Abnormal activation of calpain and protein kinase Cα promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients.

Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.

Differential regulation of the calpain-calpastatin complex by the L-domain of calpastatin.

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain-calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca(2+)] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR.

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca(2+)-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.

Evidence for alteration of calpain/calpastatin system in PBMC of cystic fibrosis patients.

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.

Adaptive modifications in the calpain/calpastatin system in brain cells after persistent alteration in Ca2+ homeostasis.

Persistent dysregulation in Ca(2+) homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in -G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca(2+)](i) elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca(2+) levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca(2+)](i), 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca(2+)](i). Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca(2+) homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.

Role of calpain in the regulation of CFTR (cystic fibrosis transmembrane conductance regulator) turnover.

The level of the mature native 170 kDa form of CFTR (cystic fibrosis transmembrane conductance regulator) at the plasma membrane is under the control of a selective proteolysis catalysed by calpain. The product of this limited digestion, consisting of discrete fragments still associated by strong interactions, is removed from the plasma membrane and internalized in vesicles and subject to an additional degradation. This process can be monitored by visualizing the accumulation of a 100 kDa fragment in a proliferating human leukaemic T-cell line and in human circulating lymphocytes. In reconstructed systems, and in intact cells, the conversion of native CFTR into the 100 kDa fragment linearly correlated with calpain activation and was prevented by addition of synthetic calpain inhibitors. A reduction in Ca2+ influx, by blocking the NMDA (N-methyl-D-aspartate) receptor Ca2+ channel, inhibited the conversion of the native 170 kDa fragment into the 100 kDa fragment, whereas an endosome acidification blocker promoted accumulation of the digested 100 kDa CFTR form. An important role in calpain-mediated turnover of CFTR is exerted by HSP90 (heat-shock protein 90), which, via association with the protein channel, modulates the degradative effect of calpain through a selective protection. Taken together these results indicate that CFTR turnover is initiated by calpain activation, which is induced by an increased Ca2+ influx and, following internalization of the cleaved channel protein, and completed by the lysosomal proteases. These findings provide new insights into the molecular mechanisms responsible for the defective functions of ion channels in human pathologies.

Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with ß-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation.

BACKGROUND: To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol. METHODS: To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging. RESULTS AND CONCLUSIONS: By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation. GENERAL SIGNIFICANCE: The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.

Protein kinase C-dependent alpha-secretory processing of the amyloid precursor protein is mediated by phosphorylation of myosin II-B.

A substantial body of evidence indicates that protein kinase C (PKC) is involved in the alpha-secretory processing of the amyloid precursor protein (APP), an event that reduces the formation of the pathogenic amyloid-beta peptide. Recently, we have shown that trafficking and processing of APP are both impaired by knockdown of myosin II-B, one of the major neuronal motor proteins. Here, we provide evidence that the alpha-secretory processing of APP is mediated by PKC-dependent phosphorylation of myosin II-B. This signaling pathway provides an important link between APP and the neuronal cytoskeleton and might be crucial for the understanding of the biological and pathological roles of APP.

Calpain-mediated activation of NO synthase in human neuroblastoma SK-N-BE cells.

In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.

Functional role of the charge at the T538 residue in the control of protein kinase Ctheta.

We show that protein kinase C (PKC) theta localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCtheta, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCtheta does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCtheta molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCtheta.

Cell protection from Ca2+-overloading by bioactive molecules extracted from olive pomace.

We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca2+-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca2+-induced cell damages by reducing the activation of the Ca2+-dependent protease calpain, acting possibly through the modification of the permeability to Ca2+ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca2+ homeostasis.

In vivo degradation of nitric oxide synthase (NOS) and heat shock protein 90 (HSP90) by calpain is modulated by the formation of a NOS-HSP90 heterocomplex.

We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion. In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs. In addition, the extent of NOS degradation is a function of the level of HSP90 expression. Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein. In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecular-mass (130 kDa) products takes place. In contrast, in aorta, which contains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity. On the basis of these findings, we propose a novel role of the HSP90-NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain. The efficiency of this effect is directly related to the level of intracellular HSP90 expression, generating a high HSP90 to NOS ratio, which favours both the formation and stabilization of the HSP90-NOS heterocomplex. This condition seems to occur in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS.

Role of the calpain-calpastatin system in the density-dependent growth arrest.

In dividing cells calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain-calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a calpastatin form permanently localized in cytosol. In calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of alpha- and theta-PKC isoforms, as well as FAK and talin occurs. In calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.

Unexpected role of the L-domain of calpastatin during the autoproteolytic activation of human erythrocyte calpain.

Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca2+], through the conversion of the 80-kDa catalytic subunit into a 75-kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75 kDa calpain-1 form also in vivo, in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of the present study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 µM) and low (5 µM) [Ca2+]. Here we demonstrate that the L-DOM binds the 80 kDa proenzyme in the absence of Ca2+ Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca2+] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75 kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75 kDa form. Taken together, our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physiopathological conditions having altered Ca2+ homeostasis.

Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells.

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.

Biotechnological traps for the reduction of inflammation due to cardiopulmonary bypass operations.

Cardiopulmonary bypass induces a systemic inflammatory response (SIR), characterized by the activation of cellular and humoral elements, with concomitant release of neutrophil elastase and matrix-metallo proteinases. In the present study, the protease release during extracorporeal circulation in 28 patients undergoing cardiac surgical operations was monitored using casein zymography. A peak in protease activity was found in all patients at the end of cardiopulmonary bypass. Plasma samples of patients were allowed to interact with different traps obtained by immobilizing different protease inhibitors on specific carriers. alpha1-Antitrypsin, Bovine Pancreatic Trypsin Inhibitor, Elastatinal or Leupeptin were used as inhibitors and were covalently immobilized by diazotization or by condensation. A reduction in the proteolytic activity of the plasma samples was observed after interaction with the different traps. The most efficient traps, i.e. the ones displaying greatest power to inhibit protease activity, were those obtained by immobilizing Bovine Pancreatic Trypsin Inhibitor and Leupeptin. The biocompatibility of traps was also tested. Results show that protease activity in blood can be decreased by our protease traps.

Activation of A431 human carcinoma cell motility by extracellular high-mobility group box 1 protein and epidermal growth factor stimuli.

HMGB1 (high-mobility group box 1) protein, a pleiotropic cytokine released by several cell types under physiological and pathological conditions, has been identified as a signal molecule active on A431 cells. Although extracellular HMGB1 itself does not trigger any detectable signalling effect on these cells, it induces an increased susceptibility to EGF (epidermal growth factor) stimulation. Specifically, at concentrations of EGF which promote undetectable or limited cell responses, the addition of sub-nanomolar concentrations of HMGB1 potentiates the effect of EGF by specifically activating a downstream pathway that leads to enhanced cell motility through an increase in Ca2+ influx, activation of extracellular-signal-regulated kinase 1/2 and remodelling of the actin cytoskeleton. These results, which identify extracellular HMGB1 as an activator of human tumour cell migration operating in concert with EGF, have important implications in the search for novel strategies to control tumour progression and metastatic invasion.

Inhibition of calpains prevents neuronal and behavioral deficits in an MPTP mouse model of Parkinson's disease.

The molecular mechanisms mediating degeneration of midbrain dopamine neurons in Parkinson's disease (PD) are poorly understood. Here, we provide evidence to support a role for the involvement of the calcium-dependent proteases, calpains, in the loss of dopamine neurons in a mouse model of PD. We show that administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) evokes an increase in calpain-mediated proteolysis in nigral dopamine neurons in vivo. Inhibition of calpain proteolysis using either a calpain inhibitor (MDL-28170) or adenovirus-mediated overexpression of the endogenous calpain inhibitor protein, calpastatin, significantly attenuated MPTP-induced loss of nigral dopamine neurons. Commensurate with this neuroprotection, MPTP-induced locomotor deficits were abolished, and markers of striatal postsynaptic activity were normalized in calpain inhibitor-treated mice. However, behavioral improvements in MPTP-treated, calpain inhibited mice did not correlate with restored levels of striatal dopamine. These results suggest that protection against nigral neuron degeneration in PD may be sufficient to facilitate normalized locomotor activity without necessitating striatal reinnervation. Immunohistochemical analyses of postmortem midbrain tissues from human PD cases also displayed evidence of increased calpain-related proteolytic activity that was not evident in age-matched control subjects. Taken together, our findings provide a potentially novel correlation between calpain proteolytic activity in an MPTP model of PD and the etiology of neuronal loss in PD in humans.