Calpain-calpastatin System Research Articles

Distinct Changes in Calpain and Calpastatin during PNS Myelination and Demyelination in Rodent Models.

Myelin forming around axons provides electrical insulation and ensures rapid and efficient transmission of electrical impulses. Disruptions to myelinated nerves often result in nerve conduction failure along with neurological symptoms and long-term disability. In the central nervous system, calpains, a family of calcium dependent cysteine proteases, have been shown to have a role in developmental myelination and in demyelinating diseases. The roles of calpains in myelination and demyelination in the peripheral nervous system remain unclear. Here, we show a transient increase of activated CAPN1, a major calpain isoform, in postnatal rat sciatic nerves when myelin is actively formed. Expression of the endogenous calpain inhibitor, calpastatin, showed a steady decrease throughout the period of peripheral nerve development. In the sciatic nerves of Trembler-J mice characterized by dysmyelination, expression levels of CAPN1 and calpastatin and calpain activity were significantly increased. In lysolecithin-induced acute demyelination in adult rat sciatic nerves, we show an increase of CAPN1 and decrease of calpastatin expression. These changes in the calpain-calpastatin system are distinct from those during central nervous system development or in acute axonal degeneration in peripheral nerves. Our results suggest that the calpain-calpastatin system has putative roles in myelination and demyelinating diseases of peripheral nerves.

Sustained Effects of Muscle Calpain System Genotypes on Tenderness Phenotypes of South African Beef Bulls during Ageing up to 20 Days.

Simple SummaryWhen searching for genetic markers for the selection of more tender beef, it is important to maintain minimal environmental variation from pre-slaughter, right through to the ageing process, to ensure the accuracy of the obtained phenotypes. This is because beef quality traits have a large environmental component that can greatly alter the characteristics of the meat, which would not reflect a true genetic effect. We propose that variable ageing times are especially important in determining whether markers are associated with tenderization or not. Our analyses included candidate genes for the protein degrading enzyme system for calpains, because they contribute the most to tenderization. We were able to validate these markers in South African beef cattle, where they could be useful for selection. The timing of the collection of tenderness data was critical, as only a few (6/134) genetic markers sustained their association with tenderization over ageing to 20 days. A larger tenderization effect earlier in ageing, as shown here for the capn1_187 and capn1_4751 markers, would decrease the length of ageing. This would not only increase profits, but also decrease the energy needed during the storage and refrigeration of aged beef, decreasing the carbon footprint of beef production.The most important factor that determines beef tenderness is its proteolytic activity, and the balance between calpain-1 protease activity and calpastatin inhibition is especially important, while contributions can also arise from calpain-2 and, possibly, calpain-3. The meat ageing process itself affects these processes. To determine whether genotypes in the calpain–calpastatin system can enhance tenderness through a 20-day ageing period, South African purebred beef bulls (n = 166) were genotyped using the Illumina BovineHD SNP BeadChip through a gene-based association analysis targeting the cast, capn3, capn2 and capn1 genes. The Warner–Bratzler shear force (WBSF) and myofibril fragment length (MFL) of Longissimus thoracis et lumborum (LTL) steaks were evaluated between d 3 and d 20 of ageing, with protease enzyme activity in the first 20 h post-mortem. Although several of the 134 SNPs are associated with tenderness, only seven SNP in the cast, capn2 and capn1 genes sustained genetic associations, additive to the ageing-associated increases in tenderness for at least three of the four ageing periods. While most genomic associations were relatively stable over time, some genotypes within the SNP responded differently to ageing, resulting in altered genomic effects over time. The level of ageing at which genomic associations are performed is an important factor that determines whether SNPs affect tenderness phenotypes.

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.

The investigation of calpain in human placenta with fetal growth restriction.

The mechanism of fetal growth restriction (FGR) is not fully understood. In this study, we explored the contribution of the calpain-calpastatin system and the activated states of calpains in human FGR placenta. The placentas were collected from patients of FGR (n=17) and controls (n=23) at elective cesarean sections in Nagoya City University Hospital and used for experiments upon informed consent. The existence and the expression of calpains and calpastatin in human placenta were compared between FGR and controls using immunohistochemistry, SDS-PAGE, and Western blotting. Staining of calpains (pre-, post-μ-calpain, pre-, post-m-calpain, and calpain-6) and calpastatin was observed in cytoplasm of trophoblast cells, both in FGR and control placenta. Pre-μ-calpain was located in the cytoplasm, and post-μ-calpain was located mainly in proximity to the cytoplasmic membrane. The expression of pre-μ-calpain was significantly higher (P<.001) and calpain-6 was significantly lower (P=.01) in FGR placentas. The inactive μ-calpain (80kDa) was significantly elevated (P<.01), and active μ-calpain (76kDa) was significantly decreased (P=.01) in FGR placentas. The results demonstrate that activation of μ-calpain is suppressed in FGR placentas and that calpain-6 in human placenta is involved in the pathology of FGR.

CT-162: Ageing of Long-term Allogeneic Hematopoietic Cell Recipients Compared to Their Donors

Context Although allogeneic hematopoietic cells transplantation (allo-HCT) is a well-established, curative option in a variety of hematologic malignancies, its influence on ageing remains poorly understood. Therefore, we launched a comprehensive study, aiming at assessing age-related changes in allo-HCT long-term survivors, compared to their donors. Objective To investigate ageing-associated changes in main lymphocyte subpopulations (CD4, CD8, CD19, CD56), by assessing telomeric shortening and abnormalities in immunophenotype. We hypothesize, that allo-HCT survivors experience, as healthy elderly, telomeric shortening, premature immunosenescence, and inflammaging, caused by clonal expansion required for rebuilding of hematopoiesis. Design Planned duration of the project: August 2019 to August 2020. Material: venous blood. All participants' PBMCs samples undergo telomeric length measurement (qPCR) and immunophenotype assessment. The results will be tested for any correlation with ageing-associated morphological features (such as Klotho protein and calpaincalpastatin system). Patients Population: 20 recipient-donor pairs. To date, we have examined 9 out of 20 scheduled pairs. Allo-HCT recipients had undergone the engraftment due to a variety of hematological malignancies (i.e., AML, ALL, CML, PNH). Median current age of participants — 51.5 years (11 males, 7 females). Enrolled patients are at least 10 years after allo-HCT with intrinsic control of living donors. Main outcome measures The preliminary results suggest an existence of differences in telomeric length between recipients and donors in all lymphocyte subpopulations (2 pairs examined). Results Telomeric length measurement in 2 recipient (R) - donor (D) pairs (average length per chromosome): TCD4+ (R – 11kb, D – 2kb), TCD8+ (R – 8.5kb, D – 6.5kb), CD19+ (R – 9kb, D – 13kb), CD 56+ (R – 4.5kb, D - 9.5kb). Immunophenotype analysis of 9 R-D pairs has shown no statistically significant (p Conclusions The data suggest the differences in telomeric length in all lymphocyte subpopulations tested. We have not found, as yet, any statistically significant differences in immunophenotype. Observed increase in the percentage of NKT-like subpopulation in allo-HTC recipients, similar to that observed in individuals with autoimmune disorders, though interesting, requires further studies. National Science Center, Poland grants: PRELUDIUM (2018/31/N/NZ3/01035), MINIATURA (2019/03/X/NZ3/01848).

Effects of crowding on the three main proteolytic mechanisms of skeletal muscle in rainbow trout (Oncorhynchus mykiss)

BackgroundSkeletal muscle is one of the tissues most affected by stress conditions. The protein degradation in this tissue is vital for the supply of energy mediated by different proteolytic pathways such as the ubiquitin-proteasome (UPS), autophagy-lysosome (ALS) and the calpain/calpastatin system (CCS). Nevertheless, the regulation of this proteolytic axis under stress conditions is not yet completely clear. Chile is the main producer of rainbow trout (Oncorhynchus mykiss) in the world. This intensive fish farming has resulted in growing problems as crowding and stress are one of the major problems in the freshwater stage. In this context, we evaluated the crowding effect in juvenile rainbow trout kept in high stocking density (30 kg/m3) for 15, 45 and 60 days, using a control group of fish (10 kg/m3).ResultsPlasmatic cortisol and glucose were evaluated by enzyme immunoassay. The mRNA levels of stress-related genes (gr1, gr2, mr, hsp70, klf15 and redd1), markers of the UPS (atrogin1 and murf1) and CCS (capn1, capn1, cast-l and cast-s) were evaluated using qPCR. ALS (LC3-I/II and P62/SQSTM1) and growth markers (4E-BP1 and ERK) were measured by Western blot analysis. The cortisol levels increased concomitantly with weight loss at 45 days of crowding. The UPS alone was upregulated at 15 days of high stocking density, while ALS activation was observed at 60 days. However, the CCS was inactivated during the entire trial.ConclusionAll these data suggest that stress conditions, such as crowding, promote muscle degradation in a time-dependent manner through the upregulation of the UPS at early stages of chronic stress and activation of the ALS in long-term stress, while the CCS is strongly inhibited by stress conditions in the rainbow trout muscle farmed during freshwater stage. Our descriptive study will allow perform functional analysis to determine, in a more detailed way, the effect of stress on skeletal muscle physiology as well as in the animal welfare in rainbow trout. Moreover, it is the first step to elucidate the optimal crop density in the freshwater stage and improve the standards of Chilean aquaculture.

Assessment of the occurrence of gene polymorphisms CAPN316 and UoGCAST in the population of cattle

To solve the problem of increasing the rate of breeding, but reduce the breeding work with farm animals, it is necessary to form herds with the desired level of productivity, that are adapted to specific regions of breeding, resistant to various diseases, with a decrease in the time for the breeding process. The active use of molecular genetic methods has contributed to the expansion of the list of DNA markers for farm animals, which are candidate genes for economically useful traits. Among these genes are widely known members of the calpain-calpastatin system, which is associated with postmortem proteolysis and tenderisation of muscles. The calpain system consists of an actively expressed μ-calpain (CAPN1) and m-calpain (CAPN2) and a single endogenous inhibitor, CAPN1 and CAPN2-calpastatin (CAST). The study of polymorphisms of these genes contributes to the expansion of marker characterisation in breeding. DNA samples (n=139) obtained from the blood of cattle were used in the work. Real-time PCRs were carried out using a ANK-32 programmable thermocycler (Synthol, Moscow, Russia). The CAPN316 gene polymorphism was present in 15% of animals tested, with allele G being found in 85% of animals. Similar calculations on the occurrence of the UoGCAST gene polymorphism in this sample of animals found the desirable allele G in 22% of animals and allele C in the remaining 78%. From the analysis of the occurrence of both genes and their polymorphisms in the study population, animals that had a combination of both desirable genotypes (CC*GG*) of CAPN316 and UoGCAST genes were not identified. There were also no animals that had the desired CAPN316 CC genotype and the heterozygous state of the UoGCAST gene (CC*GC). While 7.6% of the animals were CC*GG for the desirable expression of gene CAPN316, they contained the homozygous form of the gene UoGCAST.

Brahman genetics influence muscle fiber properties, protein degradation, and tenderness in an Angus-Brahman multibreed herd

The objective of this study was to determine the influence of Brahman genetics on muscle contractile and metabolic phenotype and postmortem proteolysis. Cattle used in this study represent a continuous spectrum of Angus-Brahman genetic variation. Steers were harvested and Longissimus samples were collected at 1.5h, 24h, and 14d postmortem. Proteolysis during the 14d aging period was evaluated, along with Warner-Bratzler shear force (WBSF) and trained sensory panel tenderness. Myosin heavy chain composition and enzymatic activity were used to evaluate fiber type characteristics. As Brahman influence increased, WBSF increased and sensory tenderness decreased. Calpain-1 autolysis decreased as Brahman percentage increased, and corresponded with reduced degradation of troponin-T, desmin, and titin. Increasing Brahman percentage was associated with greater citrate synthase activity and greater cross-sectional area of type IIx fibers. Brahman-influenced cattle produced tougher steaks and exhibited decreased protein degradation. Thus, Brahman genetics impacted not only the calpain-calpastatin system, but also muscle fiber size and metabolic properties.

Roles of calpain-calpastatin system (CCS) in human T cell activation.

The immune response is determined by the speed of the T cell reaction to antigens assured by a state of readiness for proliferation and cytokine secretion. Proliferation, apoptosis and motion of many cell types are controlled by cytoplasmic proteases - μ- and m-calpain - and their inhibitor calpastatin, together forming the “calpain-calpastatin system” (CCS), assumed to modify their targets only upon activation-dependent cytoplasmic Ca2+ increase. Contrastingly to this notion, using quantitative real time PCR and semiquantitative flow cytometry respectively, we show here that the CCS genes are constitutively expressed, and that both calpains are constitutively active in resting, circulating human CD4+ and CD8+ lymphocytes. Furthermore, we demonstrate that calpain inhibition in the resting T cells prevents them from proliferation in vitro and greatly reduces secretion of multiple cytokines. The mechanistic reason for these effects of calpain inhibition on T cell functions might be the demonstrated significant reduction of the expression of active (phosphorylated) upstream signalling molecules, including the phospholipase C gamma, p56Lck and NFκB, in the inhibitor-treated cells. Thus, we propose that the constitutive, self-regulatory calpain-calpastatin system activity in resting human T cells is a necessary, controlling element of their readiness for complex and effective response to antigenic challenge.

Zilpaterol hydrochloride improves beef yield, changes palatability traits, and increases calpain-calpastatin gene expression in Nellore heifers

This research aimed to evaluate the effects of the beta-agonist zilpaterol hydrochloride (ZH) on carcass traits, subprimal yield, meat quality, palatability traits, and gene expression in Nellore heifers. Zilpaterol increased Longissimus lumborum area and did not change back fat thickness, meat color, and cooking loss. Heifers fed ZH had greater hindquarter weight and carcass percentage. Muscles from hindquarter were heavier for animals fed ZH. Forequarter (% of carcass) decreased and brisket did not change with ZH supplementation. There were no differences between treatments for steak aroma, beef flavor, and off-flavor. However, tenderness and juiciness were reduced by ZH, depending on postmortem aging. Zilpaterol increased Calpain-1, Calpain-2, and calpastatin mRNA expression, with no effect of day of slaughter or ZH×Day interaction. In conclusion, ZH supplementation improved hypertrophy, meat production, and debone yield in Nellore heifers, which led to decreased tenderness and to increased mRNA expression in the calpain-calpastatin system.

Increased μ-Calpain Activity in Blasts of Common B-Precursor Childhood Acute Lymphoblastic Leukemia Correlates with Their Lower Susceptibility to Apoptosis.

Childhood acute lymphoblastic leukemia (ALL) blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS) composed of cytoplasmic proteases—calpains—performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor—calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher μ-calpain (CAPN1) gene transcription, protein amounts and activity (but not those of m-calpain), with calpastatin amount and transcription of its gene (CAST) greatly varying were observed in CD19+ ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk) displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL.

Expression of calpain-calpastatin system (CCS) member proteins in human lymphocytes of young and elderly individuals; pilot baseline data for the CALPACENT project.

BackgroundUbiquitous system of regulatory, calcium-dependent, cytoplasmic proteases – calpains – and their endogenous inhibitor – calpastatin – is implicated in the proteolytic regulation of activation, proliferation, and apoptosis of many cell types. However, it has not been thoroughly studied in resting and activated human lymphocytes yet, especially in relation to the subjects’ ageing process. The CALPACENT project is an international (Polish-Italian) project aiming at verifying the hypothesis of the role of calpains in the function of peripheral blood immune cells of Polish (Pomeranian) and Italian (Sicilian) centenarians, apparently relatively preserved in comparison to the general elderly population. In this preliminary report we aimed at establishing and comparing the baseline levels of expression of μ- and m-calpain and calpastatin in various, phenotypically defined, populations of human peripheral blood lymphocytes for healthy elderly Sicilians and Poles, as compared to these values observed in young cohort.ResultsWe have found significant differences in the expression of both μ- and m-calpain as well as calpastatin between various populations of peripheral blood lymphocytes (CD4+, CD8+ and CD19+), both between the age groups compared and within them. Interestingly, significantly higher amounts of μ- and m-calpains but not of calpastatin could be demonstrated in the CD4+CD28- and CD8+CD28- lymphocytes of old subjects (but not in the cells of young individuals), as compared to their CD28+ counterparts. Finally, decreased expression of both calpains in the elderly T cells is not related to the accumulation of effector/memory (CD45RO+) cells in the latter, as the expression of both calpains does not differ significantly between the naïve and memory T cells, while is significantly lower for elderly lymphocytes if both populations are taken separately.ConclusionsObserved differences in the amounts of CCS member proteins between various populations of lymphocytes of young and elderly subjects may participate in the impaired proliferative activity of these cells in the elderly.

Single APP locus knock-in mouse models of Alzheimer's disease: Distinguishing facts from artifacts

Transgenic (Tg) mice overexpressing amyloid precursor protein (APP) have made tremendous contributions to the experimental understanding of Alzheimer's disease (AD) pathophysiology. These 1st generation models however suffer from the following intrinsic problems. [1] APP is expressed to an unphysiologically high extent. Because APP interacts (indirectly) with kinesin, the axonal transport is disturbed, possibly generating artificial phenotype(s). [2] APP fragments other than Abeta, i.e. APPs, AICD and CTF-beta, are also overproduced and could also generate artifact(s). No perfect negative controls exist. [3] The transgene is inserted into a gene locus of the host animal and affect endogenous gene expression. [4] Expression of the transgene varies from line to line and from time to time. [5] APP-Tg mice often die of unknown cause(s). [6] Cross-breeding APP-Tg mice with other mutant mice is likely to generate even more complicated artifacts. Indeed, calpastatin deficiency caused overt early lethality in APP-transgenic mice. In order to overcome these drawbacks, we generated genomic knock-in (KI) mice which harbor the Swedish and Beyreuther/Iberian mutations with the Abeta sequence humanized by homologous recombination (NL-F mice). As a negative control, KI mice without the Beyreuther/Iberian mutation (NL mice) were produced. We also generated KI mice that carry the Arctic mutation in addition to the Beyreuther/Iberian mutation (NL-G-F mice). These 2 nd generation (NL-F mice) mice start accumulating Abeta at about 6 months and showed exponential deposition up to 24 months. Calpastatin deficiency accelerated Abeta pathology and cognitive dysfunction without causing early lethality. Our results indicate that the early lethality observed in the calpastatin-deficient APP-Tg mice is likely an experimental artifact and that the calpain-calpastatin system plays a mechanistic role in the pathophysiology of AD. Another line of knockin mice carrying the Arctic mutation (NL-G-F mice) displayed more aggressive Abeta deposition not only in cortex but also in subcortical regions, which may represent characteristic pathology of this particular form of familial AD. Our KI mice will be a valuable rule for identifying the mechanisms that lead to tauopathy and neurodegeneration with minimum artifact. We propose that the knockin mice be used for global collaboration for identification of therapeutic targets.

Detecting Loci under Recent Positive Selection in Dairy and Beef Cattle by Combining Different Genome-Wide Scan Methods

As the methodologies available for the detection of positive selection from genomic data vary in terms of assumptions and execution, weak correlations are expected among them. However, if there is any given signal that is consistently supported across different methodologies, it is strong evidence that the locus has been under past selection. In this paper, a straightforward frequentist approach based on the Stouffer Method to combine P-values across different tests for evidence of recent positive selection in common variations, as well as strategies for extracting biological information from the detected signals, were described and applied to high density single nucleotide polymorphism (SNP) data generated from dairy and beef cattle (taurine and indicine). The ancestral Bovinae allele state of over 440,000 SNP is also reported. Using this combination of methods, highly significant (P<3.17×10−7) population-specific sweeps pointing out to candidate genes and pathways that may be involved in beef and dairy production were identified. The most significant signal was found in the Cornichon homolog 3 gene (CNIH3) in Brown Swiss (P = 3.82×10−12), and may be involved in the regulation of pre-ovulatory luteinizing hormone surge. Other putative pathways under selection are the glucolysis/gluconeogenesis, transcription machinery and chemokine/cytokine activity in Angus; calpain-calpastatin system and ribosome biogenesis in Brown Swiss; and gangliosides deposition in milk fat globules in Gyr. The composite method, combined with the strategies applied to retrieve functional information, may be a useful tool for surveying genome-wide selective sweeps and providing insights in to the source of selection.

Single Nucleotide Polymorphisms in Caprine Calpastatin Gene

The calpains and calpastatin (CAST) make up a major cytosolic proteolytic system, the calpain-calpastatin system, found in mammalian tissues. The relative levels of the components of the calpain-calpastatin system determine the extent of meat tenderization during postmortem storage. Calpastatin (CAST) is a protein inhibitor of the ubiquitous calcium-dependent proteases-micro-calpain and m-calpain. Polymorphisms in the bovine, ovine and pig CAST gene have been associated with meat tenderness but little is known about how caprine CAST gene may affect goat meat quality traits. In this study we selected different parts of the CAST gene: 1) that have been previously reported to be polymorphic, intron 5 and 12 and 3'UTR; 2) first time explored (exon 3, 7 and 8 and part of intron 7 and 8) to investigate polymorphic status of caprine CAST gene. Using comparative sequencing ten novel SN Ps located in exon 3 and intron 5, 7 and 8 were identified. Previously reported SNPs in intron 5, 3'UTR and intron 12 were absent. Sequence analysis revealed a non synonymous amino acid variation in exon 3, which would result in Lys/Arg substitution in the corresponding protein sequence. Considerable variation was detected in intronic regions. Twenty-four InDel were also recognized in intronic regions (15) and 3'UTR (9). All the sequences shared high homology with published bovine and ovine sequences. Three PCR-RFLP loci have been established for further analyzing genetic polymorphism in indigenous goats.

Effect of Calpastatin Gene Polymorphism on Lamb Growth and Muscling

Effect of Calpastatin Gene Polymorphism on Lamb Growth and MusclingThe calpain-calpastatin system plays a key role in skeletal muscle growth and development processes. It is known that calpastatin activity correlates highly with muscle growth rate, which implies that skeletal muscle growth is likely to result from reduced protein degradation due to compromised activity of calpains or substantial increase of calpastatin activity. The objective of the present research was to identify polymorphic forms in intron 12 of the ovine calpastatin gene in sheep of synthetic lines BCP and SCP, followed by determination of the correlation between each polymorphic form of CAST gene and chosen traits characterizing lamb growth and muscling. The present study's results suggest that lambs carrying the "b" and "e" alleles and the "ae" genotype could be preferred when selecting for growth rate. The breeding efforts to obtain substantial increases in sheep muscle mass could focus on animals carrying the "ac" genotype. No statistically significant relationship was observed in any of the analysed cases between values of the traits and the CAST genotype. However, a highly significant effect of the study year on the analysed traits was found. The results should be regarded as preliminary. The research should be continued on a larger group of animals to ensure the adequate number of each genotype. The trends mentioned and discussed above may also serve as an indicator showing the future direction of further studies in this area.

Mechanistic involvement of the calpain‐calpastatin system in Alzheimer neuropathology

The mechanism by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.

Vital Role of the Calpain-Calpastatin System for Placental-Integrity-Dependent Embryonic Survival

Although the calpain-calpastatin system has been implicated in a number of pathological conditions, its normal physiological role remains largely unknown. To investigate the functions of this system, we generated conventional and conditional calpain-2 knockout mice. The conventional calpain-2 knockout embryos died around embryonic day 15, preceded by cell death associated with caspase activation and DNA fragmentation in placental trophoblasts. In contrast, conditional knockout mice in which calpain-2 is expressed in the placenta but not in the fetus were spared. These results suggest that calpain-2 contributes to trophoblast survival via suppression of caspase activation. Double-knockout mice also deficient in calpain-1 and calpastatin resulted in accelerated and rescued embryonic lethality, respectively, suggesting that calpain-1 and -2 at least in part share similar in vivo functions under the control of calpastatin. Triple-knockout mice exhibited early embryonic lethality, a finding consistent with the notion that this protease system is vital for embryonic survival.

Evidence for alteration of calpain/calpastatin system in PBMC of cystic fibrosis patients

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain–calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40–60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100 kD + 70 kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na +/H + exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35 kD and 20 kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.

56 The calpain-calpastatin system in cystic fibrosis: physiopathological implications

56 The calpain-calpastatin system in cystic fibrosis: physiopathological implications