Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates

Because of the paucity of primate experimental models, the precise molecular mechanism of ischemic neuronal death remains unknown in humans. This study focused on nonhuman primates to determine which cascade necrosis or apoptosis is predominantly involved in the development of delayed (day 5) neuronal death in the hippocampal CA1 sector undergoing 20 min ischemia. We investigated expression, activation, and/or translocation of micro-calpain, lysosome-associated membrane protein-1 (LAMP-1), caspase-3, and caspase-activated DNase (CAD), as well as morphology of the postischemic CA1 neurons and DNA electrophoresis pattern. Immunoblotting showed sustained (immediately after ischemia until day 5) and maximal (day 3) activation of micro-calpain. The immunoreactivity of activated micro-calpain became remarkable as coarse granules at lysosomes on day 2, while it translocated throughout the perikarya on day 3. The immunoreactivity of LAMP-1 also showed a dynamic and concomitant translocation that was maximal on days 2-3, indicating calpain-mediated disruption of the lysosomal membrane after ischemia. In contrast, immunoblotting demonstrated essentially no increase in the activated caspase-3 at any time points after ischemia, despite upregulation of pro-caspase-3. Although expression of CAD was slightly upregulated on day 1 or 2, or both, it was much less compared with lymph node or intestine tissues. Furthermore, light and electron microscopy showed eosinophilic coagulation necrosis and membrane disruption without apoptotic body formation, while DNA electrophoresis did not show a ladder pattern, but rather a smear pattern. Sustained calpain activation and the resultant lysosomal rupture, rather than CAD-mediated apoptosis, may cause ischemic neuronal necrosis in primates.     

Calpain‐dependent cleavage of SHP‐1 and SHP‐2 is involved in the dephosphorylation of Jurkat T cells induced by Entamoeba histolytica

Host cell death induced by Entamoeba histolytica is an important mechanism for both host defence and microbial immune evasion during human amoebiasis. However, the signalling pathways underlying cell death induced by E. histolytica are not fully understood. This study investigated the involvement of the protein tyrosine phosphatases (PTPs) SHP‐1 and SHP‐2 in the dephosphorylation associated with E. histolytica‐induced host cell death. Incubation with E. histolytica resulted in a marked decrease in protein tyrosine phosphorylation levels and degradation of SHP‐1 or SHP‐2 in Jurkat cells. Pre‐treatment of cells with a calpain inhibitor, calpeptin, impeded the amoeba‐induced dephosporylation and cleavage of SHP‐1 or SHP‐2. Additionally, inhibition of PTPs with phenylarsine oxide (PAO) attenuated Entamoeba‐induced dephosphorylation and DNA fragmentation in Jurkat T cells. These results suggest that calpain‐dependent cleavage of SHP‐1 and SHP‐2 may contribute to protein tyrosine dephosphorylation in Jurkat T cell death induced by E. histolytica.     

Calpastatin overexpression in the skeletal muscle of mice prevents clenbuterol-induced muscle hypertrophy and phenotypic shift

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Cerebral calpain in fatal falciparum malaria

Disruption of axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria (CM). Calpains are calcium (Ca2+)-activated cysteine proteases which have been implicated in axonal injury in neurological diseases of various aetiologies. In this study we examined the association between mu- and m-calpain, the specific inhibitor calpastatin, and axonal injury in post mortem brain tissue from patients who died from severe malaria. Calpains were associated with axons labelled for the beta-amyloid precursor protein that detects impaired axonal transport. Elevated levels of calpastatin were rarely observed in injured axons. There were increased numbers of neurones with mu-calpain in the nuclear compartment in severe malaria cases compared with non-neurological controls, and increased numbers of glia with nuclear mu-calpain in CM patients compared with non-CM malaria cases and non-neurological controls. There was marked redistribution of calpastatin in the sequestered Plasmodium falciparum-infected erythrocytes. Responses specific to malaria infection were ascertained following analysis of brain samples from fatal cases with acute axonal injury, HIV encephalitis, and progressive multifocal leucoencephalopathy. Our findings implicate a role for calpains in the modulation of disease progression in CM.     

Calpain-mediated cleavage of collapsin response mediator protein(CRMP)-2 during neurite degeneration in mice

Axon or dendrite degeneration involves activation of the ubiquitin-proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two-dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)-2 and CMRP-4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP-2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP-2 or -4 occurred in NGF-deprived neurites from Wld(s) (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1-specific siRNA, we have demonstrated that CRMP-2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down-regulation of caplain 1 expression suppressed CRMP-2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP-2 and -4 play a pivotal role in axonal growth and transport, and the C-terminus region of CRMP-2 is essential for its binding to kinesin-1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.     

过敏毒素C5a通过Calpain途径致VEC凋亡损伤

目的 探索C5a是否与LPS、毒源性大肠杆菌O157产生的外毒素-V毒素(verotoxin)具有相似的可以导致血管内皮细胞(VEC)凋亡的作用.方法 流式细胞仪榆测细胞凋亡发生率,首先探讨rhC5a刺激的质量浓度,分别为0.2μg/mL、0.5μg/mL、1μg/mL和1.5μg/mL刺激12 h检测VEC凋亡发生率;在rhC5a质量浓度均为1 μg/mL条件下,时间分别为2 h、6 h、12 h、18 h和24 h,检测VEC凋亡发生率.Western blot方法检测凋亡相关蛋白.结果 在rhCSa质量浓度为0.2μg/mL、0.5 μg/mL、1 μmL和1.5μg/mL刺激12 h诱导的凋亡发生率分别为4.58%、7.87%、17.94%和19.03%.在确定rhC5a质量浓度1μg/mL的条件下,VEC发生凋亡呈时间依赖关系,在2 h,6 h、12 h、18 h和24 h的时相,细胞凋亡发生率分别为4.58%、12.78%、18.12%、19.08%和19.96%.rhC5a刺激VEC细胞,calpain-2蛋白表达呈时间依赖性,而calpain-1蛋白的表达为浓度依赖性.结论 C5a可以导致VEC细胞发生凋亡,calpain参与了这类细胞的凋亡进程.     

钙激活中性蛋白酶-2对肝癌细胞阿霉素耐药性的影响

目的观察阿霉素作用下,钙激活中性蛋白酶-2(cal-pain-2)在肝癌细胞(HepG2)中的表达及其对细胞存活率的影响,评价其作为抗癌药靶的可能性。方法以倍比稀释的ADM作用于HepG2细胞,用四甲基偶氮唑盐比色(MTT)实验检测阿霉素(Adriamycin,ADM)72 h的半数致死量(LD50);用蛋白质印迹(Western blot)检测的ADM(LD50)作用下,calpain-2和cyclin E的表达和酶解情况;给予calpain-2抑制剂(calpastatin peptide)干预后,用MTT法观察ADM对HepG2细胞存活率的影响。结果 MTT实验结果显示,ADM对HepG2的LD50为2.5μmol.L-1;Western blot发现,随ADM作用时间的延长,calpain-2的表达水平增高(P<0.01),而且被剪切的程度增加(P<0.05);MTT检测ADM对HepG2的作用发现,calpastatin peptide干预的细胞存活率明显低于对照组(P<0.01),Cyclin E被剪切的程度减轻(P<0.01)。结论肝癌细胞HepG2在ADM作用下发生凋亡的同时,也上调calpain-2的表达水平和活性,并通过其信号通路降低对ADM的敏感性,而针对calpain-2的活性抑制可以提高HepG2对ADM敏感性,提示calpain-2可以作为降低肿瘤化疗耐药性的潜在靶点。