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. Author manuscript; available in PMC: 2022 Sep 8.
Published in final edited form as: Neurotox Res. 2020 Aug 6;38(3):640–649. doi: 10.1007/s12640-020-00264-3

Cell permeable calpain inhibitor SJA6017 provides functional protection to spinal motoneurons exposed to MPP+

Supriti Samantaray a, Varduhi H Knaryan a, Angelo M Del Re b, John J Woodward b, Donald C Shields a, Mitsuyoshi Azuma c,d, Jun Inoue c, Swapan K Ray e, Naren L Banik a,*
PMCID: PMC9453439  NIHMSID: NIHMS1618765  PMID: 32761446

Abstract

Extra-nigral central nervous system sites have been found to be affected in Parkinson’s disease (PD). In addition to substantia nigra, degeneration of spinal cord motor neurons may play a role in the motor symptoms of PD. To this end, hybrid rodent VSC 4.1 cells differentiated into motoneurons were used as a cell culture model following exposure to Parkinsonian neurotoxicant MPP+. SJA6017, a cell-permeable calpain inhibitor, was tested for its neuroprotective efficacy against the neurotoxicant. SJA6017 attenuated MPP+-induced rise in intracellular free Ca2+ and concomitant increases in the active form of calpain. It also significantly prevented increased levels of proteases and their activities, as shown by reduced levels of 145 kDa calpain specific and 120 kDa caspase-3 specific spectrin breakdown products. Exposure to MPP+ elevated the levels of reactive oxygen species in VSC 4.1 motoneurons; this was significantly diminished with SJA6017. The motor proteins in spinal motoneurons, i.e. dynein and kinesin, were also impaired following exposure to MPP+ through calpain-mediated mechanisms; this process was partially ameliorated by SJA6017 pre-treatment. Cytoprotection provided by SJA6017 against MPP+-induced damage to VSC 4.1 motoneurons was confirmed by restoration of membrane potential via whole cell patch clamp assay. This study demonstrates that calpain inhibition is a prospective route for neuroprotection in experimental PD; moreover, calpain inhibitor SJA6017 appears to be an effective neuroprotective agent against MPP+-induced damage in spinal motoneurons.

Keywords: calpain, Parkinson’s, motor proteins, apoptosis, oxidative stress

1. Introduction

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder that affects 1% of the U.S. population above the age of 65. While current L-DOPA therapy provides temporary relief to patients, it does not ultimately halt disease progression. Many PD symptoms are related to dopaminergic substantia nigra cell death and the resulting loss of domaminergic inputs to various CNS structures. Histological evaluation of these neurons in postmortem PD samples reveals gliosis, neurofibrillary tangles, and Lewy body formation. The latter are composed of alpha-synuclein protofibrils which are rare in normal brain samples (Warner et al. 2003; Banerjee et al. 2009; Tanner 2003). Several extra-nigral and non-dopaminergic sites in CNS have also been shown to undergo degeneration in PD. In addition to midbrain structures, involvement of spinal cord (SC) motoneurons may be crucial in regulating the motor symptoms of PD (Samantaray et al. 2015). Furthermore, transplanted stem cells (much younger than host cells and of different genetic make-up) into Parkinsonian patients developed alpha-synuclein protofibril formation (Desplats et al. 2009). Thus, the extracellular milieu and neuronal connections related to neuronal degeneration in PD require further study.

The use of an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD animal model allows for effective dopaminergic neuron analysis in both rodents and non-human primates. MPTP administration in these animals results in behavioral, biochemical, and pathological deficits as a result of nigral dopaminergic neuron destruction (Kass et al. 1988; Mizuno et al. 1987). An MPTP metabolite, MPP+, impairs the mitochondrial electron transport chain through irreversible inhibition of Complex I with a resulting increase in cytosolic intracellular calcium levels (Mandavilli et al. 2000). Cytosolic calcium levels are normally decreased by the plasma membrane Na+/Ca2+ exchanger, plasma Ca2+-ATPases, and calcium binding proteins such as calbindin (Hof et al. 1999). Moreover, intracellular calcium levels can be altered with transport into and release from mitochondria (Nicholls et al. 2003).

Intracellular calcium levels are typically well controlled by the neuron, such that calcium fluxes during cell depolarization last only a few seconds to minutes, with no pernicious cellular effects. However, in some pathological conditions, effective cellular buffering of calcium fluctuations is compromised. Prior to cell death, significant intracellular calcium accumulation can be associated with Na+/Ca2+ exchanger dysfunction. Bano et al. 2005 demonstrated that cleavage of this plasma membrane protein by a calcium-activated neutral proteinase, calpain, is a critical step in calcium dysregulation. Calpain is a nonlysosomal calcium-activated neutral protease which selectively cleaves multiple diverse cellular proteins without complete degradation. Multiple calpain isoforms have been isolated in various organ systems. This highly conserved family of cysteine proteases include the ubiquitous classic isoforms μ- and m-calpain. The former requires 3–50μmol/L calcium concentrations for half-maximal activity in vitro, while m-calpain is similarly activated at 400–800μmol/L calcium concentrations (Goll et al. 2003). While the above isoforms and calpain 10 (believed to be specific to mitochondria) are ubiquitously expressed, some calpain isoforms are found only in selective tissues (Smith & Schnellmann 2012). Although mice μ-calpain knockout models survive with defective platelet function, an m-calpain knockout is lethal in embryos (Bevers & Neumar 2008). Under normal physiological conditions, calpain degrades a wide variety of cellular proteins (cytoskeletal, receptor, mitochondrial, myofibrillar, enzymes, histones, and myelin proteins) as required for normal cellular signaling and metabolism; however, in the presence of increased intracellular calcium levels, calpain activity is poorly regulated by the endogenous inhibitor, calpastatin (Vosler et al. 2008). Excessive calpain activation has been linked to multiple neuroinflammatory mechanisms (e.g., T cell activation and chemotaxis) in various neurodegenerative disorders, including multiple sclerosis and PD (Butler et al. 2009; Levesque et al. 2010; Samantaray et al. 2015). Thus, calpain isoforms play a critical role in normal cellular function, but in pathological conditions with increased calcium concentrations, elevated calpain activity can have multiple deleterious effects leading to cell death.

In addition to Na+/Ca2+ exchanger cleavage (Bano et al. 2005), excessive calpain activation further contributes to cellular calcium dysregulation by degrading the plasma membrane calcium ATPase, L-type calcium channel, inositol (1,4,5) triphosphate receptor and inositol triphosphate kinase B (DeJongh et al. 1994; Inserte et al. 2005; Kopil et al. 2012). Calpain also cleaves alpha-synuclein into truncated subunits which are prone to aggregate, as observed in Lewy bodies (Duffy et al. 2007). Impaired axonal transport associated with reduction of microtubule motor proteins (dynein and kinesin) has also been linked to alpha-synuclein aggregation in dopaminergic neurons in PD (Chu et al. 2012). Moreover, calpain inhibition has been shown to prevent cytoskeletal protein degradation, preserve membrane integrity, and block apoptosis (Smith & Schnellmann 2012). Thus, intracellular calcium dysregulation and associated calpain over-activation results in alpha-synuclein protofibril deposition, mitochondrial/plasma membrane calcium permeability, and ultimate dopaminergic neuronal cell death.

Selective neurodegeneration of SC motoneurons in vivo following MPTP-administration was previously demonstrated (Samantaray et al. 2015). This was ameliorated by administration of calpain inhibitors. Likewise, in vitro studies demonstrate that MPP+ induces programmed cell death in various neuronal cell line (Callizot et al. 2019). The apoptotic mechanism involves the proteolytic activity of one or more cysteine proteases, such as calpain and caspase-3 (Zhao et al. 2017). We have therefore investigated the susceptibility of motoneuron cells (VSC 4.1) to MPP+ in vitro, and the findings indicate that MPP+ treatment caused increased intracellular calcium levels, calpain activation, and motoneuron death that was attenuated by calpain inhibitor SJA6017 [N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal] administration.

2. Materials and Methods

2.1. Materials

Hybrid cell line VSC 4.1 was generously gifted by Dr. Stanley H. Appel (Houston, TX, USA). VSC 4.1 is a motoneuron-neuroblastoma hybrid cell line derived by somatic fusion of rat parental embryonic day-15 ventral spinal neuron preparation and mouse N18TG2 neuroblastoma culture; hybrid selections were performed as previously described (Crawford et al. 1992). Dulbecco’s modified Earle’s medium (DMEM)/Ham’s F12 50/50 Mix with l-glutamine and 15 mM HEPES was procured from Cellgro (Mediatech, Manassas, VA, USA). Heat-inactivated fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). Penicillin (100 IU/ml), streptomycin (100 μg/ml), dibutyryl cAMP, aphidicolin, poly-L-ornithine, dimethyl sulphoxide (DMSO), 3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and MPP+ were obtained from Sigma-Aldrich (St. Louis, MO, USA). Neurotoxic compound MPP+ was handled according to the regulations of the institutional Health and Biosafety Committee. Fluorescent CM-H2DCFDA dye was purchased from LifeTechnologies (Grand Island, NY, USA), and ratiometric Fura-2 AM dye was from Molecular Probes (Carlsbad, CA, USA). Vectashield™ was obtained from Vector Laboratories (Burlingame, CA, USA). Precast sodium dodecyl sulfate–polyacrylamide gels were obtained from Bio-Rad Laboratories (Hercules, CA, USA), and Immobilon™-P polyvinylidene fluoride micro-porous membranes were from Millipore (Bedford, MA, USA). Calpain inhibitor SJA6017 was procured from EMD Biosciences (Gibbstown, NJ, USA).

The primary IgG antibodies: rabbit anti-caspase-3 polyclonal (1:250), were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-DYNLT3 antibody polyclonal (1:100) and rabbit anti-kinesin polyclonal (1:500) were from Abcam Discoveries (Cambridge, MA USA). Rabbit anti-m-calpain polyclonal (1:500) was raised in Dr. Banik’s laboratory (Banik et al., 1983). Mouse monoclonal anti-β-actin (1:15,000) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and mouse monoclonal anti-α-spectrin (1:10,000) was from Biomol International (Plymouth Meeting, PA, USA). Peroxidase-conjugated goat anti-rabbit and anti-mouse secondary IgG antibodies (1:2000) were obtained from MP Biomedicals (Solon, OH, USA).

2.2. Cell culture, differentiation, and treatments

Early passages VSC 4.1 cells were cultured in 75-cm2 flasks (Corning, NY, USA) coated with 0.01% poly-l-ornithine in 0.6% boric acid solution (pH 8.4) using complete medium of DMEM)/Ham’s F12 50/50 Mix with l-glutamine and 15 mM HEPES supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml), 2% Sato’s components and 2% of heat-inactivated FBS. VSC 4.1 cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was refreshed every other day. At 3–4 days cells that attained 60–70% confluence were redistributed at a density of 106 cells in 75-cm2 flasks and treated with differentiating agent cAMP (0.5 mM). Bystander killing was attained with aphidicolin (0.4 μg/ml) on every other day. Cells were terminally differentiated into spinal motoneurons by the seventh day. Differentiated VSC 4.1. motoneurons were exposed to neurotoxicant, MPP+, or pretreated before exposure with SNJ6017. Incubation with neurotoxicant and/or SJA6017 was conducted in a low-serum containing medium (0.5% FBS). For certain experiments cells were disseminated in six-well plates at a density of 2.5–5×104 cells/well, where treatments were performed. For cytoprotective studies two doses of SJA6017 were used, from which 100 μM was chosen.

2.3. Cell viability assay

Cell viability was assessed using MTT based on the reduction of bright yellow MTT dye to dark blue formazan crystals by the mitochondrial oxidoreductase enzymes in the living cells. Thirty minutes before exposure to MPP+ cells were incubated with SJA6017 (10 and 100 μM) at 37°C, the medium was discarded, and cells were further incubated in a medium containing MPP+ (5–200 μM) for 24 hours. Following toxicant exposure the medium was replaced with low serum medium containing MTT reagent (0.1 mg/mL) and incubated at 37°C for 1 hour. Formazan crystals were precipitated by centrifugation at 1900×g (Eppendorf Centrifuge 5804R, Germany) and the medium was aspirated. Crystals were dissolved in DMSO and transferred into 96-well plates. The absorbtion of dissolved formazan was measured at 570 nm with reference wavelength set at 630 nm in Emax Precision Microplate reader supported by SoftMax Pro software Molecular Devices (Sunnyvale, CA, USA). Estimated optical density values were compared setting the control at 100%.

2.4. Measurement of intracellular Ca2+

The level of intracellular free Ca2+ or i[Ca2+] was measured spectrofluorometrically using ratiometric Fura-2 AM dye following the original method (Grynkiewicz et al., 1985) with some modifications (Butler et al., 2009). Cells were incubated in a medium, containing MPP+ (100 μM) at 37°C for 24 hours. The medium was discarded, cells were rinsed with fresh medium free of toxicant, suspended in 2 ml of modified Locke’s buffer [154 mM NaCl:, 5.6 mM KCl:, 3.4 mM NaHCO3:, 1.2 mM MgCl2:, 5.6 mM glucose:, 5 mM Hepes: (pH 7.4), and 2.3 mM CaCl2], and counted on hemecytometer. To equilibrate the cell number in the control and toxicant-exposed sets, where the cell death was higher than in control, cells were pooled at concentrations of 1×106 cells/ml, then incubated with Fura-2 AM (5 μM) at 37°C for 30 minutes. Cells loaded with fluoroprobe were centrifuged and rinsed twice with ice-cold Locke’s buffer. The fluorescence ratio (R) was measured at 510 nm emission with dual excitement at 340 nm and 380 nm wavelengths on an SLM 8000 Thermospectronic fluorometer. Concentration of i[Ca2+] was calculated by the equation i[Ca2+] =Kd(R−Rmin)/(Rmax−R). Maximal (Rmax) and minimal (Rmin) ratios were determined using 25 μM digitonin and 5 mM EGTA, respectively. The comparative changes of i[Ca2+] levels were expressed as percentages (%).

2.5. Determination of intracellular reactive oxygen species (ROS)

Production of ROS in the living cells was measured using cell-permeable ROS detecting fluorescent dye CM-H2DCFDA. Following treatment procedures, cells were gently harvested with warm Hank’s Balanced Salt Solution (HBSS, 1X, Cellgro), transferred from flasks into tubes and spun. Pellets were suspended in HBSS and incubated with 10 μM of CM-H2DCFDA at 37°C for 30 minutes. After short centrifugation the excess dye was aspirated; cells were suspended with warm HBSS and transferred into 24-well plates. The end-point arbitrary fluorescent units were recorded on an SLM 8000 Thermospectronic fluorometer, setting 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 on them and processed for total intracellular ROS assay. Fluorescent images were captured with an Olympus BH-2 microscope at 200x magnification.

2.6. Immunocytofluorescent staining

Cells were cultured and differentiated on sterile glass cover slips inserted within 6-well plates. After treatment procedures, plates were centrifuged at 150 x g for 10 minutes to settle down less adherent apoptotic cells on cover slips. Subsequently, cells were fixed with 95% ethanol for 10 minutes, rinsed twice with PBS, blocked with goat serum in PBS for 1 hour, and incubated with primary antibodies against dynein or kinesin overnight at 4°C. Immunostaining was visualized with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary IgG (green). Cover slips with cells were removed from wells and flipped onto microscopic slides containing a drop of antifade Vectashield™. Fluorescent images were viewed and captured with an Olympus BH-2 (Melville, NY) microscope at 200× magnification.

2.7. Immunoblotting

The levels of active protease expression including calpain (inactive 80 kDa and active 76 kDa), 32 kDa pro-caspase-3, 20 kDa and 12 kDa active caspase-3 forms were determined by Western blot analysis. Proteolytic activities of calpain and caspase-3 were evaluated through determination of calpain-specific spectrin breakdown products (SBDP) as 145 kDa bands, and caspase-3-specific SBDP (120 kDa band). Cells post-treatment were harvested into 15 ml tubes and centrifuged at 500 × g for 10 minutes. Cell pellets were suspended in an ice-cold homogenizing buffer [50 mM Tris–HCl (pH 7.4), 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride], sonicated (Kontes Micro Ultrasonic Cell Disrupter) and kept on ice (15 minutes) to ensure total protein extraction. Probes were diluted (1:1) with sample buffer [62.5 mM Tris–HCl, pH 6.8, 2% sodium dodecyl sulfate, 5 mM β-mercaptoethanol, 10% glycerol] and boiled (10 minutes). In between, protein concentration was measured spectrophotometrically at 595 nm (Spectronic, Rochester, NY, USA) using Coomassie plus Protein Assay Reagent (Pierce, Rockford, IL, USA). Protein was adjusted to a final concentration of 1.5 mg/ml with 1:1 v/v mix of sample and homogenizing buffer, containing bromophenol blue dye (0.01%). Protein samples were resolved in 4–20% or 7.5% (for SBDP) precast sodium dodecyl sulfate–polyacrylamide gels at 100 V for 1 hour - then transferred to the Immobilon™-P polyvinylidene fluoride micro-porous membranes in a Genie transfer apparatus (Idea Scientific, Minneapolis, MN). Membranes were consequently incubated with 5% non-fat milk in Tris-HCl buffer (20 mM Tris–HCl, pH 7.6, containing 0.1% Tween-20) with appropriate primary IgG antibodies (4°C) and horseradish peroxidase-conjugated corresponding secondary IgG antibodies at room temperature. Between incubations membranes were rinsed three times for 5 minutes in Tris-buffer. Immunoblots were then incubated with enhanced chemiluminescent reagents (ECL or ECL Plus, Amersham Biosciences, UK); reactive protein bands were visualized on Alpha-Innotech using FluorChem FC2 Imaging System (Cell Biosciences, Santa Clara, CA, USA). All immunoblots except those for spectrin were re-probed with an antibody against β-actin, which served as the corresponding protein loading controls. Optical density of the immunoreactive protein bands were analyzed with NIH ImageJ 1.45 software.

2.8. Whole-cell patch clamp electrophysiology

The functionality of VSC 4.1 cells following treatments was recorded using a whole-cell patch clamp technique. Cells were grown on 35-mm cell culture dishes and placed on the microscope stage (Olympus IX70, Melville, NY, USA) after treatment procedures. Culture media was replaced by continuously perfused recording solution containing: 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 10 mM glucose, 5 mM HEPES (pH 7.2, osmolarity 325 mOsm). The pipette recording solution contained 150 mM KCl, 2.5 mM, 4 mM Mg-ATP, 2 mM Na2ATP, 0.3 mM NaGTP, 5 mM Na2phosphocreatine, and 10 mM HEPES (pH 7.4, osmolarity 325 mOsm).

Patch clamp recordings were made using an Axopatch 200B amplifier equipped with a CV203BU thermal cooled headstage (Axon Instruments, Union City, CA, USA) and viewed using AxoGraph software. Cells were clamped at −60 mV initially; then the resisting membrane potential was directly read from the amplifier by setting the clamp control to current = 0 setting. Membrane capacitance was read from AxoGraph and at the cell’s resting membrane potential. Membrane capacitance was used to determine relative cell size, as this value reflects the size of the neuron, indicating the total number of ion channels in the membrane.

2.9. Statistics

Each assay was performed in triplicate. Results were assessed in Stat View software (Abacus Concepts, Berkley, CA) and compared by using one-way analysis of variance (ANOVA) with Fisher’s protected least significant difference (PLSD) post hoc test at 95% confidence interval. Data were expressed as mean ±SEM; the difference between groups was considered significant at p≤0.05 at n ≥ 3.

3. Results

3.1. SJA6017 exhibits cytoprotective effects in differentiated VSC 4.1 motoneurons (dVSC 4.1) following MPP+ exposure

Cultured differentiated VSC4.1 cells were divided into sets, including controls, and cells were exposed to different doses of MPP+ (5, 10, 50, 100, 200 μM); some cells were pretreated with two doses of SJA6017 (10 and 100 μM) for 30 minutes before exposure to MPP+. MTT assay, performed after 24 hours of incubation, showed decreasing cell viability in MPP+-exposed cells in a dose-dependent manner (Fig. 1). About 50% of cell viability decrease was attained at 100 μM of MPP+ compared to controls considered 100% viable (*p ≤ 0.05). Cells pretreated with the lower concentration of SJA6017 (10 μM) were significantly resistant to toxicity induced by MPP+ at concentrations of 5, 10, and 50 μM. Pretreatment of cells with SJA6017 at a concentration of 100 μM significantly increased their viability, when exposed to high doses of MPP+ (100 and 200 μM) (#p ≤ 0.05). For subsequent experiments SJA6017 was used at a concentration of 100 μM, which was considered effective against 100 μM MPP+.

Fig. 1.

Fig. 1

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Cytoprotective effects of SJA6017 against MPP+-induced toxicity in VSC 4.1 cells. Viability of VSC 4.1 cells was measured by MTT assay after 24-hour exposure to MPP+ (5, 10, 50, 100, 200 μM). Cell viability diminished in a dose-dependent manner with the two doses of SJA6017; 100 μM was more effective. Bar graphs represent data expressed as mean±SEM of cell viability (%), n≥4; (*p≤0.05, significantly different from viability of control cells; #p ≤ 0.05, significantly different from viability of MPP+-exposed cells)

3.2. SJA6017 diminished MPP+-induced i[Ca2+] load, increased cell viability, and reduced ROS production in VSC 4.1 cells

Differentiated VSC 4.1 cells were divided into 4 sets: control, SJA6017-treated cells, cells exposed to MPP+, and cells pretreated with SJA6017 (100μM) then exposed to MPP+ (100μM). Incubation of cells with SJA6017 alone did not cause significant changes in the level of i[Ca2+] compared to the control group (Fig. 2, A). Exposure to MPP+ for 24 hours led to a significant rise of the i[Ca2+] compared to control (*p ≤ 0.05), and the measured i[Ca2+] was 4-fold higher than i[Ca2+] in control cells. Parallel monitoring of cell viability in these experiments showed that cell treatment with SJA6017 alone did not affect cell survival - similar to the control cells. Upon exposure to MPP+ cell viability was reduced by 50% (*p ≤ 0.05), concomitant with MPP+-induced i[Ca2+] rise. However, cells pretreated with SJA6017 were highly resistant to MPP+ cytotoxicity and showed nearly 50% survival compared to the MPP+-exposed cells (#p ≤ 0.05) (Fig. 2, B). Total ROS production in VSC 4.1 cells was detected following incubation with dye CM-H2DCFDA. Representative fluorescent images showed discernible ROS production in the cells exposed to MPP+ for 24 hours; ROS fluorescent signals were significantly diminished (>50%) in the cells pretreated with SJA6017 (Fig. 2, C).

Fig. 2.

Fig. 2

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Effects of SJA6017 (100 μM) on the i[Ca2+] load (A), cell viability (B) and ROS production (C) in VSC 4.1 motoneurons following exposure to MPP+ (100 μM). (A, B) Bar graphs represent data expressed as mean±SEM of i[Ca2+] (nM) and cell viability (%), respectively; n≥4; (*p≤0.05, significantly different from control; #p ≤ 0.05, significantly different from MPP+-exposure effect). (C) Representative fluorescent images of ROS in control, MPP+-exposed, and SJA6017+MPP+ VSC 4.1 motoneurons (200x magnification)

3.3. Efficacy of SJA6017 against MPP+-induced upregulation of calpain and caspase-3 expression/activity.

We evaluated the predicted upregulation of calpain and caspase-3 vis-à-vis the MPP+-induced rise in i[Ca2+] by Western blot analysis. Representative immunoblots (Fig. 3, A, B) and densitometric analysis of reactive immune bands showed that exposure of dVSC 4.1 motoneurons to MPP+ (10, 50, and 100 μM) caused significant (*p ≤ 0.05) dose-dependent increase of calpain (80 kDa) and pro-caspase-3 (32 kDa) expression, compared to control and SJA6017-treated cells. Likewise, significant (*p ≤ 0.05) dose-dependent changes were found in the levels of active 20 kDa and 12 kDa caspase-3 forms, as well as that of 145 kDa calpain and 120 kDa caspase-3 specific SBDP, compared to controls (Fig. 3, C, D). However, pretreatment of cells with SJA6017 (100 μM) before exposure to MPP+ significantly (#p ≤ 0.05)) diminished changes in calpain, pro-caspase-3 and active caspase-3 levels, compared to the MPP+-exposed cells. Down regulation of proteases by SJA6017, in turn, significantly (#p ≤ 0.05) reduced formation of calpain- and caspase-3 specific SBDP, compared to the MPP+-exposed cells. This agrees with previously reported dose-dependent elevations in i[Ca2+] in ChAT-positive VSC 4.1 cells exposed to MPP+ or rotenone (Samantaray et al. 2011).

Fig. 3.

Fig. 3

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MPP+-induced upregulation of calpain and caspase-3 in VSC 4.1 motoneurons and effects of SJA6017. (A) Representative immunoblots of 80 kDa calpain, 32 kDa procaspase-3, 20 kDa and 12 kDa active caspase-3, 145 kDa calpain- and 120 kDa caspase-3-specific SBDP. (B, C, D) Bar graphs represent changes (%) of protease and SBDP levels; data expressed as mean±SEM; n≥4; (*p≤0.05, significantly different from control; #p ≤ 0.05, significantly different from MPP+-exposure effect).

Experimental design: 1: control; 2: SJA6017 (100 μM); 3: MPP+ (10 μM); 4: SJA6017 (100 μM)+MPP+ (10 μM); 5: MPP+ (50 μM); 6: SJA6017 (100 μM)+MPP+ (50 μM); 7: MPP+ (100 μM); 8: SJA6017 (100 μM)+MPP+ (100 μM)

3.4. Amelioration of MPP+ motor protein toxicity in VSC 4.1 motoneurons with SJA6017

Exposure of VSC 4.1 motoneurons to MPP+ (100 μM) reduced the levels of motor proteins kinesin and dynein as evidenced by immunofluorescent staining. Reduction of kinesin and dynein immunoreactivity (IR) in the motoneurons exposed to MPP+ compared to control cells is shown in the upper and lower panels, respectively (Fig. 4). However, pre-treatment with SJA6017 (100 μM) partially prevented kinesin and dynein loss compared to the low IR in the population of MPP+-exposed cells.

Fig. 4.

Fig. 4

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MPP+-induced deterioration of motor proteins in VSC 4.1 motoneurons and effects of SJA6017. Representative fluorescent images show immunoreactivity (IR) of kinesin and dynein in control cells, reduced IR following exposure to MPP+ and partial restoration of IR in SJA6017-pretreated cells (200x magnification)

3.5. Functional restoration of VSC 4.1 motoneurons with SJA6017

The electrophysiological functionality of the VSC 4.1 motoneurons was measured using whole-cell patch clamp techniques. Two cell types were used in these experiments: those differentiated with cAMP and undifferentiated VSC 4.1 cells. Resting membrane potentials were measured in controls, cells exposed to MPP+, and MPP+ exposed cells pretreated with SJA6017. Whole cell patch-clamp recordings were performed after 18 hours of exposure to MPP+ (50μM) and SJA6017 (100μM). A limited number of surviving cells was found in the MPP+-exposed group (MTT assay); nevertheless, measurements showed that resting membrane potentials in control differentiated VSC 4.1 cells were lower than in undifferentiated cells (Fig. 5). A significant decline (*p ≤ 0.05) of the resting membrane potentials was found in both types of VSC 4.1 cells exposed to MPP+ compared to control. Pre-treatment with SJA6017 effectively maintained the cellular membrane potential and electrophysiological functionality of VSC 4.1 cells exposed to MPP+ toxicity.

Fig. 5.

Fig. 5

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MPP+-induced decline of the resting membrane potential of VSC 4.1 motoneurons. Bar graphs show significant reduction of the resting membrane potential in undifferentiated (A) and differentiated (B) VSC 4.1 cells after exposure to MPP+ compared to those with SJA6017 pretreatment. Data expressed as mean±SEM; n=3; (*p≤0.05, significantly different from control; #p ≤ 0.05, significantly different from MPP+-exposure effect)

4. Discussion

Calpain inhibitor SJA6017 is a water–soluble and cell permeable synthetic peptide aldehyde that reversibly binds to the active site of m-calpain (Inoue et al. 2003). SJA6017 is known as a potential therapeutic agent in eye disorders, including lens opacification (Biswas et al. 2004) and retinal pathologies (Azuma & Shearer 2008). Moreover, the neuroprotective efficacy of SJA6017 has been demonstrated in experimental traumatic brain injury (Kupina et al. 2001) and spinal cord injury (Akdemir et al. 2008; Kumar et al. 2014). With respect to mechanism of action, inhibition of calpain activity by SJA6017 reduces proteolytic breakdown of calpain substrates: crystallins, alpha-spectrin and microtubule-associated tau proteins (Warren et al. 2007).

In the present study administration of SJA6017 ameliorated large intracellular calcium spikes, reduced apoptotic cell death and maintained cell functionality in motoneuron cells in vitro following MPP+ administration. These findings are in part related to the role of calpain in neuronal mitochondria and the effects of MPTP on this system. Among other functions, mitochondria are involved in cellular apoptosis regulation, calcium buffering, and energy production. The latter is achieved through the five-complex electron transport chain traversing the inner mitochondrial membrane. With normal physiological conditions, a small percentage of electrons can leak from Complexes I and III; superoxide is thus formed when the electrons react with oxygen in the cell (Takeshige & Minakami 1979; Beyer 1992). Mitochondrial antioxidants including manganese superoxide dismutase can convert the superoxide to hydrogen peroxide, which glutathione converts to water. Thus, under normal conditions, the small amount of ROS can be rendered harmless to the cell, but this may not be true in neurons of Parkinsonian patients. As they have significant energy requirements and relatively large numbers of mitochondria, neurons in general are sensitive to mitochondrial dysfunction. Analyses of postmortem substantia nigra samples from Parkinsonian patients revealed a deficiency of Complex I – also observed in frontal cortex and peripheral tissues of these patients (Schapira et al. 1990; Mizuno et al. 1989; Parker et al. 2008). Indeed MPTP has been linked with Complex I inhibition (Nicklas et al. 1987). This molecule crosses the blood brain barrier to be converted to MPP+ by monoamine oxidase B in astrocytes. Once in neurons, MPP+ enters the mitochondria via passive transport where the toxic metabolite inhibits Complex I, leading to: ROS generation, cellular energy deficits, and ultimately cell death (Przedborski et al. 2000; Cleeter et al. 1992).

In the setting of mitochondrial Complex I dysfunction, resulting cellular ATP deficits cause partial depolarization of the cell (via reduced sodium/potassium ATPase activity) (Sherer et al. 2002). The resulting excitotoxicity from over-activation of NMDA receptors is also related to increased cytosolic calcium concentrations. In addition, dysfunctional mitochondria store calcium less effectively, further destabilizing cytosolic calcium equilibrium. In such an environment of elevated intracellular calcium, calpain is excessively activated with many potential deleterious effects. Of note, cleavage of the transmembrane sodium/calcium exchanger by μ-calpain causes cellular calcium elevation and release of apoptosis inducing factor (Ozaki et al. 2007; Kar et al. 2010). Both μ- and m-calpain have been localized in mitochondria in some tissues (Arrington et al. 2006; Kar et al. 2010). Likewise, one of the atypical calpains (calpain 10) is characterized by an N-terminus mitochondrial targeting sequence, and has been found in all mitochondrial compartments (Smith & Schnellmann 2012). Calpain 10 has also been found to cleave Complex I and ATP synthase (Arrington et al. 2006). Calpain 10 may therefore be involved normally in electron transport chain protein regulation, while overexpression of calpain 10 causes decreased mitochondrial respiration and ultimately cell death (Smith & Schnellmann 2012).

As suggested by the results in this study, calpain has been shown to cleave several proteins that regulate apoptosis. Multiple Bcl2 family proteins including Bid are calpain substrates; calpain degrades the antiapoptotic Bcl-xL protein resulting in a proapoptotic substrate (Bevers & Neumar 2008). Calpain activation also appears to precede caspase-3 activation, as active calpain cleaves caspase-3 (with possible enzyme activation) with resulting cellular DNA damage and ultimately apoptosis (Bevers & Neumar 2008; Smith & Schnellmann 2012). Of note, calpain also cleaves p35, and the corresponding p25 substrate binds with cyclin dependent kinase 5 to become proapoptotic (Smith et al. 2006).

In addition to apoptosis, the cause of elevated intracellular calcium in response to MPTP administration is likely multifactorial, but MPP+ dependent inhibition of complex I may lead indirectly to mitochondrial release of calcium into the cytoplasm. SJA6017 inhibition of calpain likely prevented excessive activation of calpain, thereby limiting degradation of the transmembrane sodium/calcium exchanger, which plays a major role in expelling calcium from the cell. However, the mechanism of intracellular calcium regulation in the setting of calpain inhibition remains poorly understood, as Butler et al., 2009 demonstrated treatment with calpain inhibitors (calpeptin and PD150606) results in elevated intracellular free calcium in a concentration dependent manner in a Jurkat E6–1 T cell line.

Thus, the calpain mediated degradation of multiple substrates (especially related to mitochondria dysfunction and apoptosis) following MPTP administration plays an important role in neuronal cell viability. The limitations of this study in cell culture do not allow for evaluation of astrocytic and microglial participation in neuronal cell death. Moreover, SJA6017 selective inhibition of various calpain isoforms is not completely understood, so the specific involvement of ubiquitous calpains versus the atypical Calpain 10 will require further studies with isoform-specific inhibitors and μ-calpain knockout animal models. Nonetheless, these results support the potential role of calpain inhibition for prevention of dopaminergic cell death in Parkinsonian patients.

Acknowledgments

This work was supported in part by NIH-NINDS (R01NS062327-01A2), Veterans Administration (1I01BX004269-01), and the South Carolina State Spinal Cord Research Fund (SCIRF-2015P-01, SCIRF-2015P-04, SCIRF-2015-I-01, SCIRF-2016 I-03, and SCIRF #2018 I-01). The hybrid cell line VSC 4.1 was a gift from Dr. Stanley H. Appel (Houston, TX, USA).

Abbreviations:

dVSC 4.1

differentiated ventral spinal cord cells

MTT

3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide

IR

immunoreactivity

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

PD

Parkinson’s disease

ROS

reactive oxygen species

SBDP

spectrin breakdown products

SC

spinal cord

Footnotes

Ethics Approval: No human or animal subjects were included in this study.

Consent to Participate: Not applicable

Consent for Publication: Not applicable

Code availability: Not applicable

Conflicts of Interest: The authors have no conflicts of interest related to this study.

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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