Calcium-dependent Neutral Protease Research Articles

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.

Inhibition of Calpain Attenuates Degeneration of Substantia Nigra Neurons in the Rotenone Rat Model of Parkinson's Disease.

In the central nervous system (CNS), calcium homeostasis is a critical determinant of neuronal survival. Calpain, a calcium-dependent neutral protease, is widely expressed in the brain, including substantia nigra (SN) dopaminergic (DA) neurons. Though calpain is implicated in human Parkinson's disease (PD) and corresponding animal models, the roles of specific ubiquitous calpain isoforms in PD, calpain-1 and calpain-2, remain poorly understood. In this study, we found that both isoforms are activated in a nigrostriatal pathway with increased phosphorylated synuclein following the administration of rotenone in Lewis rats, but calpain isoforms played different roles in neuronal survival. Although increased expression of calpain-1 and calpain-2 were detected in the SN of rotenone-administered rats, calpain-1 expression was not altered significantly after treatment with calpain inhibitor (calpeptin); this correlated with neuronal survival. By contrast, increased calpain-2 expression in the SN of rotenone rats correlated with neuronal death, and calpeptin treatment significantly attenuated calpain-2 and neuronal death. Calpain inhibition by calpeptin prevented glial (astroglia/microglia) activation in rotenone-treated rats in vivo, promoted M2-type microglia, and protected neurons. These data suggest that enhanced expression of calpain-1 and calpain-2 in PD models differentially affects glial activation and neuronal survival; thus, the attenuation of calpain-2 may be important in reducing SN neuronal loss in PD.

Controlled cortical impact-induced neurodegeneration decreases after administration of the novel calpain-inhibitor Gabadur

One aspect of secondary injury in traumatic brain injury is the marked increase in intracellular calcium and resultant over-activation of the calcium-dependent neutral cysteine protease calpain. Gabadur is a novel protease inhibitor with calpain-inhibition properties formulated from the classic protease inhibitor leupeptin linked to a pregabalin carrier. This construction allows the entire compound to cross the blood-brain barrier after peripheral administration to better target the site of injury. In this study, a single intraperitoneal dose of Gabadur was administered immediately following controlled cortical impact injury in rats. Neocortical slices were examined at 48 h post-injury via Fluoro-Jade B staining, revealing an improvement in cortical neurodegeneration in Gabadur treated rats. Levels of detrimental active calpain-2 measured via western blot were also decreased in rats receiving Gabadur. This data supports the benefit of targeted protease inhibition in the treatment of traumatic brain injury.

CAPN1 is a novel binding partner and regulator of the tumor suppressor NF1 in melanoma.

Neurofibromin 1 (NF1), a tumor suppressor that negatively regulates RAS through its GTPase activity, is highly mutated in various types of sporadic human cancers, including melanoma. However, the binding partners of NF1 and the pathways in which it is involved in melanoma have not been characterized in an in depth manner. Utilizing a mass spectrometry analysis of NF1 binding partners, we revealed Calpain1 (CAPN1), a calcium-dependent neutral cysteine protease, as a novel NF1 binding partner that regulates NF1 degradation in melanoma cells. ShRNA-mediated knockdown of CAPN1 or treatment with a CAPN1 inhibitor stabilizes NF1 protein levels, downregulates AKT signaling and melanoma cell growth. Combination treatment of Calpain inhibitor I with MEKi Trametinib in different melanoma cells is more effective in reducing melanoma cell growth compared to treatment with Trametinib alone, suggesting that this combination may have a therapeutic potential in melanoma. This novel mechanism for regulating NF1 in melanoma provides a molecular basis for targeting CAPN1 in order to stabilize NF1 levels and, in doing so, suppressing Ras activation; this mechanism can be exploited therapeutically in melanoma and other cancers.

Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet-induced Obese Mice

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Calpain-GRIP Signaling in Nucleus Accumbens Core Mediates the Reconsolidation of Drug Reward Memory.

Exposure to drug-paired cues causes drug memories to be in a destabilized state and interfering with memory reconsolidation can inhibit relapse. Calpain, a calcium-dependent neutral cysteine protease, is involved in synaptic plasticity and the formation of long-term fear memory. However, the role of calpain in the reconsolidation of drug reward memory is still unknown. In the present study, using a conditioned place preference (CPP) model, we found that exposure to drug-paired contextual stimuli induced the activation of calpain and decreased the expression of glutamate receptor interacting protein 1 (GRIP1) in the nucleus accumbens (NAc) core, but not shell, of male rats. Infusions of calpain inhibitors in the NAc core immediately after retrieval disrupted the reconsolidation of cocaine/morphine cue memory and blocked retrieval-induced calpain activation and GRIP1 degradation. The suppressive effect of calpain inhibitors on the expression of drug-induced CPP lasted for at least 14 d. The inhibition of calpain without retrieval 6 h after retrieval or after exposure to an unpaired context had no effects on the expression of reward memory. Calpain inhibition after retrieval also decreased cocaine seeking in a self-administration model and this effect did not recover spontaneously after 28 d. Moreover, the knock-down of GRIP1 expression in the NAc core by lentivirus-mediated short-hairpin RNA blocked disruption of the reconsolidation of drug cue memories that was induced by calpain inhibitor treatment. These results suggest that calpain activity in the NAc core is crucial for the reconsolidation of drug reward memory via the regulation of GRIP1 expression.SIGNIFICANCE STATEMENT Calpain plays an important role in synaptic plasticity and long-term memory consolidation, however, its role in the reconsolidation of drug cue memory remains unknown. Using conditioned place preference and self-administration procedures, we found that exposure to drug-paired cues induced the activation of calpain and decreased glutamate receptor interacting protein 1 (GRIP1) expression in the nucleus accumbens (NAc) core. The inhibition of calpain activity in the NAc core immediately after retrieval disrupted the reconsolidation of cocaine/morphine cue memory that was blocked by prior GRIP1 knock-down. Our findings indicate that calpain-GRIP signaling is essential for the restabilization process that is associated with drug cue memory and the inhibition of calpain activity may be a novel strategy for the prevention of drug relapse.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin

Calpain, a family of calcium-dependent neutral proteases, plays important roles in neurophysiology and pathology through the proteolytic modification of cytoskeletal proteins, receptors and kinases. Alpha 2 spectrin (αII spectrin) is a major substrate for this protease family, and the presence of the αII spectrin breakdown product (αΙΙ spectrin BDP) in a cell is evidence of calpain activity triggered by enhanced intracytoplasmic Ca(2+) concentrations. Astrocytes, the most dynamic CNS cells, respond to micro-environmental changes or noxious stimuli by elevating intracytoplasmic Ca(2+) concentration to become activated. As one measure of whether calpains are involved with reactive glial transformation, we examined paraffin sections of the human cerebral cortex and white matter by immunohistochemistry with an antibody specific for the calpain-mediated αΙΙ spectrin BDP. We also performed conventional double immunohistochemistry as well as immunofluorescent studies utilizing antibodies against αΙΙ spectrin BDP as well as glial fibrillary acidic protein (GFAP). We found strong immunopositivity in selected protoplasmic and fibrous astrocytes, and in transitional forms that raise the possibility of some of fibrous astrocytes emerging from protoplasmic astrocytes. Immunoreactive astrocytes were numerous in brain sections from cases with severe cardiac and/or respiratory diseases in the current study as opposed to our previous study of cases without significant clinical conditions that failed to reveal such remarkable immunohistochemical alterations. Our study suggests that astrocytes become αΙΙ spectrin BDP immunopositive in various stages of activation, and that spectrin cleavage product persists even in fully reactive astrocytes. Immunohistochemistry for αΙΙ spectrin BDP thus marks reactive astrocytes, and highlights the likelihood that calpains and their proteolytic processing of spectrin participate in the morphologic and physiologic transition from resting protoplasmic astrocytes to reactive fibrous astrocytes.

Abstract 495: Calpain Inhibition in Bone Marrow--Derived Cells Attenuates Atherosclerosis but Not Aortic Aneurysms in Hypercholesterolemic Mice Infused With Angiotensin II

Background and Objective: Calpains are calcium-dependent neutral cysteine proteases that are essential for multiple cellular functions such as cytoskeletal remodeling, cell cycle, and apoptosis. Angiotensin II (AngII)-infusion into mice profoundly increased both aortic protein and activity of calpains. In addition, pharmacological inhibition of calpain attenuated AngII-induced aortic medial macrophage accumulation, atherosclerosis, ascending and abdominal aortic aneurysm (AAs) in mice. Using mice that overexpress calpastatin, the endogenous inhibitor of calpains, the purpose of this study was to determine whether calpains in bone marrow-derived cells contribute to AngII-induced atherosclerosis and AAs in hypercholesterolemic mice. Methods and Results: Bone marrow cells were harvested from either calpastatin (CAST) overexpression transgenic (Tg) or wild type (WT) mice and injected (7.5 x 10 6 cells/mouse) into age-matched male LDL receptor -/- recipient mice that had been irradiated with a total of 900 Rads from a cesium source. Four weeks after bone marrow repopulation, recipient mice were fed a saturated fat-enriched diet and infused with either saline or AngII (1,000 ng/kg/min) for 4 weeks. The CAST Tg or WT genotypes used for repopulation had no effect on body weight, white blood cell counts and plasma cholesterol concentrations during AngII infusion. Baseline systolic blood pressure was not different, and AngII infusion increased systolic blood pressure equivalently in both genotypes. Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in WT and Tg mice (1.28 ± 0.11 versus 1.32 ± 0.08 mm, P = NS). Calpain inhibition showed no discernible effects on AngII-induced ascending AA development (11.7 ± 0.47 versus 11.1 ± 0.54 mm 2 , P = NS). Interestingly, overexpression of CAST in bone marrow-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches by en face measurement (Intimal lesions: WT = 19.9 ± 1.8 % versus Tg = 11.0 ± 1.2 %; n=14, P<0.001). Conclusion: Calpain inhibition in bone marrow-derived cells attenuates AngII-induced atherosclerosis but does not influence formation of ascending and abdominal AAs in hypercholesterolemic mice.

Abstract 81: Pharmacological Inhibition of Calpain Attenuates Adipose Tissue Apoptosis, Macrophage Accumulation and Inflammation in Diet-induced Obese Mice

Background and Objective Obesity is associated with low-grade chronic inflammation and apoptosis that contributes to development of insulin resistance and other metabolic complications. Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not defined. Calpains are calcium-dependent neutral cysteine proteases that are essential for multiple cellular functions, such as cytoskeletal remodeling and apoptotic cell death. Recent studies have demonstrated that activated calpain promotes adipocyte differentiation in vitro, and enhances macrophage recruitment during nephropathy. However, the functional role of calpain activation in adipose tissue macrophage accumulation and obesity remains to be elucidated. The purpose of this study was to define whether pharmacological inhibition of calpain influences diet-induced obesity and adipose tissue macrophage accumulation in mice. Methods and Results Male C57BL/6 mice (8 weeks old; n=10 per group) were fed either low (10% kcal) or high (60% kcal) fat diet for 12 weeks. Calpeptin, a calpain inhibitor (2.5 mg/kg/day) or vehicle (DMSO) was administered daily by osmotic mini-pumps for 12 weeks. Calpeptin administration did not influence high fat diet induced body weight and fat mass gain throughout the study. Calpain inhibition had no effect on glucose and insulin tolerance in obese mice. However, calpain inhibition highly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as evident by TUNEL, Picro Sirius and CD68 immunostaining. Real-time PCR analysis showed that calpain inhibition significantly suppressed inflammatory cytokines (TNFα, IL-6, MCP-1, F4/80) expression in adipose tissue (P<0.05 vs vehicle). In addition, Oil Red O staining revealed that calpain inhibition also suppressed accumulation of hepatic fat. Conclusion Pharmacological inhibition of calpain attenuated macrophage accumulation, adipocyte apoptosis, fibrosis and inflammation in diet-induced obese mice without influencing body weight gain and insulin tolerance.

Neuroprotection by μ-calpain and matrix metalloproteinases inhibition by Piroxicam in cerebral ischemia: an in silico study

An intervention to reduce brain injury before, during, or after an ischemic injury irrespective of the cause remains an exciting prospect. Increasing evidences suggest that the excessive activation of calcium-dependent neutral proteases, calpains, could play a key role in the pathology of cerebral ischemia. Targeting calcium-activated proteolysis could, therefore, be an alternative strategy for protecting neurons against post-ischemic injury. In addition, blood brain barrier (BBB) breakdown after stroke is linked to the up-regulation of metalloproteinases (MMPs) and inflammation. MMPs are zinc-dependent endopeptidases which are capable of degrading many types of extracellular matrix proteins and are also involved in the process of tissue remodeling in various pathologic conditions. Previous studies suggest that MMPs, in particular MMP-2 and MMP-9, are deleterious in the brain after stroke. In acute stage after ischemic stroke, the effects of MMP activity is correlated to the degradation of neurovascular matrix and opening of the BBB which promotes vasogenic edema and results in neurological deficits. Hence, calpain and MMPs are considered to be a potential drug target to develop novel therapeutics against cerebral ischemia. To explore such, we have tried to study the interaction of Piroxicam with calpain and MMPs by exploring molecular docking studies. The in silico studies revealed that Piroxicam occupied the active site of μ-calpain, MMP-2, and MMP-9. The binding energy of the complexes was −5.27, −9.75, and −6.47 kcal/mol for μ-calpain, MMP-2, and MMP-9, respectively. On the basis of these in silico studies collectively, we hypothesize for the first time that Piroxicam might have a strong potential to inhibit μ-calpain, MMP-2, and MMP-9 concomitantly which can be explored as a potent therapeutic target for treating cerebral ischemia in future.

Regulation of Calpain-2 in Neurons: Implications for Synaptic Plasticity

The family of calcium-dependent neutral proteases, calpains, was discovered more than 30 years ago, but their functional roles in the nervous system under physiological or pathological conditions still remain unclear. Although calpain was proposed to participate in synaptic plasticity and in learning and memory in the early 1980s, the precise mechanism regarding its activation, its target(s) and the functional consequences of its activation have remained controversial. A major issue has been the identification of roles of the two major calpain isoforms present in the brain, calpain-1 and calpain-2, and the calcium requirement for their activation, which exceeds levels that could be reached intracellularly under conditions leading to changes in synaptic efficacy. In this review, we discussed the features of calpains that make them ideally suited to link certain patterns of presynaptic activity to the structural modifications of dendritic spines that could underlie synaptic plasticity and learning and memory. We then summarize recent findings that provide critical answers to the various questions raised by the initial hypothesis, and that further support the idea that, in brain, calpain-2 plays critical roles in developmental and adult synaptic plasticity.

17-Beta-estradiol increases neuronal excitability through MAP kinase-induced calpain activation.

17-Beta-estradiol (E2) is a steroid hormone involved in numerous brain functions. E2 regulates synaptic plasticity in part by enhancing NMDA receptor function and spine density in the hippocampus, resulting in increased long-term potentiation and facilitation of learning and memory. As the calcium-dependent neutral protease, calpain, is also involved in these processes, we tested whether E2 could activate calpain and examined the functional consequences of E2-mediated calpain activation in hippocampus. Calpain activity was analyzed by a fluorescence resonance energy transfer (FRET)-based assay that allows both quantitative determination and spatial resolution. E2 rapidly activated calpain in cultured cortical and hippocampal neurons, prominently in dendrites and dendritic spines. E2-induced calpain activation was mediated through mitogen-activated protein kinase (MAPK), as it was completely blocked by MEK inhibitors. It was also calcium-independent, as it was still evident in presence of the calcium chelator, BAPTA-AM. Activation of ERalpha and ERbeta receptors by specific agonists stimulated calpain activity. Finally, the rapid E2-mediated increase in excitability in acute hippocampal slices was prevented by a membrane-permeable calpain inhibitor. Furthermore, E2 treatment of acute hippocampal slices resulted in increased actin polymerization and membrane levels of GluR1 but not GluR2/3 subunits of AMPA receptors; both effects were also blocked by a calpain inhibitor. Our results indicate that E2 rapidly stimulates calpain activity through MAP kinase-mediated phosphorylation, resulting in increased membrane levels of AMPA receptors. These effects could be responsible for E2-mediated increase in neuronal excitability and facilitation of cognitive processes.

Calpain Regulates ADAM17-Mediated Platelet Glycoprotein Ibalpha Ectodomain Shedding.

Abstract 2996Poster Board II-974 Introduction:The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Calpain, the calcium-dependent neutral protease, has been thought to play a key role in GPIbalpha shedding, whereas it still remains unclear the role of calpain in ADAM17-mediated GPIbalpha shedding. Methods:Washed platelets isolated from vein blood of healthy donors were pre-incubated with various reagents. GPIbalpha fragments were detected by Western blot with anti- GPIbalpha C- or N-terminal antibodies. GPIbalpha surface expression was determined by flow cytometry with anti-GPIbalpha N-terminal antibody. Results:Western blot and flow cytometry results showed that cell permeable calpain inhibitors, MDL 28170, calpain inhibit I and calpain inhibitor II, significantly reduced calmodulin inhibitor W7- and calcium ionophore A23187-induced GPIbalpha shedding. Dibucaine induced GPIbalpha shedding was potently inhibited by calpain inhibitors. W7 and A23187 induced talin cleavage was abolished by calpain inhibitors. Calpain inhibitors had no obvious effect on N-ethylmaleimide-induced GPIbalpha shedding. In order to investigate the role of protein kinase C (PKC) in calpain-mediated GPIbalpha shedding, calpain inhibitors or PKC inhibitor (bisindolylmaleimide I hydrochloride, BIM) were incubated with platelets respectively, and then platelets were further incubated with PKC activator phorbol-ester (PMA) or dibucaine. PMA-induced or dibucaine-mediated GPIbalpha shedding did not inhibit by calpain inhibitors or PKC inhibitor respectively, indicating that PKC is not involved in calpain-mediated GPIbalpha shedding. In addition, two CHO cell lines expressing wild-type GPIb-IX and GPIb-IX mutant with truncated GPIbalpha lacking filamin A binding site were used to test the role of filamin A in calpain-regulated GPIbalpha shedding. Dibucaine-induced GPIbalpha shedding was inhibited by MDL28170 in both of the cell lines. Conclusion:The data indicate that calpain plays key roles in ADAM17-mediated GPIbalpha ectodomain shedding and the calpain-regualted GPIbalpha shedding is independent of PKC activity or filamin A cleavage. Disclosures:No relevant conflicts of interest to declare.

Calpain inhibitor AK 295 inhibits calpain-induced apoptosis and improves neurologic function after traumatic spinal cord injury in rats

An increase in the level of intracellular calcium activates the calcium-dependent neutral protease calpain, which in turn leads to cellular dysfunction and cell death after an insult to the central nervous system. In this study, we evaluated the effect of a calpain inhibitor, AK 295, on spinal cord structure, neurologic function, and apoptosis after spinal cord injury (SCI) in a murine model. Thirty albino Wistar rats were divided into 3 groups of 10 each: the sham-operated control group (group 1), the spinal cord trauma group (group 2), and the spinal cord trauma plus AK 295 treatment group (group 3). After having received a combination of ketamine 60 mg/kg and xylazine 9 mg/kg to induce anesthesia, the rats in groups 2 and 3 were subjected to thoracic trauma by the weight drop technique (40 g-cm). One hour after having been subjected to that trauma, the rats in groups 2 and 3 were treated with an intraperitoneal injection of either dimethyl sulfoxide 2 mg/kg or AK 295 2 mg/kg. The effects of the injury and the efficacy of AK 295 were determined by an assessment of the TUNEL technique and the results of examination with a light microscope. The neurologic performance of 5 rats from group 2 and 5 from group 3 was assessed by means of the inclined plane technique and the modified Tarlov's motor grading scale 1, 3, and 5 days after spinal cord trauma. Light-microscopic examination of spinal cord specimens from group 2 revealed hemorrhage, edema, necrosis, and vascular thrombi 24 hours after trauma. Similar (but less prominent) features were seen in specimens obtained from group 3 rats. Twenty-four hours after injury, the mean apoptotic cell numbers in groups 1 and 2 were zero and 4.57 +/- 0.37 cells, respectively. In group 3, the mean apoptotic cell number was 2.30 +/- 0.34 cells, a value significantly lower than that in group 2 (P < .05). Five days after trauma, the injured rats in group 2 demonstrated significant motor dysfunction (P < .05). In comparison, the motor scores exhibited by group 3 rats were markedly better (P < .05). AK 295 inhibited apoptosis via calpaindependent pathways and provided neuroprotection and improved neurologic function in a rat model of SCI. To our knowledge, this is the first study to evaluate the use of AK 295, a calpain inhibitor, after SCI. Our data suggest that AK 295 might be a novel therapeutic compound for the neuroprotection of tissue and the recovery of function in patients with a SCI.

ERp57-associated mitochondrial μ-calpain truncates apoptosis-inducing factor

Calpains, calcium-dependent neutral cystein proteases, are involved in a variety of cellular processes. We have previously shown the characteristics of mitochondrial μ-calpain even though calpastatin, a specific endogenous inhibitor of cytosolic calpains, was not present in the mitochondria. This suggested that the regulatory system of mitochondrial calpains differs from that of cytosolic calpains, and endogenous regulatory molecule(s) must exist in the mitochondria. In this study, we have identified ERp57 in partially purified mitochondrial μ-calpain using peptide mass fingerprinting based on MALDI-TOFMS. ERp57 is a member of the protein-disulfide isomerase (PDI) family and functions as a molecular chaperone within the ER. We showed that ERp57 was present in the mitochondria and was associated with mitochondrial μ-calpain. PDI inhibitors, such as DTNB and PAO, caused a degradation of the mitochondrial μ-calpain large subunit. The release of apoptosis-inducing factor (AIF) from the mitochondrial inner membrane was inhibited by treatment of the isolated mitochondria with DTNB and immunoprecipitation of ERp57-associated mitochondrial μ-calpain. Mitochondrial μ-calpain band in casein zymography disappeared by treatment with anti-ERp57 antibody. Our results demonstrate that ERp57 forms complexes with mitochondrial μ-calpain, and ERp57-associated mitochondrial μ-calpain cleaves AIF to a truncated form.

Genetic variability in beta-lactoglobulin, calpastain and calpain loci in Kurdi sheep

DNA-based molecular methods have made possible genotyping of animals of any age and sex for milk and meat genes, thus providing a potentially more efficient and flexible selection tool. Among specific genes that may affect economically important traits in sheep, the Beta-lactoglobulin (BLG) locus has been extensively studied [Barillet et al., 2005]. The genotype BB of BLG seems to be associated with higher milk yield; on the other hand genotypes AA and AB seem to be superior in protein and casein content and crude yield [Garzon et al., 1992]. Another genes intensively investigated in farm animal are that of calpastatin (CAST) and calpain (CAPN). Calpastatin and calpain (CAPN) deserves special attention because of their major role in meat production. The calpain-calpastatin system (CCS) comprises a family of calcium dependent neutral proteases. The CAPNs have been shown to play the major role in post mortem tenderization in beef, lamb, and pork by degrading specific muscle structural proteins. The aim of the present study was to identify genotypes of BLG, CAST and CAPN genes in Kurdi sheep breed by PCR.

Diminished degradation of myelin basic protein by anti-sulfatide antibody and interferon-γ in myelin from glia maturation factor-deficient mice

In this study we show the effect of anti-sulfatide (RmAb) antibodies and inflammatory cytokines, TNF-α and IFN-γ in inducing myelin basic protein (MBP) degradation in myelin isolated from control wild type (WT) and glia maturation factor (GMF)-deficient (GMF-KO) mice. GMF was not detected in isolated myelin from WT and GMF-KO mice although it is present in brains of WT mice. Our results show that calcium-dependent neutral protease activity caused significantly elevated degradation of 18.5 and/or 17.5 kDa isoforms of MBP in WT myelin treated with RmAb or IFN-γ. In contrast, MBP degradation in isolated myelin from GMF-KO mice remained unaffected following treatment with RmAb, IFN-γ, or GM-CSF. Neither the 14 kDa isoform of MBP nor proteolipid protein (PLP) showed an elevated degradation compared to controls. A virtual absence of GM-CSF, TNF-α and IFN-γ in GMF-KO brain compared to WT was also evident when the animals were challenged with MOG 35-55. Additionally, the myelin from GMF-KO mice showed difference in distribution of myelin oligodendrocyte glycoprotein (MOG) and β-tubulin in a sucrose density gradient myelin–axolemmal fractions compared to WT. Taken together, our data suggests a role for GMF in the biochemical organization of myelin and thereby its effect on MBP degradation induced by RmAb and IFN-γ.

Identification of a novel gill-specific calpain from rainbow trout (Oncorhynchus mykiss)

Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as mu-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the mu- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca(2+)-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization, Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.