Cell-Permeable Calpain Inhibitor SJA6017 Provides Functional Protection to Spinal Motoneurons Exposed to MPP.

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 pretreatment. 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.

Calpain mediated expansion of CD4+ cytotoxic T cells in rodent models of Parkinson's disease.

Parkinson's disease (PD), a debilitating progressive degenerative movement disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra (SN), afflicts approximately one million people in the U.S., including a significant number of Veterans. Disease characteristics include tremor, rigidity, postural instability, bradykinesia, and at a cellular level, glial cell activation and Lewy body inclusions in DA neurons. The most potent medical/surgical treatments do not ultimately prevent disease progression. Therefore, new therapies must be developed to halt progression of the disease. While the mechanisms of the degenerative process in PD remain elusive, chronic inflammation, a common factor in many neurodegenerative diseases, has been implicated with associated accumulation of toxic aggregated α-synuclein in neurons. Calpain, a calcium-activated cysteine neutral protease, plays a pivotal role in SN and spinal cord degeneration in PD via its role in α-synuclein aggregation, activation/migration of microglia and T cells, and upregulation of inflammatory processes. Here we report an increased expression of a subset of CD4+ T cells in rodent models of PD, including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mice and DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride]/6-hydroxydopamine rats, which produced higher levels of perforin and granzyme B - typically found in cytotoxic T cells. Importantly, the CD4+ cytotoxic subtype was attenuated following calpain inhibition in MPTP mice, suggesting that calpain and this distinct CD4+ T cell subset may have critical roles in the inflammatory process, disease progression, and neurodegeneration in PD.

Chronic intermittent ethanol induced axon and myelin degeneration is attenuated by calpain inhibition.

Chronic alcohol consumption causes multifaceted damage to the central nervous system (CNS), underlying mechanisms of which are gradually being unraveled. In our previous studies, activation of calpain, a calcium-activated neutral protease has been found to cause detrimental alterations in spinal motor neurons following ethanol (EtOH) exposure in vitro. However, it is not known whether calpain plays a pivotal role in chronic EtOH exposure-induced structural damage to CNS in vivo. To test the possible involvement of calpain in EtOH-associated neurodegenerative mechanisms the present investigation was conducted in a well-established mouse model of alcohol dependence - chronic intermittent EtOH (CIE) exposure and withdrawal. Our studies indicated significant loss of axonal proteins (neurofilament light and heavy, 50-60%), myelin proteins (myelin basic protein, 20-40% proteolipid protein, 25%) and enzyme (2', 3'-cyclic-nucleotide 3'-phosphodiesterase, 21-55%) following CIE in multiple regions of brain including hippocampus, corpus callosum, cerebellum, and importantly in spinal cord. These CIE-induced deleterious effects escalated after withdrawal in each CNS region tested. Increased expression and activity of calpain along with enhanced ratio of active calpain to calpastatin (sole endogenous inhibitor) was observed after withdrawal compared to EtOH exposure. Pharmacological inhibition of calpain with calpeptin (25 µg/kg) prior to each EtOH vapor inhalation significantly attenuated damage to axons and myelin as demonstrated by immuno-profiles of axonal and myelin proteins, and Luxol Fast Blue staining. Calpain inhibition significantly protected the ultrastructural integrity of axons and myelin compared to control as confirmed by electron microscopy. Together, these findings confirm CIE exposure and withdrawal induced structural alterations in axons and myelin, predominantly after withdrawal and corroborate calpain inhibition as a potential protective strategy against EtOH associated CNS degeneration.

Inhibition of Calpain Activation Protects MPTP-Induced Nigral and Spinal Cord Neurodegeneration, Reduces Inflammation, and Improves Gait Dynamics in Mice.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, resulting in dopaminergic (DA) neuronal loss in the substantia nigra pars compacta (SNpc) and damage to the extranigral spinal cord neurons. Current therapies do not prevent the disease progression. Hence, developing efficacious therapeutic strategies for treatment of PD is of utmost importance. The goal of this study is to delineate the involvement of calpain-mediated inflammation and neurodegeneration in SN and spinal cord in MPTP-induced parkinsonian mice (C57BL/6 N), thereby elucidating potential therapeutic target(s). Increased calpain expression was found localized to tyrosine hydroxylase (TH(+)) neurons in SN with significantly increased TUNEL-positive neurons in SN and spinal cord neurons in MPTP mice. Inflammatory markers Cox-2, caspase-1, and NOS-2 were significantly upregulated in MPTP mouse spinal cord as compared to control. These parameters correlated with the activation of astrocytes, microglia, infiltration of CD4(+)/CD8(+) T cells, and macrophages. We found that subpopulations of CD4(+) cells (Th1 and Tregs) were differentially expanded in MPTP mice, which could be regulated by inhibition of calpain with the potent inhibitor calpeptin. Pretreatment with calpeptin (25 µg/kg, i.p.) attenuated glial activation, T cell infiltration, nigral dopaminergic degeneration in SN, and neuronal death in spinal cord. Importantly, calpeptin ameliorated MPTP-induced altered gait parameters (e.g., reduced stride length and increased stride frequency) as demonstrated by analyses of spatiotemporal gait indices using ventral plane videography. These findings suggest that calpain plays a pivotal role in MPTP-induced nigral and extranigral neurodegenerative processes and may be a valid therapeutic target in PD.

SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP(+) and rotenone.

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 Ca(2+) 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. SH-SY5Y cells, differentiated as dopaminergic (TH positive) and cholinergic (ChAT positive), were used as in vitro models for Parkinson's disease. MPP+ and rotenone induced up-regulation of calpain, expression, and activity as a common mechanism of neurodegeneration. SNJ-1945, a novel calpain inhibitor, protected both the cell phenotypes against MPP+ and rotenone.

Critical role of calpain in spinal cord degeneration in Parkinson's disease.

While multiple molecular mechanisms contribute to midbrain nigrostriatal dopaminergic degeneration in Parkinson's disease (PD), the mechanism of damage in non-dopaminergic sites within the central nervous system, including the spinal cord, is not well-understood. Thus, to understand the comprehensive pathophysiology underlying this devastating disease, postmortem spinal cord tissue samples (cervical, thoracic, and lumbar segments) from patients with PD were analyzed compared to age-matched normal subjects or Alzheimer's disease for selective molecular markers of neurodegeneration and inflammation. Distal axonal degeneration, relative abundance of both sensory and motor neuron death, selective loss of ChAT(+) motoneurons, reactive astrogliosis, microgliosis, increased cycloxygenase-2 (Cox-2) expression, and infiltration of T cells were observed in spinal cord of PD patients compared to normal subjects. Biochemical analyses of spinal cord tissues revealed associated inflammatory and proteolytic events (elevated levels of Cox-2, expression and activity of µ- and m-calpain, degradation of axonal neurofilament protein, and concomitantly low levels of endogenous inhibitor - calpastatin) in spinal cord of PD patients. Thus, pathologically upregulated calpain activity in spinal cords of patients with PD may contribute to inflammatory response-mediated neuronal death, leading to motor dysfunction. We proposed calpain over-activation and calpain-calpastatin dysregulation driving in a cascade of inflammatory responses (microglial activation and T cell infiltration) and degenerative pathways culminating in axonal degeneration and neuronal death in spinal cord of Parkinson's disease patients. This may be one of the crucial mechanisms in the degenerative process.

Calpain inhibition prevents ethanol-induced alterations in spinal motoneurons.

Long-term exposure of ethanol (EtOH) alters the structure and function in brain and spinal cord. The present study addresses the mechanisms of EtOH-induced damaging effects on spinal motoneurons in vitro. Altered morphology and biochemical changes of such damage were demonstrated by in situ Wright staining and DNA ladder assay. EtOH at low to moderate (25-50 mM) concentrations induced damaging effects in the motoneuronal scaffold which involved activation of proteases like µ-calpain and caspase-3. Caspase-8 was seen only at higher (100 mM) EtOH concentration. Further, pretreatment with calpeptin, a potent calpain inhibitor, confirmed the involvement of active proteases in EtOH-induced damage to motoneurons. The lysosomal enzyme cathepsin D was also elevated in the motoneurons by EtOH, and this effect was significantly attenuated by inhibitor treatment. Overall, EtOH exposure rendered spinal motoneurons vulnerable to damage, and calpeptin provided protection, suggesting a critical role of calpain activation in EtOH-induced alterations in spinal motoneurons.

Tracking extranigral degeneration in animal models of Parkinson's disease: quest for effective therapeutic strategies.

Sporadic Parkinson's disease (PD) is now interpreted as a complex nervous system disorder in which the projection neurons are predominantly damaged. Such an interpretation is based on mapping of Lewy body and Lewy neurite pathology. Symptoms of the human disease are much widespread, which span from pre-clinical non-motor symptoms and clinical motor symptoms to cognitive discrepancies often seen in advanced stages. Existing symptomatic treatments further complicate with overt drug-irresponsive symptoms. PD is better understood by assimilation of extranigral degenerative pathways with nigrostriatal degenerative mechanisms. The term 'extranigral' appeared first in the 1990s to more rigorously define the nigral pathology by process of elimination. However, as clinicians progressively identified PD symptoms unresponsive to the gold standard drug l-DOPA, definitions of PD symptoms were redefined. Non-motor symptoms prodromal to motor symptoms just as pre-clinical to clinical, and conjointly emerged the concept of nigral versus extranigral degeneration in PD. While nigrostriatal degeneration is responsible for the neurobiological substrates of extrapyramydal motor features, extranigral degeneration corroborates a vast majority of other changes in discrete central, peripheral, and enteric nervous system nuclei, which together account for global symptoms of the human disease. As an extranigral site, spinal cord degeneration has also been implicated in PD progression. Interconnected to the upper CNS structures with descending and ascending pathways, spinal neurons participate in movement and sensory circuits, controlling movement and reflexes. Several clinical and in vivo studies have demonstrated signs of parkinsonism-related degenerative processes in spinal cord, which led to recent consideration of spinal cord as an area of potential therapeutic target. In a nutshell, this review explores how the existing animal models can actually reflect the human disease in order to facilitate PD research. Evolution of extranigral degeneration studies has been succinctly revisited, followed by a survey on animal models in light of recent findings in clinical PD. Together, it may help to develop effective therapeutic strategies for PD.

Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats.

Multiple investigations in vivo have shown that melatonin (MEL) has a neuroprotective effect in the treatment of spinal cord injury (SCI). This study investigates the role of MEL as an intervening agent for ameliorating Ca(2+)-mediated events, including activation of calpain, following its administration to rats sustaining experimental SCI. Calpain, a Ca(2+)-dependent neutral protease, is known to be involved in the pathogenesis of SCI. Rats were injured using a standard weight-drop method that induced a moderately severe injury (40 g.cm force) at T10. Sham controls received laminectomy only. Injured animals were given either 45 mg/kg MEL or vehicle at 15 min post-injury by intraperitoneal injection. At 48 hr post-injury, spinal cord (SC) samples were collected. Immunofluorescent labelings were used to identify calpain expression in specific cell types, such as neurons, glia, or macrophages. Combination of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and double immunofluorescent labelings was used to identify apoptosis in specific cells in the SC. The effect of MEL on axonal damage was also investigated using antibody specific for dephosphorylated neurofilament protein (dNFP). Treatment of SCI animals with MEL attenuated calpain expression, inflammation, axonal damage (dNFP), and neuronal death, indicating that MEL provided neuroprotective effect in SCI. Further, expression and activity of calpain and caspse-3 were examined by Western blotting. The results indicated a significant decrease in expression and activity of calpain and caspse-3 in SCI animals after treatment with MEL. Taken together, this study strongly suggested that MEL could be an effective neuroprotective agent for treatment of SCI.

Spinal cord degeneration in C57BL/6N mice following induction of experimental parkinsonism with MPTP.

We examined neurodegeneration in spinal cord (SC) and role of such extra-nigral degeneration in MPTP-induced experimental parkinsonism in C57BL/6N mice. HPLC-photodiode array analysis confirmed presence of the active neurotoxin MPP+ in SC after single injection of MPTP (25 mg/kg, i.p.). Mitochondrial enzyme monoamine oxidase-B (MAO-B) responsible for in vivo conversion of MPTP to MPP+ was inhibited in SC by pre-treatment with l-deprenyl, a specific inhibitor of MAO-B. Besides in vitro conversion of MPTP to MPP+ occurred by SC mitochondrial preparation, which was inhibited by l-deprenyl implicating SC as a specific target of MPTP-neurotoxicity. Double immunofluorescent labeling and spectrofluorimetric assay via kynuramine oxidation showed MAO-B expression and activity in SC neurons. Localization of dopamine transporter immunoreactivity in SC along with specific uptake of (3)H-MPP+ by SC synaptosomal preparation further confirmed SC as target of MPTP-neurotoxicity. Compared with control, increased neuronal death on the seventh day in SC of mice injected with MPTP (2 x 25 mg/kg, at 6 h interval) strongly suggested SC degeneration in pre-symptomatic phase of MPTP-induced experimental parkinsonism. Such extra-nigral neurodegeneration in Parkinson's disease indicated novel molecular mechanism preceding nigrostriatal degeneration and suggested designing broad therapeutic intervention for this complex movement disorder.

Synthetic bovine proline-rich-polypeptides generate hydroxyl radicals and fail to protect dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity in mice.

Proline-rich-polypeptides (PRPs) isolated from bovine hypothalamus have been shown to render protection against neuronal injury of the brain and spinal cord. We examined two PRPs containing 15 and 10 amino acid residues (PRP-1 and PRP-4 synthetic polypeptide) for their effect, if any, on dopaminergic neuronal damage caused by the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Effects of these PRPs on hydroxyl radical ((*)OH) generation in a Fenton-like reaction as well as from isolated mitochondria were monitored, employing a sensitive salicylate hydroxylation procedure. Balb/c mice treated (i.p., twice, 16 h apart) with MPTP (30 mg/kg) or PRP-1 (1.6 mg/kg), but not PRP-4 (1.6 mg/kg) showed significant loss of striatal dopamine and norepinephrine as assayed by an HPLC-electrochemical procedure. Pretreatment with the PRPs, 30 min prior to the neurotoxin administration failed to attenuate MPTP-induced striatal dopamine or norepinephrine depletion, but significantly attenuated the MPTP-induced decrease in dopamine turnover. A significant increase in the generation of (*)OH by the PRPs in a Fenton-like reaction or from isolated mitochondria suggests their pro-oxidant action, and explains their failure to protect against MPTP-induced parkinsonism in mice.

A synthetic human proline-rich-polypeptide enhances hydroxyl radical generation and fails to protect dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced toxicity in mice.

Some of the proline-rich-polypeptides (PRPs) are shown to afford protection against spinal cord transection or crush syndrome-induced neurodegeneration in the brain. In the present study a synthetic proline-rich-polypeptide of human hypothalamus origin (h-PRP) has been examined for its potency to protect against dopaminergic neuronal damage caused by the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Effect of h-PRP on hydroxyl radical (*OH) generation in a Fenton-like reaction was monitored, employing a sensitive salicylate hydroxylation procedure. Balb/c mice treated twice with MPTP (30 mg/kg. i.p., twice, 16 h apart) or h-PRP (20 microg/animal, twice, 16 h apart) showed significant loss of striatal dopamine as assayed by HPLC with electrochemical detection. h-PRP pretreatment failed to attenuate MPTP-induced striatal dopamine depletion. A dose-dependent increase in the generation of *OH by h-PRP suggests its pro-oxidant action, and explains its failure to protect against MPTP-induced parkinsonism in mice.