Abstract
We have recently demonstrated neuroprotective abilities of nimodipine, an L-type voltage dependent calcium channel (VDCC) blocker in cellular and animal models of Parkinson’s disease (PD). To understand the calcium regulatory mechanisms in the disease pathogenesis, the present study examined calcium regulatory proteins calbindin and calpain mRNA and protein levels employing quantitative PCR and western blot in 1-methyl-4-phenyl pyridiniumion (MPP+)-treated SH-SY5Y cell lines and in the striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). mRNA and protein levels of calbindin were lower, while that of calpain were higher in MPP+-treated SH-SY5Y cells and MPTP-treated mousestriatum as compared to theirrespective controls. Nimodipine pretreatment significantly attenuated these effects in the parkinsonian neurotoxin treated SH-SY5Y cell line and in the mouse striatum. The activities of the apoptotic mediator, caspase-3 and calpain were increased in the neurotoxin-treated groups as compared to theirrespective controls, which Osteogenic biomimetic porous scaffolds was ameliorated by nimodipine pretreatment. These results suggest that parkinsonian neurotoxin-mediated dopaminergic neuronal death might involve defects in calcium regulatory proteins that control intracellular calcium homeostasis , and these could be corrected by inhibiting L-type VDCC activity. These findings support the notion that hypertensive patients who are on long-term intake of dihydropyridine have reduced risk for PD.
Keywords: dopaminergic neuroprotection, calcium regulatory proteins, caspase 3 activity,apoptosis.
1. Introduction
Parkinson’s disease (PD) is caused by the demise of dopaminergic neurons from substantia nigra pars compacta (SNpc) area of the midbrain, and is characterized by rigidity, akinesia and tremor resulting from severe loss of striatal dopamine (DA) content (Dauer and Przedborski 2003;Dorsey et al. 2007). Major mechanisms involved in the pathogenesis of PD are mitochondrial
dysfunction, pro-apoptotic events, oxidative and nitrosative stresses, genetic predisposition,protein aggregation and defects in proteasome function (Chandra et al., 2017; Double et al. 2010;Kazlauskaite and Muqit 2015; Parsanejad etal. 2014; Rio and Velez-Pardo 2008; Ryan et al.2013). Both cellular and animal models of the disease are employed extensively to investigate
cellular, molecular and biochemical mechanisms involved in PD pathogenesis and for drug screening, which involve treatment of dopaminergic neurons with parkinsonian neurotoxins such
as 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) or its active metabolite 1-methyl-4-phenyl pyridiniumion (MPP+), 6-hydroxydopamine (6-OHDA), rotenone, paraquat, etc.(Przedborski et al. 2001; Soto-Otero etal. 2000; Tanner et al. 2011) as well as transgenic cells and animals (Chenn et al. 2015; Wu et al. 2016).
Besides major pathologic markers as stated above, deregulated calcium homeostasis in cellular and animal models of the disease brought more attention recently following epidemiological
findings on a decreased risk for PD in humans who were treated for hypertension with dihydropyridine (DHP) calcium channel antagonists (Becker et al. 2008; Pasternack etal. 2012;
Ritz et al. 2010). The SNpc dopaminergic neurons are autonomously active, with prominent transmembrane calcium currents that generate regular, slow, broad action potentials (2-4 Hz) in
the absence of synaptic input (Surmeier et al. 2011). This pace-making neuronal activity is accompanied by sustained calcium entry through L-type voltage-dependent calcium channels
(VDCC) (Grace and Bunney 1984). DHP moieties are suggested appropriate candidates for slowing down or curbing PD pathogenesis as their binding site to L-type calcium channels are preserved in PD brain (Sen etal. 1993). 6-OHDA-induced increase in calcium level is curbed by treatment with VDCC blockers such asnimodipine, amlodipine and nitrendipine (Antonyet al.2012), and nimodipine or isradipine, protected against MPTP- or 6-OHDA-induced DA-ergic nigral neuronal death (Ilijic etal. 2011; Kupsch etal. 1995, 1996; Singh etal. 2016). Recently we have reported increases in intracellular calcium, which is regulated by L-type VDCC, in cellular and animal models of PD, and nimodipine offered effective dopaminergic neuroprotection from PD pathologies (Singh etal., 2016). Therefore we investigated the levels of calcium binding proteins and their mRNA to understand the mechanism of neuroprotection imparted by the L-type VDCC blocker, nimodipine in MPP+-induced or MPTP-mediated dopaminergic pathology, respectively in SH-SY5Y cell line or in mice.
2. Materials and methods
2.1 Chemicals
Human neuroblastoma cell line SH-SY5Y was obtained from ATCC, VA, USA. Penicillin,streptomycin and amphotericin B, trypsin and Dulbecco’smodified Eagle’s medium (DMEM) were procured from GIBCO, Invitrogen Corporation, CA, USA. Characterized fetal bovine serum was obtained from Hyclone, UT, USA. MPTP, MPP+, N-acetyl-Asp-Glu-Val-Asp-7 amido-4-methyl coumarin, N-Suc-Leu-Tyr-amido-4-methyl coumarin and nimodipine were obtained from Sigma, St. Louis, MO, USA. 3,3’-Diaminobenzidine was purchased from MP Biomedicals (Solon, OH, USA). Bovine serum albumin and sucrose were bought from SRL,Mumbai, India. Absolute alcohol, iso-propyl alcohol, acrylamide and MgCl2 were procured from Merck, Germany. Dimethyl sulfoxide (DMSO) was obtained from Merck, Mumbai, India.
Chloroform was obtained from MP Biomedicals, California, USA. Taq buffer, Oligo DT, Taq DNA polymerase, RNAase inhibitor and reverse transcriptase were procured from Thermo Scientific, Massachusetts, USA. Trypsin and glycine were purchased from Calbiochem, San Diego, USA. SYBR Mix was purchased from Kappa Biosystems, MA, USA. Ammonium per sulphate and sodium dodecyl sulphate were purchased from ICN Biomedicals, Ohio, USA.Polyvinylidene fluoride membrane was obtained from Millipore, Bangalore, India. Tris (NH2C(CH2OH)3) was purchased from Amersham Life Sciences, USA. Skim milk powder was obtained from LOBA Chemie Mumbai, India. Primary antibodies against Calpain and Calbindin (raised in rabbits) were purchased from Cell Signaling Technology, Massachusetts, USA.Secondary antibodies, anti-rabbit IgG raised in goat were obtained from Genei, Bengaluru, India. Primers for realtime PCR were procured from Integrated DNA Technologies, Coralville, Iowa,United States.
2.2 Maintenance of cells
SH-SY5Y cells were maintained in DMEM supplemented with 10% FBS, 50 Units/ml penicillin,50 μg/ml streptomycin and 0.25 μg/ml amphotericin B. Cells were trypsinized and passaged when they reached confluency.
2.3 Cell treatment paradigm
MPP+ and nimodipine were dissolved in Milli-Q water and 2% DMSO, respectively to make stock solutions, and of desired concentrations. These were prepared fresh for each application. Specific numbers of SH-SY5Y cells were plated in appropriate culture dishes. Next day, the cells were treated with nimodipine 10 µM for 15 min. The media was aspirated off and cells were maintained in fresh medium containing MPP+ (1 mM) or without MPP+ for 24 h.Adult male Balb/c mice were inbred, weighing 22-25 g. Four mice were housed per cage at controlled temperature of 22 ± 1 °C, humidity 60 ± 5%, and illumination (12-h light-dark cycles). Water and food pellets were made available to them freely. Institutional animal ethics committee appointed by the ‘Committee for the Purpose of Control and Supervision of Experimentation in Animals’ of the Animal Welfare Division under the Ministry of Environment & Forests, Govt. of India approved the protocols for animal experimentation. We have in the past demonstrated equal sensitivity of Balb/c and C57/Bl6 mice strainsto MPTP, and compared the neurodegeneration and striatal dopamine depletion (Mitra et al., 1994). We have also demonstrated comparable effects of MPTP-treatment on neurobehaviors in C57/Bl6 mice and Balb/c mice (Haobam et al., 2005). In the present study, too we have used Balb/cmice following the same protocol of MPTP administration. The animal model we have employed helped us to understand the PD pathology and the beneficial effect of the neuroprotective drug, nimodipine.
2.5 Treatment protocol
MPTP selleck and nimodipine were dissolved in 0.9% saline and 2% DMSO respectively. These solutions were prepared fresh for each administration cycle. MPTP (30 mg/kg body weight, i.p.) was administered in mice twice, 16 h apart (Mitra et al., 1994; Naskaretal., 2013). Nimodipine (10 mg/kg body weight) was injected intraperitoneally in mice once, 30 min before the first injection of MPTP, or saline. Nimodipine in vitro and in vivo doses were standardized in our recent study (Singh etal., 2016) and the optimal doses were selected for the present study. On the 7th day, when the animals were found to have parkinsonian neurotoxicity, these mice were sacrificed by cervical dislocation and the brains were dissected out for obtaining the striatal tissue used for various assays.
2.6 Quantitative PCR
For in vitro experiments, SH-SY5Y cells were scraped into Trizol. For in vivo experiments,striata were dissected out and homogenized in Trizol. The homogenate was centrifuged at 12000xg for 10 min at 4 °C. The supernatant was collected and chloroform (1/5th volume of Trizol) was added and shaken vigorously for 15 sec. The solution was kept at room temperature for 15 min and centrifuged at 12000xg for 15 min at 4 °C. The upper colorless aqueous layer was transferred to a fresh tube and isopropanol (1/2 volume of Trizol) was added. The solution was mixed properly and kept at room temperature for 5 min followed by centrifugation at 12000xg for 10 min at 4 °C. The pelleted RNA thus isolated was washed with 75% ethanol and air dried.The pellet was dissolved in diethylpyrocarbonate water. Five µg total RNA was reverse transcribed using Thermo Scientific kit into cDNA following the manufacturer’s instructions.Forty ng of cDNA was analyzed using SYBR-green master mix and PCR was performed for 40 cycles. PCR conditions were an initial denaturation of 94 °C for 2 min followed by 40 cycles of 94 °C for 20 sec of denaturation, 58 °C for 20 sec of annealing and 72 °C for 20 sec of elongation. A single cycle of 45 °C to 95 °C with 4 °C per sec of dissociation was given at the end of PCR to analyze the product. Cycle of Threshold (CT) values were noted down at the crossing point to determine the changes in gene expression. The calculations were made according to Livak and Shmittgen (2001). Primer sequences used are listed in Table 1.
2.7 Immunoblot
SH-SY5Y cells or striatal tissue samples were processed to extract the protein and quantified subsequently as described by Lowry et al. (1951), using bovine serum albumin as the standard.
An aliquot of 50 µg of protein was electrophoresed on a 10% sodium dodecyl sulphate acrylamide gel and transferred to polyvinylidene fluoride membrane. The membrane was then blocked with 10% (w/v) skimmed milk for 1 h, and incubated overnight with primary antibody at 4 °C. The blots were incubated with horseradish peroxidase conjugated secondary antibody for 2 h, and developed with 3,3’-diaminobenzidine and H2O2 or enhanced chemiluminiscence, and densitometry was performed using ImageJ software.
2.8 Calpain activity
Calpain activity was assayed fluorimetrically according to Bizat etal. (2003). SH-SY5Y cells or striata were homogenized in 10 volumes of ice-cold homogenization buffer. Lysate was
centrifuged at 15000xg, 15 min, at 4 °C. Calpain activity was measured by the cleavage of the substrate N-Suc-Leu-Tyr-AMC and by deriving the difference between calcium-dependent and calcium-independent fluorescence. Thirty µg of sample protein was incubated with 50 µM of the respective substrate buffers for 30 min at 37 °C. The fluorescence was measured at excitation of 340 nm and emission at 460 nm in a Perkin Elmer Spectrofluorimeter.
2.9 Caspase 3 activity
Fluorimetric assay for caspase 3 activity was performed according to Jeon etal. (1999). SH-SY5Y cells or striatum was homogenized in 10 volumes of ice-cold homogenization buffer.Lysate was spun at 15000xg, 15 min, 4 °C. The supernatant so obtained (30 µg protein equivalent) was incubated with caspase substrate buffer containing 50 µM of caspase 3 substrate N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin for 1 h at 37 °C. After the incubation,reaction was stopped by adding 100 µl ethanol. The fluorescence was measured at excitation of 340 nm and emission at 460 nm in a Perkin Elmer Spectrofluorimeter.
2.10 Statistics
One-way ANOVA followed by Dunnett’s post-hoc test was used for determining significant differences among different treatment groups. Values of p ≤ 0.05 were considered significant.
3. Results
3.1 Alterations in the mRNA expression levels of calpain and calbindin in SH-SY5Y and mouse striatum
Nimodipine and MPP+-treated SH-SY5Y cells or MPTP-treated mousestriatal RNA were isolated and cDNA prepared. Quantitative realtime PCR was performed to monitor the RNA expression levels of calcium regulatory protein, calbindin. Calbindin expression was reduced to one-tenth level after MPP+ (1 mM, 24 h) treatment,which was reverted to control level by nimodipine pretreatment (Fig. 1A). Calbindin expression was reduced by one-tenth after MPTP treatment, which was restored back to control values by nimodipine pretreatment (Fig. 1B).MPP+-treatment caused an increase in calpain expression by eight-folds as compared to control,which were significantly attenuated by nimodipine (10 µM, 15 min) pretreatment (Fig. 1C).MPTP (30 mg/kg, i.p. twice 16 h apart) caused increases in calpain by seventeen-folds as compared to control, which was significantly attenuated by one-time nimodipine pretreatment (10 mg/kg, i.p., once 30 min before the first MPTP dose) (Fig. 1D).
3.2 Effect of nimodipine on MPTP- or MPP+-induced alteration in protein expression levels of calcium related proteins in the mouse striata or SH-SY5Y cells
Whole cell protein was isolated from MPP+- and/or nimodipine-treated SH-SY5Y cells and Western blot was performed to monitor the expression levels of calcium related proteins.Calbindin expression was reduced by about 40% after MPP+-treatment that was partially attenuated by nimodipine (Fig. 2A). Whole cell protein was isolated from saline, MPTP and/or nimodipine treated mouse striata. MPTP-treated striatum showed about 40% reductions in calbindin level, which was corrected by nimodipine treatment (Fig. 2B). MPP+-treatment (1 mM) caused an increase in calpain expression by 1.5-folds as compared to control, which was significantly attenuated by nimodipine (10 µM, 15 min) pretreatment (Fig. 2C). A similar effect was observed in the striatum of mice treated with MPTP, which was corrected to control value by nimodipine treatment (10 mg/kg, i.p.) (Fig. 2D).
3.3 Effect of nimodipine on MPTP-induced alterations in the activity of calpain and caspase 3 in SH-SY5Y cells and mouse striata.
SH-SY5Y cells were processed for calpain activity assay as per Bizat etal. (2003) after toxin and nimodipine treatment. The calcium activated protease i.e., calpain activity was three-times higher in MPP+-treated group that was significantly attenuated by nimodipine treatment (Fig. 3A). Calpain activity was eight-folds higher in MPTP-treated group, which was effectively attenuated by nimodipine pretreatment (Fig. 3B).The SH-SY5Y cell extracts were processed for caspase 3 activity assay after MPP+ and/or nimodipine treatment. The apoptosis effector,caspase 3 activity was two-folds higher in MPP+- treated group, which was significantly attenuated by nimodipine treatment (Fig. 3C). Caspase 3 activity was two-folds higher in MPTP-treated group that was significantly attenuated by nimodipine treatment (Fig. 3D).
4. Discussion
Most of the experimental models of PD are based on mitochondrial toxins and this most consistent neurodegenerative movement disorder could be recapitulated in animals by injecting MPTP, apotent mitochondrial ETC complex 1 inhibitor. Mitochondrial dysfunction and aberrant intracellular Ca2+ homeostasis is linked and probably associated with dopaminergic neuronal demise in PD (Imai and Lu 2011; Lezi and Swerdlow 2012; Mohanakumar and Muralikrishnan 2000; Mohanakumaretal. 2002). The hypothesis is based on the notion that midbrain DA neurons will be resilient, if these contain calcium buffering proteins, so that these neurons could sequester excessive calcium without the use of ATP (Damier etal. 1999; German etal. 1992;Yuan et al. 2013). It is interesting to observe in literature that media analysis the expression of calbindin, the calcium buffering protein is higher in ventral tegmental area, while relatively lower in the PD susceptible SNpc region of the midbrain of postmortem human brains or MPTP-treated monkeys or C57/Bl mice. This suggested enhanced susceptibility of SNpc dopaminergic neurons to calcium-induced neuro-degeneration (German et al., 1992). It has been further demonstrated that the dopamine containing, calbindin-rich neurons of the SNpc ‘matrix’ are least affected than the calbindin-poor zones (‘nigrosomes’) of SNpc in postmortem human brains, and that the spatiotemporal progressive demise of these neurons is disease-duration-dependent (Damier et al.,1999). In this regard, an examination of voltage-gated calcium channel Ca(V)1 subtypes, and calcium binding proteins such as calbindin, calmodulin and calreticulin in PD postmortem brains when compared to control brains could not support such contentions (Hurley et al. 2013).However, the study clearly revealed an increased use of Ca(V)1.3 subtype neurons that resulted in increased metabolic burden for electrical activity resulting in more susceptibility of these neurons (Hurley et al., 2013). In these lines, in the past we have shown MPP+, the active metabolite of MPTP, to inhibit L-type voltage regulated calcium channel currents in cultured dorsal root ganglia neurons containing DA, and a significant increase in intracellular Ca2+ levels (Mohanakumaretal. 2002). The information available in literature suffices to the hypothesis that mitochondrial dysfunction and calcium homeostasis aberrations are central to the pathogenesis of PD. However, the exact molecular mechanism involved in MPTP-induced dopaminergic neurodegeneration has not been brought to light, yet.
We have demonstrated that the parkinsonian neurotoxin, MPP+ affects Ca2+ levels in the cells or in the mitochondria, which could be influenced by the L-type VDCC nimodipine. This led to the protection against experimental parkinsonism in vitro and in vivo (Singh etal. 2016). Dysregulated calcium homeostasis is one of the major factors implicated in the pathogenesis of PD (Nedergaard etal. 1993; Rivero-Ríos et al. 2014; Singh etal. 2016). In continuation with our finding, the present study investigated the calcium regulatory protein status that may influence the Ca2+ channel mechanism aiding in nimodipine-mediated neuroprotection. Calcium plays a very important role in cell signaling, particularly in neuronal transmission since membrane depolarization leads to calcium-mediated neurotransmitter flux at synapses, and in postsynaptic responses (Burnashev and Rozov 2005; Hartmann and Konnerth 2005; Mendez et al. 2011;Yuste etal. 2000). In the present study, we found that nimodipine ameliorates MPP+- or MPTP-induced increases in the calcium binding protein, calbindin and a decrease in the calcium-
dependent, cysteine protease, calpain in SH-SY5Y cells or in the striatum of mice, both at mRNA and protein levels, and restores calpain and caspase 3 activities.
Calpain is a calcium activated cysteine protease with two subunits: a small regulatory 30 KDa subunit and a large 80 KDa subunit. In neurons, there are two specific isoforms: µ -calapin
and m-calpain that are activated by µM and mM concentration of calcium, respectively (Croall and Demartino 1991). In response to toxic neuronal insults, intracellular calcium levels are
elevated. This leads to calpain activation and cleavage of target proteins (Ray etal. 2000).Calpastatin, an endogenous inhibitor of calpain, also becomes the substrate, thus causing an
imbalance in the cellular proteolytic mechanisms (Pontremoli etal. 1991). Cytoskeletal elements that maintain cellular integrity and act as pathway for vesicle trafficking also get degraded by activated calpain (Martin et al. 1995). Thus, neurotransmitters carrying vesicles would not reach the synaptic cleft, hindering the neuronal signaling. Neurotoxin-induced calpain activation leads to neuronal death (Rayetal. 2000), and a calpain inhibitor, SNJ 1945 was effective in rendering protection against MPP+- or rotenone-mediated dopaminergic neuronal death (Knaryanet al.2014).
The increase in the levels of Ca2+-activated neutral protease, calpain as observed in the present study, is analogous to increased expression and activity of calpain observed in dopaminergic neurons of postmortem PD brains (Mouatt-Prigent et al. 2000). Therefore, it is interesting that nimodipine could effectively block the MPP+- and MPTP-induced increases in calpain mRNA and protein levels, and its activity both in vitro and in vivo in dopaminergic neurons, thereby restricting dysregulated calcium increase in cells and block caspase 3 activity, a mitochondrial calcium-mediated apoptotic pathway signal. We have observed a significant increase in calpain activity following MPTP treatment in mice, which was partially blocked by nimodipine. The present results suggest that nimodipine’s ability to reduce the neurotoxin-induced increases in calpain levels and its activity could be instrumental in bringing out the neuroprotective effect. In this regard, it is interesting to observe that differentiated PC12 neurons, when treated with MPP+ resulted in activation of BimEL expression, affecting an increase in calpain I activity that in turn, caused apoptotic inducing factor release from the mitochondria, leading to the cell degeneration (Liou etal. 2005). Knocking down BimEL expression resulted in a decrease in calpain I activity,as well as apoptotic inducing factor release from the mitochondria and cell death (Liou etal.2005). In our studies, a similar mechanism might be operating, since caspase 3 activation could have stimulated apoptotic inducing factor release following MPP+ treatment.
Overall data from the present study further strengthen the hypothesis that perturbed calcium homeostasis underlies the pathophysiology of SNpc dopaminergic neuronal death leading to PD,
and nimodipine influences the calcium binding proteins and calcium regulated proteases in addition to the antagonistic action at L-type VDCC, and the related mitochondrial effects (Singh
et al 2016). We propose that the present findings have strong potential to use VDCC as a prophylactic agent or as a drug for PD patients. Specially, nimodipine, a commonly used anti-
hypertensive agent, can serve as a prophylactic medicine in subclinical and drug naïve PD patients to prevent the progression of PD. Furthermore, it will also be beneficial to individuals
with genetic predisposition and with family history of PD incidents.