Ca2+-dependent proteases in ischemic neuronal death: a conserved 'calpain-cathepsin cascade' from nematodes to primates

From rodents to primates, transient global brain ischemia is a well known cause of delayed neuronal death of the vulnerable neurons including cornu Ammonis 1 (CA1) pyramidal cells of the hippocampus. Previous reports using the rodent experimental paradigm indicated that apoptosis is a main contributor to such ischemic neuronal death. In primates, however, the detailed molecular mechanism of ischemic neuronal death still remains obscure. Recent data suggest that necrosis rather than apoptosis appear to be the crucial component of the damage to the nervous system during human ischemic injuries and neurodegenerative diseases. Currently, necrotic neuronal death mediated by Ca2+-dependent cysteine proteases, is becoming accepted to underlie the pathology of neurodegenerative conditions from the nematode Caenorhabditis elegans to primates. This paper reviews the role of cysteine proteases such as caspase, calpain and cathepsin in order to clarify the mechanism of ischemic neuronal death being triggered by the unspecific digestion of lysosomal proteases.     

Calpains and Delayed Calcium Deregulation in Excitotoxicity

Overactivation of glutamate receptors results in neurodegeneration in a variety of brain pathologies, including ischemia, epilepsy, traumatic brain injury and slow-progressing neurodegenerative disorders. In all these pathologies, it is well accepted that the calcium-dependent cysteine proteases calpains are key players in the mechanisms of neuronal cell death. Many research groups have been actively pursuing to establish a link between the deregulation of intracellular Ca²⁺ homeostasis associated with excitotoxicity and calpain activity. It is well established that these two events are connected and interact synergistically to promote neurodegeneration, but whether calpain activity depends on or contributes to Ca²⁺ deregulation is still under debate.     

Cathepsin K knockout alleviates aging‐induced cardiac dysfunction

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload‐induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age‐dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca²⁺ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura‐2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24‐month‐old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross‐sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release compared to young (6‐month‐old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged‐cathepsin K knockout mice compared to their wild‐type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age‐induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis‐inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin‐induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age‐related decline in cardiac function via suppressing caspase‐dependent and caspase‐independent apoptosis.     

1,25-Dihydroxyvitamin D3 induces Ca-mediated apoptosis in adipocytes via activation of calpain and caspase-12

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

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.     

Bcl-2 siRNA Augments Taxol Mediated Apoptotic Death in Human Glioblastoma U138MG and U251MG Cells

The anti-neoplastic drug taxol binds to β-tubulin to prevent tumor cell division, promoting cell death. However, high dose taxol treatment may induce cell death in normal cells too. The anti-apoptotic molecule Bcl-2 is upregulated in many cancer cells to protect them from apoptosis. In the current study, we knocked down Bcl-2 expression using cognate siRNA during low-dose taxol treatment to induce apoptosis in two human glioblastoma U138MG and U251MG cell lines. The cells were treated with either 100 nM taxol or 100 nM Bcl-2 siRNA or both for 72 h. Immunofluorescent stainings for calpain and active caspase-3 showed increases in expression and co-localization of these proteases in apoptotic cells. Fluorometric assays demonstrated increases in intracellular free [Ca²⁺], calpain, and caspase-3 indicating augmentation of apoptosis. Western blotting demonstrated dramatic increases in the levels of Bax, Bak, tBid, active caspases, DNA fragmentation factor-40 (DFF40), cleaved fragments of lamin, fodrin, and poly(ADP-ribose) polymerase (PARP) during apoptosis. The events related to apoptosis were prominent more in combination therapy than in either treatment alone. Our current study demonstrated that Bcl-2 siRNA significantly augmented taxol mediated apoptosis in different human glioblastoma cells through induction of calpain and caspase proteolytic activities. Thus, combination of taxol and Bcl-2 siRNA offers a novel therapeutic strategy for controlling the malignant growth of human glioblastoma cells. Special issue article in honor of Dr. George DeVries.     

Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.     

Role of Caspases in N-Methyl-d-Aspartate-Induced Apoptosis in Cerebrocortical Neurons

Abstract: Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N-methyl-d -aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin-1β-converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V-ICE_inh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z-VAD·fmk) that inhibits caspase activity. Pretreatment with either V-ICE_inh or Z-VAD·fmk protects cerebrocortical neurons from NMDA-induced apoptosis, suggesting a role for caspases in NMDA-induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca²⁺ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca²⁺ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICE_inh or Z-VAD·fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICE_inh and Z-VAD·fmk. Taken together, our results suggest that Ca²⁺ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.     

Calcium,mitochondria and cell metabolism: A functional triangle in bioenergetics

The versatility of mitochondrial metabolism and its fine adjustments to specific physiological or pathological conditions regulate fundamental cell pathways, ranging from proliferation to apoptosis. In particular, Ca²⁺ signalling has emerged as a key player exploited by mitochondria to tune their activity according with cell demand. The functional interaction between mitochondria and endoplasmic reticulum (ER) deeply impacts on the correct mitochondrial Ca²⁺ signal, thus modulating cell bioenergetics and functionality. Indeed, Ca²⁺ released by the ER is taken up by mitochondria where, both in the intermembrane space and in the matrix, it regulates the activity of transporters, enzymes and proteins involved in organelles' metabolism. In this review, we will briefly summarize Ca²⁺-dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca²⁺ signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer.     

Melatonin,a calpain inhibitor in the central nervous system: Current status and future perspectives

Dysregulation of neuronal Ca²⁺ and oxidative stress plays an important role in the activation of cysteine proteases including calpains and caspases that contribute to neuronal death. In neurodegenerative diseases, traumatic brain injury, stroke, and neuropathic pain calpain activities are markedly increased. Melatonin is a beneficial supplement in the treatment of central nervous system (CNS) disorders. Melatonin is a potent antioxidant and works as a free-radical scavenger to regulate a large number of molecular pathways, including oxidative stress, inflammation, apoptosis, and cell death under different pathological conditions. However, limited studies have evaluated the inhibitory effect of melatonin on calpains. This review summarizes the current knowledge related to the effects of melatonin on calpains in some of the common CNS disorders.     

Calpain 2 is required for sister chromatid cohesion

Calpains form a family of Ca²⁺-dependent cysteine proteases involved in diverse cellular processes. However, the specific functions of each calpain isoform remain unknown. Recent reports have shown that calpain 2 (Capn2) is essential for cell viability. We have recently shown that Capn2 is a nuclear protease associated with chromosomes during mitosis in mammalian embryonic cells. We now report that Capn2 depletion impairs mitosis and induces apoptosis in murine cells. Low Capn2 levels induce chromosome alignment defects, the loss of histone H3 threonine 3 phosphorylation at centromeres, and premature sister chromatid separation. Thus, Capn2 may play a role in fundamental mitotic functions, such as the maintenance of sister chromatid cohesion.     

Effect of PK11195, a peripheral benzodiazepine receptor agonist,on insulinoma cell death and insulin secretion

Functional role of peripheral benzodiazepine receptor on mitochondrial membrane in apoptosis and insulin secretion from insulinoma cells was studied. A prototypic peripheral benzodiazepine receptor agonist PK11195 induced insulinoma cell apoptosis, while a central benzodiazepine receptor agonist did not. Death of insulinoma cells by PK11195 was inhibited by cyclosporin A,{ a blocker of mitochondrial permeability transition pore}. Caspase inhibitors further inhibited MIN6N8 cell death. PK11195 induced dissipation of mitochondrial potential and cytochrome c translocation to cytoplasm. PK11195 induced an increase in cytoplasmic [Ca^{2 +}], which was reversed by cyclosporin A. Rhod-2 staining showed decreased mitochondrial [Ca^{2 +}] after PK11195 treatment. PK11195 potentiated glucose-induced insulin secretion probably due to the increased cytoplasmic [Ca^{2 +}]. Calpain was activated following Ca^{2 +} release, and calpain inhibitors attenuated death of insulinoma cells by PK11195. These results suggest that PK11195 induces mitochondrial potential loss, cytochrome c translocation, increased insulin secretion in conjunction with an increase in cytoplasmic [Ca^{2 +}] and calpain activation, which collectively leads to apoptosis of insulinoma cells.     

Mitochondrial calpain 10 activity and expression in the kidney of multiple species

Calpains, Ca²⁺-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in Ca²⁺-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75 kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75 kDa (calpain 10a), 56 kDa (calpain 10c or 10d), and 50 kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice.     

ER Ca2+ release and store-operated Ca2+ entry – partners in crime or independent actors in oncogenic transformation?

Ca²⁺ is a pleiotropic messenger that controls life and death decisions from fertilisation until death. Cellular Ca²⁺ handling mechanisms show plasticity and are remodelled throughout life to meet the changing needs of the cell. In turn, as the demands on a cell alter, for example through a change in its niche environment or its functional requirements, Ca²⁺ handling systems may be targeted to sustain the remodelled cellular state. Nowhere is this more apparent than in cancer. Oncogenic transformation is a multi-stage process during which normal cells become progressively differentiated towards a cancerous state that is principally associated with enhanced proliferation and avoidance of death. Ca²⁺ signalling is intimately involved in almost all aspects of the life of a transformed cell and alterations in Ca²⁺ handling have been observed in cancer. Moreover, this remodelling of Ca²⁺ signalling pathways is also required in some cases to sustain the transformed phenotype. As such, Ca²⁺ handling is hijacked by oncogenic processes to deliver and maintain the transformed phenotype. Central to generation of intracellular Ca²⁺ signals is the release of Ca²⁺ from the endoplasmic reticulum intracellular (ER) Ca²⁺ store via inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Upon depletion of ER Ca²⁺, store-operated Ca²⁺ entry (SOCE) across the plasma membrane occurs via STIM-gated Orai channels. SOCE serves to both replenish stores but also sustain Ca²⁺ signalling events. Here, we will discuss the role and regulation of these two signalling pathways and their interplay in oncogenic transformation.     

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum

Ca²⁺ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca²⁺ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca²⁺‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca²⁺ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca²⁺ ATPase by increasing the ER Ca²⁺ permeability. This results in diminished cytosolic and mitochondrial Ca²⁺ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca²⁺ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca²⁺ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca²⁺ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.     

Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium

Calcium (Ca²⁺) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca²⁺ level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca²⁺ triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca²⁺ agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.     

Calpain–calpastatin system and cancer progression

The calpain system is required by many important physiological processes, including the cell cycle, cytoskeleton remodelling, cellular proliferation, migration, cancer cell invasion, metastasis, survival, autophagy, apoptosis and signalling, as well as the pathogenesis of a wide range of disorders, in which it may function to promote tumorigenesis. Calpains are intracellular conserved calcium-activated neutral cysteine proteinases that are involved in mediating cancer progression via catalysing and regulating the proteolysis of their specific substrates, which are important signalling molecules during cancer progression. μ-calpain, m-calpain, and their specific inhibitor calpastatin are the three molecules originally identified as comprising the calpain system and they contain several crucial domains, specific motifs, and functional sites. A large amount of data supports the roles of the calpain–calpastatin system in cancer progression via regulation of cellular adhesion, proliferation, invasion, metastasis, and cellular survival and death, as well as inflammation and angiogenesis during tumorigenesis, implying that the inhibition of calpain activity may be a potential anti-cancer intervention strategy targeting cancer cell survival, invasion and chemotherapy resistance.     

Effects of calpain inhibitor on the apoptosis of hepatic stellate cells induced by calcium ionophore A23187

We previously showed that changes in calcium concentrations were related to cell apoptosis in vitro. The endoplasmic reticulum (ER) is the main component of calcium storage and signal transduction, and disrupting the balance of intracellular Ca²⁺ can cause endoplasmic reticulum stress (ERS). In this process, the ER releases stored Ca ²⁺ into the cytoplasm and activates calpain-2. To further investigate the effect of calpain in hepatic stellate cells (HSCs), in the current study, we examine the effect of N-acetyl-leu-leu-norleucinal (ALLN) on apoptosis resulting from calcium ionophore A23187–induced ERS. Our findings indicate that calpain inhibition reduces calcium ionophore A23187–induced apoptosis of HSCs and decreases the expression of ER stress proteins that may be related to the calpain/caspase signaling pathway.     

Role of calpains in diabetes mellitus-induced cataractogenesis: A mini review

Premature visual impairment due to lens opacification is a debilitating characteristic of untreated diabetes. Lens opacification is primarily due to the insolubilization of crystallins, proteins essential for lens optical properties, and recent studies have suggested that a major cause of this insolubilization may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of calcium (Ca²⁺) and elevated levels of lens Ca²⁺ is a condition associated with both diabetic cataractogenesis and other forms of the disorder. A number of calpains have been identified in the lens, including calpain 2, calpain 10 and two isozymes of calpain 3:Lp82 and Lp85. The use of animal hereditary cataract models have suggested that calpain 2 and/or Lp82 may be the major calpains involved in murine cataractogenesis with contributions from calpain 10 and Lp85. However, calpain 2 appears to be the major calpain involved in murine diabetic cataractogenesis and the strongest candidate of the calpains for a role in human types of cataractogenesis. Here, we present an overview of recent evidence on which these observations are based with an emphasis on the ability of calpains to proteolyse lens crystallins and calpain structural features, which appear to be involved in the Ca²⁺-mediated activation of these enzymes. (Mol Cell Biochem 261: 151–159, 2004)