Knockdown of m-calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and μ-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain ( capn2 ) was found to be 50-fold higher than for the μ-calpain catalytic subunit ( capn1 ) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively. Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro . Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2–4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

Postischemic Reperfusion Induces α-Fodrin Proteolysis by m-Calpain in the Synaptosome and Nucleus in Rat Brain

Abstract: A membrane cytoskeletal protein, fodrin, is a substrate for a Ca²⁺-dependent protease, calpain. It remains unknown whether μ-calpain or m-calpain is involved in the proteolysis of either α- or β-fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ-calpain or a significant change of m-calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca²⁺-dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE-cellulose column. α- and β-fodrins and m-calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α-fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µ M , 1 ml, i.v.). The μ-calpain-specific fragment of β-fodrin was not generated during ischemia-reperfusion, supporting the possibility of the involvement of m-calpain rather than μ-calpain in the α-fodrin proteolysis.     

EXTRA-LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca²⁺-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca²⁺did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and μ-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas μ-calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.     

Calpain对细胞骨架蛋白tau降解作用的研究(英文)

Calpain是钙依赖性中性蛋白酶 ,根据其对钙敏感性的不同 ,可分为m 和 μ calpain两型 .分别用不同浓度CaCl2 溶液孵育Wistar大鼠脑皮质匀浆液 ,并用蛋白质印迹和定量图像分析技术检测不同亚型calpain对tau蛋白的降解作用 .研究发现 :在 3 7℃用 1mmol/LCa2 孵育底物 15min ,可见tau蛋白明显降解 ,并在分子质量为 2 9ku处出现tau蛋白降解片段 ;当Ca2 浓度为 5mmol/L时 ,tau蛋白几乎全部被降解 ;这种tau蛋白降解可被calpain特异性抑制剂完全逆转 .进一步的研究发现 ,分别用 μ calpain抑制剂 (0 0 5μmol/Lcalpastatin) ,m calpain抑制剂 (10 0 μmol/LcalpaininhibitorⅣ )或总calpain抑制剂 (552 μmol/Lcalpeptin)与 1mmol/LCa2 共同孵育Wistar大鼠脑皮质匀浆液 ,Ca2 激活的tau蛋白降解分别被抑制8 6% ,92 5%和 97 8% .结果表明一定浓度的Ca2 可同时激活 μ calpain和m calpain ,这两种亚型calpain均参与降解tau蛋白 ,但m calpain的作用比 μ calpain更强     

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."     

μ-Calpain Activation and Calpain-Mediated Cytoskeletal Proteolysis Following Traumatic Brain Injury

Abstract: Increasing evidence suggests that excessive activation of the calcium-activated neutral protease μ-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of μ-calpain to its 80-kDa precursor was measured by western blotting. This μ-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ-calpain activation on proteolysis of the neuronal cytoskeletal protein α-spectrin. Immunoreactivity for α-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ-calpain autolysis, calpain-mediated α-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.     

Mechanisms of calpain mediated proteolysis of voltage gated sodium channel α-subunits following in vitro dynamic stretch injury

Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.     

Synthesis and investigation of dihydroxychalcones as calpain and cathepsin inhibitors

In order to identify potential calpain and cathepsin inhibitors we prepared 12 dihydroxychalcone analogues and tested their ability to inhibit μ-calpain, m-calpain, cathepsins B and L. In the calpain inhibition test, compound 10 exhibited the most active inhibitory activity against m-calpain with an IC₅₀ value of 25.25 ± 0.901 μM. With respect to inhibition of cathepsins B and L, compound 13 exhibited the most potent inhibitory activity on cathepsin L and moderate inhibitory activity on cathepsin B with IC₅₀ values of 2.80 ± 0.100 and 11.47 ± 0.087 μM, respectively. Our results suggest the possibility of developing dual calpain and cathepsin inhibitors by properly modulating structures and/or combining the essential aspects of the functional group effective for specific calpain and cathepsin inhibition.     

M-calpain levels increase during fusion of myoblasts in the mutant muscular dysgenesis (mdg) mouse

Previous studies have led to the hypothesis of a possible role for the calcium-dependent neutral protease m-calpain in myoblast fusion in culture. To evaluate this hypothesis, we chose as our model, the "muscular dysgenesis" mouse (mdg), which presents in vivo and in vitro characteristics of an elevated process of fusion (Yao and Essien, 1975; Dussartre, 1993; Ashby et al., 1993, Joffroy et al., 1999). The aim of this study was to demonstrate using myoblast cell lines and muscle biopsies from this mdg mutant, that the amount of m-calpain increases significantly as multinucleated myotubes are formed. Using immunoblot analysis, it was shown that the m-calpain concentration in a dysgenic cell line (GLT) increased 3-fold compared to what it was upon the introduction of the differentiation medium. On the other hand, in a normal cell line (NLT), the concentration of m-calpain did not vary significantly. Thus, when the transition from myoblasts to myotubes was slow, and the absolute level of fusion was reduced, as in the NLT cell line, the level of m-calpain was stable. In contrast, when the process of fusion was precocious and fast, and the level of fusion was elevated, such as in the GLT cell line, the concentration of m-calpain increased during fusion. Moreover, when myoblast fusion was prevented by the addition of calpain inhibitor II, the process was reduced by approximately 93%. Taking into account these observations, it is clear from our data that the muscular dysgenesis mouse provides a relevant model to study myoblast fusion and that m-calpain is involved in this process.     

Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimer''s disease) and other tauopathy diseases such as CTE (chronic traumatic encephalopathy). Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury). In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform) as Ser¹³⁰↓Lys¹³¹, Gly¹⁵⁷↓Ala¹⁵⁸ and Arg³⁸⁰↓Glu³⁸¹. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product) and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-d-aspartate)], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine), dual fragmentation by calpain (TauBDP-35K) and caspase-3 (TauBDP-45K) was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45–42 kDa (minor), 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.     

Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.     

Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle

The present study investigatesthe role of two major proteolytic systems in transforming rabbit andrat muscles. The fast-to-slow transformation of rabbit muscle bychronic low-frequency stimulation (CLFS) induces fast-to-slowtransitions of intact, mature fibers and replacement of degeneratingfibers by newly formed slow fibers. Ubiquitination, an indicator of theATP-dependent proteasome system, and calpain activity were measured inhomogenates of control and stimulated extensor digitorum longusmuscles. Calpain activity increased similarly (~2-fold) in stimulatedrat and rabbit muscles. CLFS had no effect on protein ubiquitination inrat muscle but led to elevations in ubiquitin protein conjugates inrabbit muscle. Immunohistochemistry was used to study the distributionof µ-calpain and m-calpain and of ubiquitinated proteins in myosinheavy chain-based fiber types. The findings suggest that bothproteolytic systems are involved in fiber transformation andreplacement. Transforming mature fibers displayed increases inµ-calpain and accumulation of ubiquitin protein conjugates. Themajority of these fibers were identified as type IIA. Enhancedubiquitination was also observed in degenerating and necrotic fibers.Such fibers additionally displayed elevated m-calpain levels.Conversely, p94, the skeletal muscle-specific calpain, decayed rapidlyafter stimulation onset and was hardly detectable after 4 days of CLFS.

     

Effect of exercise training on calpain systems in lean and obese Zucker rats

Exercise training plays a major role in the improving physiology of diabetes. Herein we aimed to investigate the influence of exercise upon the calcium-dependent calpain-isoform expressions of lean or obese Zucker rats, a model of obesity and type II diabetes (NIDDM). Five-month-old rats were divided: (1) obese sedentary (OS, n=7); (2) obese exercise (OE, n=7); (3) lean sedentary (LS, n=7); (4) lean exercise (LE, n=7). After 2-month exercise (treadmill running), the body weight (BW) and expression of calpain 10, μ-calpain, and m-calpain in skeletal muscles were determined by RT-PCR, using β-actin as internal standard. We found exercise is useful for BW lossing, especially in the obese rats. The BW difference between OS and OE rats (69 g vs. 18.2 g) was more significantly than that between LS and LE rats (41.8 g vs. 28.7g). The calpain 10 expression of LS rats (0.965) was lower than that of LE rats (1.006), whereas those of OS and OE were comparable. The μ- or m-calpain expressions of sedentary groups (OS, LS) was significantly higher than those of exercise groups (OE, LE). The μ-calpain expression (1.13/0.92) and m-calpain expression (1.01/0.99) of OS/LS rats was significantly higher than those of OE/LE rats [1.07/0.9 (μ-calpain); 0.97/0.95 (m-calpain)]. We concluded that the μ- or m-calpains in skeletal muscle are regulated by exercise in both lean and obese Zucker rats. Exercise and BW controlling might improve the physiopathology of obesity and diabetes. Both μ- or m-calpains might become useful markers for prognoses of diabetes.     

Neuronal Nitric Oxide Synthase and Calmodulin-Dependent Protein Kinase IIα Undergo Neurotoxin-Induced Proteolysis

Abstract: Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [ N -methyl- d -aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid α-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-α (CaMPK-IIα) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified μ- and m-calpain produced fragmentation patterns for CaMPK-IIα and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-α are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIα and nNOS. The degradation of CaMPK-IIα, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.     

Calpain activity alters in rat myocardial subfractions after ischemia or reperfusion

To examine whether calpain is activated during ischemic or reperfusion injury, we measured calpain activity of the subfractions of rat myocardia after global ischemia for 60 min or the ischemia followed by 30 min reperfusion by the Langendorff procedure. The myocardial homogenate was fractionated into 600 × g, 10 000 × g and 100 000 × g pellet fractions as well as 10 000 × g supernatant fraction. The supernatant fraction was further subjected to DEAE cellulose and phenyl-Sepharose chromatographies to separate μ- and m-calpains. The m-calpain activity of the DEAE fractions after global ischemia for 60 min was higher but that after ischemia-reperfusion was lower than that of the control. On the other hand, the ischemia-reperfusion but not ischemia by itself raised the calpain activity of the phenyl-Sepharose fraction (μ-calpain) and the 10 000 × g pellet measured at 100 μM and 5 mM Ca²⁺. Treatment with verapamil but not with ryanodine during ischemia attenuated the increase in m-calpain activity. A dot-blotting analysis of calpain antigenicity showed a decrease in soluble but no change in the particulate fractions after ischemia-reperfusion. An immunoblotting technique did not detect proteolysis of the calpain 80-kDa subunit. These observations suggest that calpain is activated by Ca²⁺ influx during ischemia and reperfusion without gross changes in its amount. Some unknown processes other than translocation or autolysis are thought to be involved in the alterations.     

Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

Apoptosis, which is anti-inflammatory, and necrosis, which is pro-inflammatory, represent the extremes of the cell death spectrum. Cell death is complex and both apoptosis and necrosis can be observed in the same cells or tissues. Here, we introduce a novel combined mode of cellular demise – caspase-dependent regulated necrosis. Most importantly, it is mainly characterized with release of marked amount of oligo- or poly-nucleosomes and their attached damage-associated molecular patterns (DAMPs) and initiated by caspase activation. Caspase-activated DNase has dual roles in nucleosomal release as it can degrade extracellularly released chromatin into poly- or oligo-nucleosomes although it prohibits release of nucleosomes. In addition, osmotically triggered water movement following Cl⁻ influx and subsequent Na⁺ influx appears to be the major driving force for nucleosomal and DAMPs release. Finally, Ca²⁺-activated cysteine protease, calpain, is an another essential factor in nucleosomal and DAMPs release because of complete reversion to apoptotic morphology from necrotic one and blockade of nucleosomal and DAMPs release by its inhibition.Apoptosis is characterized by membrane blebbing, cellular shrinkage, nuclear condensation, nuclear fragmentations, oligo-nucleosomal DNA fragmentation and formation of apoptotic bodies. These characteristics are attributed mainly to the caspase family of cysteine proteases.^1,2 Necrosis is distinguished from apoptosis by cellular swelling, plasma membrane rupture, absence of oligo-nucleosomal degradation and, finally, rapid lysis of cells and cellular constituents including damage-associated molecular patterns (DAMPs) are massively exuded extracellularly to activate inflammatory and immune responses. ^{3, 4, 5}Calpains are a family of Ca²⁺-activated cysteine proteases consisting of 15 genes. Among them, μ-calpain (calpain I) and m-calpain (calpain II) are ubiquitously expressed in most cells as a heterodimer consisting of a large subunit (80 kDa; calpain 1 of μ-calpain and calpain 2 of m-calpain) and a common small subunit (29 kDa; calpain S1), which is processed into a smaller heterodimer (18–78 kDa) upon activation by Ca²⁺. Ubiquitous calpains are regulated by an endogenous inhibitor, calpastatin.⁶It has long been observed that both apoptosis and necrosis can be simultaneously detected in tissues or cell culture. Therefore, apoptosis and necrosis have been assumed to be two extremes of the cell death spectrum capable of inter-conversion by key regulators.^5,7 In this study, we introduce a novel mode of cell death involving the combination of apoptosis and necrosis, being a caspase-dependent process with necrotic morphology, involving the active release of DAMPs bound to nucleosomes.     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.