediated protection against proteases Next the profiles of proteases caspase-3, -8 expression and 120 kDa caspase-3 specific SBDP and 145 kDa calpainspecific SBDP were examined. In SH-SY5Y-DA cells, caspase-3 expression remained unaltered; the active bands (20, 12 kDa) were not expressed at 24 h time point (Fig. 7). Likewise, there was no neurotoxicant-induced up-regulation of caspase-8 as well in these cells (data not presented). However, 145 kDa calpain specific SBDP were significantly induced following MPP+ or rotenone exposure. SNJ-1945 pre-treatment could successfully attenuate calpain activity as marked by the diminished levels of 145 kDa band (Fig. 7a and b) and the corresponding densitometric analysis on% change (bar graphs). In SH-SY5Y-ChAT cells procaspase-3 was 40–65% up-regulated compared with control (Fig. 8a and b). Pre-treatment with SNJ-1945 (50, 100, or 250 lM) could dose-dependently attenuate the increase of procaspase-3. Importantly, active caspase-3 bands (20 and 12 kDa) remained unaltered throughout the treatment groups (Fig. 8a). Further MPP+ and rotenone exposure elevated the levels of intermediate caspase-8 in SH-SY5Y-ChAT

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Fig. 3 SNJ-1945-mediated protection of SH-SY5Y-DA cell morphology. In situ Wright staining assay was used to distinguish apoptotic cells following 24 h exposure to MPP+ (100 lM) or rotenone (50 nM) with/without pre-treatment with SNJ-1945 (100 or 250 lM). Representative images show healthy control and cells treated with SNJ-1945

(250 lM; a, b), which were significantly altered (shrunken) with MPP+ (100 lM; c) or rotenone (50 nM; d); Pre-treatment with SNJ-1945 (100 or 250 lM) attenuated MPP+-induced (d, e, respectively) and rotenone-induced (g, h, respectively) alterations in cell morphology. Images were captured by light microscope at 2009 magnification.

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Fig. 4 Rise in reactive oxygen species (ROS) in SH-SY5Y-DA cells. (a) In situ profiles of ROS in cells following MPP+ (100 lM) or rotenone (50 nM) exposure for 24 h with respect to control in upper panel and pre-treatment with SNJ-1945 (250 lM) in lower panel. (b) Cumulative

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levels of ROS after prolonged 72 h exposure to neurotoxicants and SNJ-1945-mediated protection against elevated levels of ROS in SHSY5Y-DA cells. *p < 0.05 compared to control and @p < 0.05 compared to MPP+-exposed cells.

cells; SNJ-1945 pre-treatment dose-dependently attenuated it (Fig. 8a and c). Both 145 kDa and 120 kDa SBDP levels were enhanced by MPP+ and rotenone in these cells, which could be dose-dependently attenuated by SNJ-1945 pretreatment (Fig. 8a, d, and e). Post-treatment of SNJ-1945 demonstrated partial protection (Figure S2 and S3). Fig. 5 Immunofluorecent images of tyrosin hydroxylase (TH) staining in SH-SY5Y cells. The TH staining was performed to confirm dopaminergic phenotype of SH-SY5Y cells differentiated with retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA). Greater levels of TH immunoreactivity (IR) (red) were observed in SH-SY5Y cells differentiated with RA and PMA, compared to the low intensity in undifferentiated SH-SY5Y cells and cells differentiated with RA only. Images were captured by fluorescent microscope at 2009 magnification.

Discussion Present study conducted in vitro in human neuroblastoma cells SH-SY5Y compared the probable mechanisms of degeneration in the dopaminergic versus cholinergic neuronal phenotypes, following exposure to the parkinsonian neurotoxicants MPP+ and rotenone. Our salient findings include rise in [Ca2+]i, with concomitant activation of calpain in both the phenotypes.

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Fig. 6 Induction of inflammatory mediators in SH-SY5Y-ChAT cells. Significantly elevated levels of caspase-1, p10 cleaved fragment of caspase-1 and Cox-2 was found in SH-SY5Y-ChAT cells; SNJ-1945 dosedependently protected against the effects of both MPP+ (a, b) and rotenone (c, d); quantifications are represented in the right panels correspondingly (b, d). Representative immunoblots from three independent experiments (n = 3) and corre-sponding bar graphs showed significantly increased levels of Cox2, caspase-1 and cleaved p10 fragment in MPP+ or rotenone-exposed SH-SY5Y-ChAT cells (*p ≤ 0.05, compared to control). SNJ1945 relatively reduced levels of Cox-2, caspase-1, and cleaved p10 fragment in SH-SY5Y-ChAT cells (@p ≤ 0.05, relative to neurotoxicant-exposed cells). SNJ-1945 (100 or 250 lM) was found effective against MPP+ or rotenone, whereas 50 lM SNJ-1945 had limited efficacy.

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Induction of oxidative stress was predominant in the dopaminergic phenotype, whereas inflammatory mediators were significantly elevated in the cholinergic phenotype after a 24 h time period. Importantly, the novel water-soluble calpain inhibitor SNJ-1945 could significantly protect against damaging pathways including oxidative stress, inflammation, calpain-calpastatin dysregulation, and proteolysis. Progressive neurodegeneration in PD involves CNS locations that are scattered much beyond the dopaminergic neuronal loss in midbrain substantia nigra and the paucity of neurotransmission in striata (Olanow et al. 2011; Giza et al. 2012). Indeed, several parkinsonian symptoms are attributed to degeneration in spinal cord, which was also implicated by the presence of Lewy bodies in the spinal cord (Wakabayashi and Takahashi 1997; Braak et al. 2007). Unlike previous proposition that spinal cord might be one of the earliest and consistently affected sites in PD, it was confirmed recently that brain degeneration always precedes that of spinal cord (Del Tredici and Braak 2012). The involvement of spinal cord degeneration and dysfunction in PD received much attention primarily from the studies in animal models of PD (Ray et al. 2000; Chera et al. 2002, 2004; Samantaray et al. 2007, 2008a; Vivacqua et al. 2011, 2012). Molecular mechanisms of dopaminergic neuronal degeneration in vivo in PD has been extensively studied in vitro using MPP+ and rotenone. These neurotoxicants were also employed to test the vulnerability of spinal motoneurons in vitro (Samantaray

et al. 2011). MPP+ and rotenone are potent mitochondrial toxins which inhibit oxidative phosphorylation, induce ATP depletion, impair mitochondrial membrane potential, elevate [Ca2+]i, generate ROS, induce inflammatory mediators, release cytochrome c, and cause several other events like in idiopathic PD. Such events are well documented in the midbrain nigrostriatal degeneration using experimental models of PD (Crocker et al. 2003; Samantaray et al. 2008b; Banerjee et al. 2009). While multiple of these detrimental pathways are operational in a cell, particularly a neuronal cell undergoing mitochondrial dysfunction will invariably activate calpain (Esteves et al. 2010). In this study, we report that both SH-SY5Y-DA and SH-SY5Y-ChAT cells when exposed to mitochondrial toxins showed calpain activation, thus underscoring the activation of calpain as a common denominator in different phenotypes in cell culture models of parkinsonism. Protective efficacy of calpain inhibition was examined in this study following exposure to MPP+ and rotenone in SHSY5Y cells differentiated into dopaminergic and cholinergic phenotypes. The study not only confirmed the previously reported MPP+ or rotenone-induced apoptotic mechanisms in VSC 4.1 cells (Samantaray et al. 2011), but also discerned distinct pathways induced by MPP+ and rotenone depending upon the cellular phenotype. Inhibition of mitochondrial complex I by MPP+ and rotenone presumably induced cascade of signaling pathways that provoked increase in

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Fig. 7 Altered calpain activity but not caspase-3 in SH-SY5Y-DA cells. Immunoblot analysis of caspase-3 failed to discern any MPP+ (100 lM) or rotenone (50 nM)induced alterations in the active bands of caspase-3; this was further confirmed by unaltered levels of 120 kDa spectrin breakdown products (SBDP) amongst different groups. Calpain-specific 145 kDa SBDP showed distinct up-regulation by MPP+ or rotenone (*p ≤ 0.05, compared with control); this effect could be significantly attenuated by SNJ-1945 pre-treatment (@p ≤ 0.05, relative to neurotoxicant-exposed cells). Differentiation regimen [RA/PMA or RA/brain derived neurotrophic factor (BDNF)] had not influence either, (n = 3).

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(c) Fig. 8 Altered profiles of protease expression and activity in SH-SY5Y-choline acetyltransferase positive (ChAT) cells. Upregulation of proteases (caspase-3, -8) levels were seen in MPP+ (100 lM) or rotenone (50 nM) exposed cells; SNJ-1945 dosedependently attenuated this response as shown in (a) and the densitometric analysis in (b, c). MPP+ and rotenone also induced activation of calpain and caspase-3 observed as elevation of 145 kDa and 120 kDa spectrin breakdown products (SBDP), respectively, in (a) and the corresponding densitometric analysis in (d, e); (n = 3); (*p ≤ 0.05, compared with control) and (@p ≤ 0.05, relative to neurotoxicantexposed cells).

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[Ca2+]i concentration giving rise to an environment conducive for up-regulation of calpain expression and activity. Anomalous Ca2+ homeostasis, calpain-calpastatin dysregulation involved in pathophysiology of PD is implicated in midbrain nigrostriatal degeneration (Samantaray et al. 2008b) and in post-mortem PD spinal cord (Samantaray et al. 2013a). Ca2+-dependent cell death mechanism has been previously demonstrated in VSC 4.1 motoneuronal cells (Samantaray et al. 2011). This study confirms elevation of intracellular free Ca2+ induced by MPP+ and rotenone, suggesting common initialization of damaging pathways in dopaminergic and cholinergic neurons. The calpain inhibitor SNJ-1945 rendered significant cytoprotection whether cells were treated before or after insult with the neurotoxicants, which further confirmed the involvement of calpain in MPP+- and rotenone-mediated apoptosis in dopaminergic and cholinergic neuronal phenotypes. These findings indicate calpain as a promising therapeutic target in PD. Novel finding in this study is that when SH-SY5Y-DA cells were exposed to mitochondrial toxins, the primary event that followed was generation of ROS, whereas the SHSY5Y-ChAT cells underwent a burst of inflammatory mediators. In this context, an important review on inflammation and neurodegeneration (Glass et al. 2010) surmises that the inducer of inflammation occurs in disease specific manner, yet, there may be convergence of pathways among sensing, transduction and amplification of inflammatory processes into neurodegenerative diseases. Thus, it would be likely to expect that the SH-SY5Y-ChAT cells if exposed longer to MPP+ or rotenone may generate ROS as neurotoxic mediators. Worthwhile to note that participation of glial cells play a prominent role in induction of inflammatory mediators in midbrain substantia nigra (Jun-ichi 2013); in absence of such events we observed persistent ROS over 72 h (Fig. 4) and no inflammatory mediators in SH-SY5Y-DA cells (Figure S1) in our study. Investigation of such mechanisms is important to elucidate the complex pathophysiology of PD as carried out in this study using SH-SY5Y cells and differentiation agents RA, PMA, and BDNF (Presgraves et al. 2004a,b; Cheng et al. 2009; Mastroeni et al. 2009). Important to note that MPP+ enters dopaminergic cells via dopamine transporters, which are reported to be up-regulated in SH-SY5Y cells upon differentiation; such transporters are not expressed in the cholinergic phenotypes. Entry of MPP+ in these cells might be through alternate pathway using cationic amino acid transporters present in neuronal cells. Mechanisms of MPP+- or rotenone-induced toxicity depend on the cell type. A major research focus has been to compare the effects of these toxins in the same cell line (Martins et al. 2013). However, in this study the focus was to discern whether calpain was a common mediator in MPP+ or rotenone-induced toxicity and the calpain inhibitor SNJ-1945 was efficacious. Indeed, SNJ-1945 was capable of attenuating destructive effects of both MPP+ and rotenone. In this

study, the protective mechanism of SNJ-1945 in dopaminergic phenotype included attenuation of ROS production, reduction of a-spectrin proteolysis, whereas in cholinergic phenotype, the inhibitor down-regulated Cox-2, caspase-1 and cleaved caspase-1 p10. Calpain was a common mediator involved in neurotoxic mechanism triggered by MPP+ or rotenone, and inhibition of calpain activation by SNJ-1945 rendered significant neuroprotection. Overall, PD therapeutics is in search for a drug that is not limited to dopaminergic replenishment, but addresses the complex PD pathophysiology. Current in vitro investigation suggests that the novel water-soluble calpain inhibitor SNJ1945 may be tested in animal models of experimental parkinsonism.

Acknowledgements and conflict of interest disclosure This study was funded in part by the RO1 grants from National Institute of Neurological Disorders and Stroke of the National Institutes of Health (NINDS-NIH; NS-62327-01A2; NS-56176 and NS-65456) and the Veterans Administration (I01 BX001262). All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflicts of interest to declare.

Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. Inflammatory mediators in SH-SY5Y-DA cells. Unaltered levels of inflammatory mediators (caspase-1, p10 cleaved fragment of caspase-1 and Cox-2) were observed in SH-SY5Y-DA cells following exposure to MPP+ (100 lM) or rotenone (50 nM). Figure S2. Restoration of calpain-calpastatin ratio by posttreatment with SNJ-1945. MPP+ or rotenone exposure induced dysregulation of calpain-calpastatin system by enhancing the ratio of active calpain to calpastatin; post-treatment with SNJ-1945 1–3 h ameliorated MPP+ or rotenone effects. Figure S3. Attenuation of calpain activity by post-treatment with SNJ-1945.

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