Oral administration of crocetin prevents inner retinal damage induced by N-methyl-D-aspartate in mice.

Crocetin, an aglycone of crocin, is found in stigmas of the saffron crocus (Crocus starus L.) and has been used in traditional medicine. We investigated the effects of oral administration of crocetin on damage induced by N-methyl-D-aspartate (NMDA) in the murine retina. Crocetin was orally administered before and after intravitreal injection of NMDA. A histological analysis was conducted by counting the cell number of ganglion cell layer (GCL). Cell apoptosis was assessed by counting cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Retinal functions were measured in terms of a- and b-wave amplitudes using an electroretinogram (ERG). Activation of caspase-3/7 and cleaved caspase-3 expression were assayed. Calpain activity was evaluated by immunoblotting assays for proteolysis of α-spectrin. NMDA injection decreased the cell number in the GCL, and crocetin at a dose of 100 mg/kg inhibited this reduction. TUNEL-positive cells were observed in both GCL and inner nuclear layer (INL) after NMDA injection, and crocetin inhibited the increase in number of TUNEL-positive cells. ERG analysis showed that both a- and b-wave amplitudes were decreased by NMDA injection. Crocetin inhibited the reduction in the b-wave amplitude, but not in the a-wave. NMDA injection activated caspase-3/7 and increased expression of cleaved caspsase-3 in the GCL and INL, but both of these processes were inhibited by crocetin. NMDA injection also induced cleavage of α-spectrin, but crocetin did not affect this process. These findings indicate that oral administration of crocetin prevented NMDA-induced retinal damage via inhibition of the caspase pathway.

Neuroprotective effect of Salvia sahendica is mediated by restoration of mitochondrial function and inhibition of endoplasmic reticulum stress.

Herein, we investigated the protective effect of Salvia sahendica against H(2)O(2)-induced cell death in rat pheochromocytoma (PC12) cells. Our data show that S. sahendica blocks apoptosis pathway by inhibition of cytochrome c release from mitochondria and leakage of calcium from endoplasmic reticulum. It also activates/inactivates two members of Bcl-2 family, Bax and Bcl-2. Bax inhibition and Bcl-2 activation suppress release of cytochrome c from mitochondria that prevents cleavage of caspase-3. Besides S. sahendica suppresses ER stress via attenuation of intracellular levels of calcium. Suppression of ER stress decreased calpain activation and subsequently cleavage of caspase-12. Altogether, these results indicate that S. sahendica protects PC12 cells treated with H(2)O(2) via suppression of upstream factors of apoptosis pathway. While oxidative stress is an early event in Alzheimer disease, it seems that S. sahendica prevents deleterious effects of reactive oxygen species by stabilizing mitochondrial membranes and inhibiting ER stress.

Calpain inhibition attenuates apoptosis of retinal ganglion cells in acute optic neuritis.

PURPOSE: Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with the pathogenesis of multiple sclerosis (MS) and is initiated by the attack of autoreactive T cells against self-myelin antigens, resulting in demyelination, degeneration of retinal ganglion cells (RGCs), and cumulative visual impairment. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats on day 0, and animals received daily intraperitoneal injections of calpain inhibitor (calpeptin) or vehicle from day 1 until killed. Retinal cell death was analyzed by DNA fragmentation, and surviving ganglion cells were quantified after double labeling of retinal tissue with TUNEL and Brn3a. The expression of apoptotic and inflammatory proteins was determined by Western blotting. RESULTS: It was demonstrated that calpain inhibition downregulates expression of proapoptotic proteins and the proinflammatory molecule nuclear factor-kappa B (NF-κB) in the retina of Lewis rats with acute EAE. Immunofluorescent labeling revealed that apoptotic cells in the RGC layer of vehicle-treated EAE animals were Brn3a positive, and a moderate dose of calpeptin dramatically reduced the frequency of apoptotic RGCs. CONCLUSIONS: These results suggest that calpain inhibition might be a useful supplement to immunomodulatory therapies such as corticosteroids in ON, due to its neuroprotective effect on RGCs.

Effects of fatiguing jumping exercise on mRNA expression of titin-complex proteins and calpains.

Eccentric exercise induced by electrostimulation increases mRNA expression of titin-complex proteins in rodent skeletal muscle. In this study, mRNA expression of titin, muscle LIM protein (MLP), cardiac ankyrin repeat protein (CARP), ankyrin repeat domain protein 2 (Ankrd2), diabetes-related ankyrin repeat protein (DARP), and calcium-activated proteinases, calpains, were investigated in human skeletal muscle after fatiguing jumping exercise. Fatiguing jumping exercise did not change mRNA expression of titin, DARP, calpain 1, or calpain 3. MLP, Ankrd2 and calpain 2 mRNA levels were increased 2 days postexercise. CARP mRNA level was already elevated 30 min and remained elevated 2 days postexercise. Increased mRNA expression of MLP, CARP, and Ankrd2, observed for the first time in human skeletal muscle, may be part of the signaling activated by physical exercise. The rapid increase in the level of CARP mRNA nominates CARP as one of the first genes to respond to exercise. The increase in the mRNA level of calpain 2 suggests its involvement in myofiber remodeling after strenuous jumping exercise.

Calpain system regulates the differentiation of adult primitive mesenchymal ST-13 adipocytes.

The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-alpha (C/EBPalpha), peroxisome proliferator-activated receptor (PPAR)-gamma, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor alpha, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca(2+)-dependent degradation of PPARgamma and C/EBPalpha but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARgamma and C/EBPalpha. Under both conditions, calpain inhibitors almost completely prevented C/EBPalpha cleavage but partially blocked the decrease of PPARgamma. Two ubiquitous forms of calpain, mu- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of mu- but not m-calpain was found in the nucleus. Finally, stable dominant-negative mu-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARgamma and C/EBPalpha during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.

Molecular cloning and RNA expression of a novel Drosophila calpain, Calpain C.

The calpains are Ca(2+)-activated cysteine proteases whose biochemical properties have been extensively characterized in vitro. Less is known, however, about the physiological role of calpains. In this respect, Drosophila melanogaster is a useful experimental organism to study calpain activity and regulation in vivo. The sequencing of the fly genome has been recently completed and a novel calpain homologue has been identified in the CG3692 gene product. We embarked on the cloning and characterization of this putative novel calpain. We demonstrate that the actual calpain is different from the predicted protein and we provide experimental evidence for the correction of the genomic annotation. This novel protein, Calpain C, must be catalytically inactive, having mutated active site residues but is otherwise structurally similar to the other known fly calpains. Moreover, we analysed Calpain C RNA expression during Drosophila development by RT-PCR and RNA in situ hybridization, which revealed strong expression in the salivary glands.

Differential effect of oestrogen on post-exercise cardiac muscle myeloperoxidase and calpain activities in female rats.

The effects of oestrogen administration on 1 h post-exercise cardiac muscle myeloperoxidase (MPO) and calpain activities were determined in female rats. Rats were ovariectomized and implanted for 2 weeks with either oestrogen (25 mg 17-oestradiol) or placebo pellets or left with ovaries intact. Rats were then run for 1 h at 21 m min-1, 12% grade, killed 1 h post-exercise and cardiac muscle and blood samples were removed. Control animals from each group were killed without prior exercise. Serum oestrogen levels in the order of the highest to lowest were; ovariectomized oestrogen replaced rats > intact ovaries rats > ovariectomized placebo rats. Oestrogen induced significant (P < 0.05) elevations in cardiac MPO activity at rest and at 1 h post-exercise in ovariectomized rats. No significant elevations in cardiac MPO activity were evident in placebo ovariectomized or normal ovary rats at rest or post-exercise. Cardiac calpain activities were similar in all unexercised groups. Ovariectomized placebo and intact ovary rats had significantly (P < 0.05) elevated cardiac calpain activities 1 h post-exercise while calpain activity was not significantly elevated in hearts from ovariectomized oestrogen rats. These results demonstrate that oestrogen supplementation in ovariectomized rats induces elevations in cardiac muscle MPO activities at rest and at 1 h post-exercise. This is opposite to the effect of oestrogen in post-exercise skeletal muscle and implies a greater neutrophil infiltration into cardiac muscle caused by oestrogen. This effect cannot be explained by changes in 1 h post-exercise cardiac muscle calpain activity, the elevation of which was suppressed by oestrogen administration. Oestrogen influences cardiac calpain activity similarly to its effect in skeletal muscle. Thus, oestrogen administration to ovariectomized rats induces elevations in cardiac MPO activity while suppressing cardiac calpain activity.

XCL-2 is a novel m-type calpain and disrupts morphogenetic movements during embryogenesis in Xenopus laevis.

We identified a novel cDNA, XCL-2, encoding an m-type calpain, a calcium-dependent intracellular protease. This protein has all characteristic structures and active sites of canonical calpains. Zygotic transcription of the gene was first detected at stage 10. It is expressed exclusively in the ventral circumblastoporal collar and the mesoderm-free zone at the most anterior tip of neural fold in late gastrulae and neurulae. In later stages, expression is only found in cement gland and proctodeum. It is also expressed in a tissue-specific manner. In adult tissues, various levels of expression were detected in brain, eye, heart, intestine, kidney, lung, stomach and testis, but not in liver, muscle, nerve, ovary, skin and spleen. Overexpression of wild-type XCL-2 suggests that this gene is involved in gastrulation movement and convergent extension during gastrulation and neurulation. Overexpression of a dominant-negative mutant caused a phenotype morphologically similar to, but histologically different from, that caused by overexpression of wild-type XCL-2. The mutant phenotype can be rescued by injection of wild-type XCL-2. These data suggest that XCL-2 plays an important role in convergent extension movements during embryogenesis in Xenopus laevis.

Characterization and expression of calpain 10. A novel ubiquitous calpain with nuclear localization.

Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to hydrolyze specific substrates important in calcium-regulated signaling pathways. Recently, an atypical member of the calpain family, calpain 10, was described, and genetic variation in this gene was associated with an increased risk of type II diabetes mellitus in humans. In the present report, a polyclonal antibody directed against rat calpain 10 was developed. This antibody was used to monitor the expression of calpain 10 protein in tissues from rats, mice, and humans. Calpain 10 protein was found to be present in all tissues examined by Western blotting including the lens, retina, brain, heart, and skeletal muscle. Although some calpain 10 was detectable in the water-soluble protein fraction of these tissues, it was preferentially found in the water-insoluble fraction. In the lens, immunohistochemistry revealed that calpain 10 was predominately located in the cytoplasm of epithelial and newly differentiating lens fibers at the transition zone. However, calpain 10 was found to be associated with the plasma membrane of differentiated lens fiber cells and the sarcolemma of skeletal muscle. In the lens epithelium-derived cell line, alphaTN4-1, the calpain 10 protein was found in a punctate distribution in the cell nucleus as well as the cytoplasm. After the elevation of intracellular calcium levels with ionomycin, calpain 10 protein levels in the nucleus of alphaTN4-1 cells increased markedly, whereas those in the cytoplasm decreased. In the lens, the elevation of intracellular calcium levels after selenite administration resulted in increased levels of calpain 10 RNA within 1 day and a loss of calpain 10 protein from the lens nucleus coincident with the onset of selenite cataract. In conclusion, calpain 10 seems to be a ubiquitous calpain, the expression level and subcellular distribution of which are dynamically influenced by calcium.

Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis.

Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.

Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis.

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Molecular cloning and characterization of a novel tissue-specific calpain predominantly expressed in the digestive tract.

In the course of the genomic cloning of nCL-2, a stomach-specific calpain, we identified a genomic clone encoding a novel member of the calpain large subunit family and designated it 'nCL-4'. First, using exon sequences, we cloned the cDNA for mouse nCL-4. Based on this sequence, we also cloned the cDNAs for rat and human nCL-4. In the case of human nCL-4, the longest open reading frame encodes 690 amino acid residues (Mr 79095) with equal sequence similarities (50-55%) to both ubiquitous and organ-specific calpain large subunits from mammals. The deduced amino acid sequence revealed that nCL-4 is highly conserved among mammals. nCL-4 can be aligned without significant deletions or insertions, and, thus, like other calpains, can be divided into four domains (I-IV). The significant similarity of domains II and IV to those in conventional calpain large subunits suggests the potential protease activity and Ca2+-binding ability of nCL-4. Northern blot analysis revealed that the mRNA for nCL-4 is expressed predominantly in stomach and small intestine but not in uterus, suggesting specialized functions of nCL-4 in the digestive tract. When overexpressed in COS-7 cells, a specific band for nCL-4 was detected. In addition, the gene coding for nCL-4 was localized on human chromosome 1.

Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit.

The complete cDNA of 3.2 kb for rat calpain II large subunit has been constructed from library- and polymerase chain reaction-derived fragments, and sequenced. The cDNA encodes a protein of 700 amino acids having 93% sequence identity with human calpain II, and 61% identity with human calpain I. The gene possesses 21 exons, of which exons 3-21 have been mapped over 33 kb of the rat genome. A new phagemid expression vector was created from pT7-7 by insertion of the f1 origin and mutation of an NdeI to an NcoI site. Rat calpain II cDNA ligated into this vector expressed in Escherichia coli an 80 kDa protein identical in size to highly purified rat calpain II; this protein was specifically recognized on immunoblots by an affinity-purified anti-rat calpain II antibody. This is the second mammalian calpain II large subunit to be fully sequenced, and the first to be artificially expressed.