Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters

Calpastatin is the specific endogenous inhibitor of calpain proteinase that is encoded by a single gene. Transient transfection assays in both a non-fusing skeletal muscle and non-muscle cell-line demonstrated that the putative porcine calpastatin promoter regions 5' to exons 1xa, 1xb, and 1u were functional. Both real-time quantitative and semi-quantitative RT-PCR on porcine skeletal muscle total RNA indicated that steady-state expression of Type I and III mRNAs containing exons 1xa and 1u, respectively, was at equivalent levels whilst the expression of Type II mRNA containing exon 1xb was significantly less (p<0.001). Immunoprobing of Western blotted muscle extracts with an antibody raised against a peptide sequence encoded by exon 1xa indicated that Type I protein was expressed and that there was significantly more Type I protein in cardiac than skeletal muscle (p<0.001). The results suggest that the expression of the single calpastatin gene was differentially controlled at several levels.     

Cross-talk Between Calpain and Caspase-3 in Penumbra and Core During Focal Cerebral Ischemia-reperfusion

Aims Some data have shown the functional connection between calpain and caspase-3. Here, we investigated the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion. Methods The activities of calpain and the levels of calpastatin, microtubule-associated protein-2 (MAP-2), and spectrin in penumbra and core at 3 or 23 h of reperfusion (R 3 h or R 23 h) after 1-h focal cerebral ischemia in rats were determined in sham- or caspase-3 inhibitor z-DEVD-CHO-treated rats.On the other hand, the determination of the activities of caspase-3 and the levels of MAP-2 and spectrin was done in sham- or calpain-inhibitor I-treated rats. Results z-DEVD-CHO (600 ng/rat, i.c.v.) markedly reduced the μ- and m-calpain activities in penumbra and the m-calpain activities in core at R 3 h and R 23 h, and enhanced the calpastatin levels in penumbra at R 3 h and in core at R 3 h and R 23 h significantly; however, it had no significant effects on the μ-calpain activities in core and the calpastatin levels in penumbra at R 23 h. Calpain inhibitor I (0.8 mg/rat, i.c.v.) markedly reduced the caspase-3 activities in core at R 3 h and R 23 h, but not in penumbra. Both calpain and caspase-3 inhibitors increased the levels of MAP-2 and spectrin in penumbra and core significantly after focal cerebral ischemia-reperfusion. Conclusions Our data provide direct evidence to demonstrate the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.     

Calpain Activation Promotes BACE1 Expression,Amyloid Precursor Protein Processing,and Amyloid Plaque Formation in a Transgenic Mouse Model of Alzheimer Disease

Abnormal activation of calpain is implicated in synaptic dysfunction and participates in neuronal death in Alzheimer disease (AD) and other neurological disorders. Pharmacological inhibition of calpain has been shown to improve memory and synaptic transmission in the mouse model of AD. However, the role and mechanism of calpain in AD progression remain elusive. Here we demonstrate a role of calpain in the neuropathology in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, an established mouse model of AD. We found that overexpression of endogenous calpain inhibitor calpastatin (CAST) under the control of the calcium/calmodulin-dependent protein kinase II promoter in APP/PS1 mice caused a remarkable decrease of amyloid plaque burdens and prevented Tau phosphorylation and the loss of synapses. Furthermore, CAST overexpression prevented the decrease in the phosphorylation of the memory-related molecules CREB and ERK in the brain of APP/PS1 mice and improved spatial learning and memory. Interestingly, treatment of cultured primary neurons with amyloid-β (Aβ) peptides caused an increase in the level of β-site APP-cleaving enzyme 1 (BACE1), the key enzyme responsible for APP processing and Aβ production. This effect was inhibited by CAST overexpression. Consistently, overexpression of calpain in heterologous APP expressing cells up-regulated the level of BACE1 and increased Aβ production. Finally, CAST transgene prevented the increase of BACE1 in APP/PS1 mice. Thus, calpain activation plays an important role in APP processing and plaque formation, probably by regulating the expression of BACE1.     

Subcellular Distribution of Calpain and Calpastatin Immunoreactivity and Fodrin Proteolysis in Rabbit Hippocampus After Hypoxia and Glucocorticoid Treatment

Abstract: Rabbits were subjected to hypoxia (5% O₂) for up to 90 min and allowed to recover for a maximum of 4 days. Hippocampus homogenate was assayed for fodrin breakdown product (BDP). After separation into a nuclear and mitochondrial fraction (NMF), a membrane and microsomal fraction (MMF), and a cytosolic fraction (CF), samples were assayed for μ-calpain, m-calpain, and calpastatin immunoreactivity. Calpain and calpastatin immunoreactivity decreased in the NMF and CF but increased in the MMF during hypoxia and short-term recovery. This translocation occurred in parallel with the increase in fodrin BDP. Because the increase in the MMF was not large enough to explain the decrease in the other two fractions, it was assumed that the translocation and activation was accompanied by a reduction in the total amounts of calpains and calpastatin. Glucocorticoid pretreatment (beta-methasone, 0.4 mg × kg⁻¹× day⁻¹) for 7 days produced a decrease in the ratio of activated μ-calpain in all three fractions in nearly all samples before, during, and after hypoxia, compared with untreated animals. Glucocorticoid pretreatment also prevented the increase in fodrin BDP that occurred in untreated animals during hypoxia and short-term recovery, indicating impairment of calpain activation.     

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.     

Characterization of the calpain/calpastatin system in human hemopoietic cell lines

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.     

Decreased expression of calpain and calpastatin mRNA during development is highly correlated with muscle protein accumulation in neonatal pigs

It is well known that rapid gain of muscle mass in neonatal pigs is highly related to protein synthesis. However, the role of protein degradation in muscle gain of the neonatal period has not been well established. Calpains and their endogenous inhibitors, calpastatins, play a significant role in early-stage myofibrillar protein degradation. To investigate the role of calpain–calpastatin system in muscle protein accumulation, we studied the expressions of their mRNA in muscle tissue sampled at days 1, 4, 6, 12, 20 and 28 from a total of 36 neonatal pigs. The steady-state mRNA levels of calpains 1A, 2 and 3A, calpastatin types 1, 2 and 3, obtained by quantitative real-time PCR analysis, decreased by 2–4 folds at the age of 4 to 6 days compared to 1-day-old piglets. Then, the relatively low expression level was maintained through 28 days of age. Expressions of calpains 1A, 3A and calpastatin type 1 were significantly correlated with the measurements of muscle protein accumulations such as muscle protein content and RNA/protein ratio. Expressions of calpain 1A, calpastatin types 1 and 3 were negatively correlated with birth weight and fractional rate of growth. The levels of calpains 1A and 2 mRNA were correspondent to their protease activities. In conclusion, decreased levels of calpain and calpastatin expressions over development in neonatal pigs are associated with high protein accumulations, suggesting that dramatic muscle growth during the neonatal period may be partially controlled by down-regulated calpain–calpastatin system.     

Developmental changes of calpain and calpastatin in rabbit brain

A major part of the Ca-activated proteolytic activity in the soluble fraction from rabbit brain could be due to the activity of the neutral thiol-proteases calpain I and II. The activity of calpains exceeded that of the endogenous inhibitor, calpastatin, at all developmental stages studied. The level of calpains increased rapidly from the prenatal stage to reach a peak 10–20 days postnatally. From this period the level of calpains decreased slowly to reach the adult levels. The level of calpastatin increased steadily from the prenatal stage to old age.     

Neuroprotective Mechanism of Taurine due to Up-regulating Calpastatin and Down-regulating Calpain and Caspase-3 during Focal Cerebral Ischemia

Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover, taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.     

Calpain activity in adult and aged human brain regions

We assayed calpain activity in 27 human brain regions from adult (43–65 years of age) and aged (66–83 years of age) postmortem tissue samples. Calpain I (M Ca-requiring) activity was 10% or less of the total activity; it was below detectable levels in a number of areas, and so data are are expressed as total (M+mM Ca-dependent) calpain activity. The distribution of the enzyme was regionally heterogeneous. Highest activity was found in the spinal cord, followed by the amygdala, and levels in mesencephalic areas and in cerebellar grey matter were also high. Levels in cerebellar white matter, tegmentum, pons, and putamen were low, and activity in cortical areas was also relatively low. Although in some areas activity seemed higher with aging, the differences were not statistically significant. We previously found that the regional distribution of cathepsin D in human and in rat brain is similar, this seems to be true for calpain activity as well. The increase of protease activity with age found in rat brain is not found in human areas, as was shown previously with cathepsin D, and in the present study with calpain.Special issue dedicated to Dr. Bernard W. Agranoff.     

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.     

Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cells

Calpain, a calcium-dependent cysteine protease, is known to associate with the T-cell plasma membrane and subsequently cleave a number of cytoskeletal-associated proteins. In this study, we report the novel observation that calpain II, but not calpain I, associates with membrane lipid rafts on human peripheral blood T-cells and Jurkat cells. Raft-associated calpain activity is enhanced with exogenous calcium and inhibited with calpeptin, a specific inhibitor of calpain activity. In addition, we demonstrate that calpain cleaves the cytoskeletal-associated protein, talin, during the first 30-min after cell stimulation. We propose that lipid raft associated-calpain II could function in early TCR signaling to facilitate immune synapse formation through cytoskeletal remodeling mechanisms. Hence, we demonstrate that the positioning of calpain II within T-cell lipid rafts strategically places it in close proximity to known calpain substrates that are cleaved during Ag-specific T-cell signaling and immune synapse formation.     

Selenoprotein K is a novel target of m-calpain, and cleavage is regulated by Toll-like receptor-induced calpastatin in macrophages

Calpains are proteolytic enzymes that modulate cellular function through cleavage of targets, thereby modifying their actions. An important role is emerging for calpains in regulating inflammation and immune responses, although specific mechanisms by which this occurs have not been clearly defined. In this study, we identify a novel target of calpain, selenoprotein K (SelK), which is an endoplasmic reticulum transmembrane protein important for Ca(2+) flux in immune cells. Calpain-mediated cleavage of SelK was detected in myeloid cells (macrophages, neutrophils, and dendritic cells) but not in lymphoid cells (B and T cells). Both m- and μ-calpain were capable of cleaving immunoprecipitated SelK, but m-calpain was the predominant isoform expressed in mouse immune cells. Consistent with these results, specific inhibitors were used to show that only m-calpain cleaved SelK in macrophages. The cleavage site in SelK was identified between Arg(81) and Gly(82) and the resulting truncated SelK was shown to lack selenocysteine, the amino acid that defines selenoproteins. Resting macrophages predominantly expressed cleaved SelK and, when activated through different Toll-like receptors (TLRs), SelK cleavage was inhibited. We found that decreased calpain cleavage was due to TLR-induced up-regulation of the endogenous inhibitor, calpastatin. TLR-induced calpastatin expression not only inhibited SelK cleavage, but cleavage of another calpain target, talin. Moreover, the expression of the calpain isoforms and calpastatin in macrophages were different from T and B cells. Overall, our findings identify SelK as a novel calpain target and reveal dynamic changes in the calpain/calpastatin system during TLR-induced activation of macrophages.     

Monoamine and amino acid content in brain regions of Brattleboro rats

Monoamine and amino acid content were measured in brain regions from 12 week old male, homozygous Brattleboro (DI,n=12) and Long-Evans control (LE,n=12) rats. Norepinephrine (NE) content was significantly elevated (16–25%) in the spinal cord, pons-medulla and anterior hypothalamus of DI rats when compared to LE controls. NE content of the neurointermediate lobe of pituitary in DI rats was almost twice that of LE controls. Serotonin content was also significantly elevated in the spinal cord, pons-medulla, anterior hypothalamus and forebrain of DI rats relative to the LE controls. Taurine content in DI rats was increased (31–42%) above that of LE rats in the anterior hypothalamus, striatum and forebrain. Glutamine content was also greater in DI rats than LE in the spinal cord, pons-medulla, anterior hypothalamus, striatum, hippocampus and forebrain. The changes in monoamine and amino acid content were discussed in relation to the cardiovascular and osmoregulatory deficits that are present in DI rats due to arginine vasopressin (AVP) deficiency. The possible role of AVP in modulating NE turnover was also discussed. The increase in brain TAU content in DI rats may be a physiological response to hypernatremia.     

XIAP decreases caspase-12 cleavage and calpain activity in spinal cord of ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons. The cause for nerve cell demise is not clear but involves activation of the caspase family of cysteine proteases. We have shown that ER stress and caspase-12 activation occur in ALS transgenic mice carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. In these mice, we found that the antiapoptotic proteins, X-linked Inhibitor of Apoptosis Protein (XIAP) and the related protein, MIAP2 were decreased. To study the role of this, we generated double transgenic mice expressing XIAP in ALS spinal cord neurons using the Thy1 promoter. Overexpression of XIAP inhibited caspase-12 cleavage and reduced calpain activity in the ALS mice. XIAP also reduced the breakdown of calpastatin that is an inhibitor of calpain. In the double transgenic mice, life span was increased by about 12%. These data support the view that XIAP has beneficial effects in ALS and extends survival. The neuroprotective effect of XIAP involves inhibition of caspases and the stabilization of the calpastatin/calpain system that is altered in the ALS mice.     

Maitotoxin Induces Calpain But Not Caspase-3 Activation and Necrotic Cell Death in Primary Septo-Hippocampal Cultures

Maitotoxin is a potent toxin that activates voltage and receptor-mediated Ca²⁺ channels, resulting in Ca²⁺ overload and rapid cell death. We report that maitotoxin-induced cell death is associated with activation of calpain but not caspase-3 proteases in septo-hippocampal cell cultures. Calpain and caspase-3 activation were examined by accumulation of protease-specific breakdown products to -spectrin. Cell death manifested exclusively necrotic-like characteristics including round, shrunken nuclei, even distribution of chromatin, absence of DNA fragmentation and failure of protein synthesis inhibition to reduce cell death. Necrotic cell death was observed in neurons and astroglia. Calpain inhibitor II inhibited calpain-specific processing of -spectrin and significantly reduced cell death. The pan-caspase inhibitor, Z-D-DCB, nominally attenuated cell death. Results suggest that: (1) calpain, but not caspase-3, is activated as a result of maitotoxin-induced Ca²⁺ influx; (2) necrotic cell death caused by maitotoxin exposure is partially mediated by calpain activation; (3) maitotoxin is a useful tool to investigate pathological mechanisms of necrosis.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

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

Background

To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol.

Methods

To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging.

Results and conclusions

By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation.

General significance

The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.     

Direct Evidence for Calpain Involvement in Apoptotic Death of Neurons in Spinal Cord Injury in Rats and Neuroprotection with Calpain Inhibitor

To demonstrate calpain involvement in neurodegeneration in rat spinal cord injury (SCI), we examined SCI segments for DNA fragmentation, neurons for calpain overexpression, neuronal death, and neuroprotection with calpain inhibitor (E-64-d). After the induction of SCI (40 g cm force) on T12, rats were treated within 15 min with vehicle (DMSO) or E-64-d. Sham animals underwent laminectomy only. Animals were sacrificed at 24 h, and five 1-cm long spinal cord segments were collected: two rostral (S1 and S2), one lesion (S3), and two caudal segments (S4 and S5). Agarose gel electrophoresis of DNA samples isolated from the SCI segments showed both random and internucleosomal DNA fragmentation indicating occurrence of necrosis as well as apoptosis mostly in the lesion, moderately in caudal, and slightly in rostral segments from SCI rats. Treatment of SCI rats with E-64-d (1 mg/kg) reduced DNA fragmentation in all segments. The lesion and adjacent caudal segments (S3 and S4) were further investigated by in situ double-immunofluorescent labelings that showed increase in calpain expression in neurons in SCI rats and decrease in calpain expression in SCI rats treated with E-64-d. In situ combined TUNEL and double-immunofluorescent labelings directly detected co-localization of neuronal death and calpain overexpressin in SCI rats treated with only vehicle while attenuation of neuronal death in SCI rats treated with E-64-d. Previous studies from our laboratory indirectly showed neuroprotective effect of E-64-d in SCI rats. Our current results provide direct in situ evidence for calpain involvement in neuronal death and neuroprotective efficacy of E-64-d in lesion and penumbra in SCI rats. Special issue in honor of Naren Banik.