Gloria G. Wilford

Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells

Calpain, a Ca2+-dependent cysteine protease, has previously been implicated in apoptosis or programmed cell death (PCD) in immune cells. Although oxidative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous system (CNS) due to these agents has not yet been defined. To explore a possible role for calpain in neuronal PCD under oxidative stress and Ca2+ influx, we examined the effects of H2O2 and A23187 on PC12 cells. Treatments caused PCD (light microscopy and TUNEL assay) with altered mRNA expression (RT-PCR) of bax (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a high bax/bcl-2 ratio. Control cells expressed 1.3-fold more microcalpain (requiring microM Ca2+) than mcalpain (requiring mM Ca2+). Expression of mcalpain was significantly increased following exposure to oxidative stress and Ca2+ influx. The mRNA levels of calpastatin (endogenous calpain inhibitor) and beta-actin (house-keeping) genes were not changed. Western analysis indicated degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibitor of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpain, degradation of 68 kDa NFP, and occurrence of PCD. These results suggest a role for calpain in PCD of PC12 cells due to oxidative stress and Ca2+ influx.

Estrogen attenuated markers of inflammation and decreased lesion volume in acute spinal cord injury in rats.

Spinal cord injury (SCI) is a devastating neurologic injury with functional deficits for which the only currently recommended pharmacotherapy is high‐dose methylprednisolone, which has limited efficacy. Estrogen is a multiactive steroid that has shown antiinflammatory and antioxidant effects, and estrogen may modulate intracellular Ca2+ and attenuate apoptosis. For this study, male rats were divided into three groups. Sham group animals received a laminectomy at T12. Injured rats received both laminectomy and 40 g · cm force SCI. Estrogen‐group rats received 4 mg/kg 17β‐estradiol (estrogen) at 15 min and 24 hr post‐injury, and vehicle‐group rats received equal volumes of dimethyl sulfoxide (vehicle). Animals were sacrificed at 48 hr post‐injury, and 1‐cm‐long segments of the lesion, rostral penumbra, and caudal penumbra were excised. Inflammation was assessed by examining tissue edema, infiltration of macrophages/microglia, and levels of cytosolic and nuclear NFκB and inhibitor of kappa B (IκBα). Myelin integrity was examined using Luxol fast blue staining. When compared to sham, vehicle‐treated animals revealed increased tissue edema, increased infiltration of inflammatory cells, decreased cytosolic levels of NFκB and IκBα, increased levels of nuclear NFκB, and increased myelin loss. Treatment of SCI rats with estrogen reduced edema and decreased inflammation and myelin loss in the lesion and penumbral areas, suggesting its potential as a therapeutic agent. Further work needs to be done, however, to elucidate the neuroprotective mechanism of estrogen.

Early induction of secondary injury factors causing activation of calpain and mitochondria-mediated neuronal apoptosis following spinal cord injury in rats.

To investigate a potential relationship between calpain and mitochondrial damage in spinal cord injury (SCI), a 40 gram‐centimeter force (g‐cm) injury was induced in rats by a weight‐drop method and allowed to progress for 4 hr. One‐centimeter segments of spinal cord tissue representing the adjacent rostral, lesion, and adjacent caudal areas were then removed for various analyses. Calcium green 2‐AM staining of the lesion and penumbra sections showed an increase in intracellular free calcium (Ca2+) levels following injury, compared with corresponding tissue sections from sham‐operated (control) animals. Western blot analysis showed increased calpain expression and activity in the lesion and penumbra segments following SCI. Double‐immunofluorescent labeling indicated that increased calpain expression occurred in neurons in injured segments. Western blot analysis also showed an increased Bax:Bcl‐2 ratio, indicating the induction of the mitochondria‐mediated cell death pathway in the lesion and penumbra. The morphology of mitochondria was altered in lesion and penumbra following SCI: mostly hydropic change (swelling) in the lesion, with the penumbra shrunken or normal. At 4 hr after induction of injury, a substantial amount of cytochrome c had been released into the cytoplasm, suggesting a trigger for apoptosis through caspase 3 activation. Neuronal death after 4 hr of injury was detected by a combined TUNEL and double‐immunofluoresence assay in the lesion and penumbra sections of injured cord, compared with sham controls. These results suggest that an early induction of secondary factors is involved in the pathogenesis of SCI. The increased Ca2+ levels could activate calpain and mediate mitochondrial damage leading to neuronal death in lesion and penumbra following injury. Thus, secondary injury processes mediating cell death are induced as early as 4 hr after the injury, and calpain and caspase inhibitors may provide neuroprotection.

Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells.

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.

Role of Calpain in Spinal Cord Injury: Effects of Calpain and Free Radical Inhibitorsa

ABSTRACT: The demonstration of increased calpain activity, immunostaining, and expression at the gene (mRNA) and protein levels concomitant with ultrastructural degeneration and loss of axon and myelin proteins in lesioned cord have implicated a pivotal role for calpain in tissue destruction in spinal cord injury (SCI). Calpain, stimulated by free radicals, also mediates apoptotic cell death. These findings suggested that the use of calpain and lipid peroxidation drugs as therapeutic agents would protect cells and maintain the axon‐myelin structural unit by preventing protein degradation. In order to examine this hypothesis, we treated SCI animals with calpain inhibitors (calpeptin) and/or methylprednisolone (MP), an antiinflammatory and free‐radical inhibitor. SCI (40 g/cm) was induced by weight‐drop, and 1 mg calpeptin or 165 mg MP/kg were given intravenously (i.v.) for 24 hours. Untreated injured animals receiving vehicle served as controls. Lesion 68‐kDa and 200‐kDa neurofilament proteins (NFPs) were analyzed by sodium dodecylsulfate polyarcylamide gel electrophoresis (SDS‐PAGE) and chemiluminescence, and the extent of protein loss was quantitated. Loss of protein in the lesion of untreated cord amounted to 47% compared to sham control, while that for calpeptin‐ or MP‐treated rats was 25–30%. Combination treatment with calpeptin and MP was slightly more effective in preventing NFP degradation, compared to either when used alone. Apopotic cell death in SCI as characterized by internucleosomal DNA fragmentation was also reduced following treatment with the inhibitors. The inhibition of cytoskeletal protein degradation suggests that calpain and free‐radical inhibitors may rescue cells and preserve and maintain membrane structure by preventing protein breakdown, preserving motor function, and being neuroprotective.

Calpeptin and methylprednisolone inhibit apoptosis in rat spinal cord injury.

ABSTRACT: Intracellular free Ca2+ and free radicals are increased following spinal cord injury (SCI). These can activate calpain to degrade cytoskeletal proteins leading to apoptotic and necrotic cell death. Primary injury triggers a cascade of secondary injury, which spreads to rostral and caudal areas. We tested calpain involvement in apoptosis in five 1‐cm segments of rat spinal cord with injury (40 g‐cm) induced at T12 by weight‐drop. Animals were immediately treated with calpeptin (250 μg/kg) and methylprednisolone (165 mg/kg) and sacrificed at 48 hr. Untreated SCI rats manifested 68‐kD neurofilament protein (NFP) degradation (indicating calpain activity), and internucleosomal DNA fragmentation (indicating apoptosis). Both calpain activity and apoptosis were highest in the lesion, and decreased with increasing distance from the lesion. Treatment decreased 68‐kD NFP degradation with reduction in apoptosis in all five areas. Thus, calpeptin and methylprednisolone are found to be neuroprotective in SCI.

Molecular evidence of apoptotic death in malignant brain tumors including glioblastoma multiforme: Upregulation of calpain and caspase-3

Cell death in the core of human brain tumors is triggered by hypoxia and lack of nutrients, but the mode of cell death whether necrosis or apoptosis is not clearly defined. To identify the role of apoptosis in brain tumor cell death, we investigated macromolecular (RNA and protein) synthesis and activity in the central to peripheral region of benign [desmoplastic infantile ganglioglioma (DIG) and transitional meningioma (TMG)] and malignant [ependymoma (END), anaplastic astrocytoma (APA), and glioblastoma multiforme (GBM)] brain tumors derived from five patients who had not received previously radiotherapy or chemotherapy. Normal brain tissue (NBT) served as control. RT‐PCR analysis of tumor tissues covering central to peripheral regions detected mRNA overexpression of pro‐apoptotic gene bax in malignant tumors, indicating a commitment to apoptosis. The mRNA expression of calpain (a Ca2+‐dependent cysteine protease) and calpastatin (endogenous calpain inhibitor) was altered resulting in an elevated calpain/calpastatin ratio. Calpain content and activity were increased, suggesting a role for calpain in cell death. In the mitochondria‐dependent death pathway, caspase‐9 and caspase‐3 were also overexpressed in tumors. The increased caspase‐3 activity cleaved poly(ADP‐ribose) polymerase (PARP). Agarose gel electrophoresis detected a mixture of random and internucleosomal DNA fragmentation in malignant brain tumors. Overexpression of pro‐apoptotic bax, upregulation of calpain and caspase‐3, and occurrence of internucleosomal DNA fragmentation are now presented indicating that one mechanism of cell death in malignant brain tumors is apoptosis, and that enhancement of this process therapeutically may promote decreased tumor growth.

E-64-d prevents both calpain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats.

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.

Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.

Abstract: Degradation of cytoskeletal proteins by calpain, a Ca2+‐dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g‐cm force) SCI in rats was induced at T12 by a standardized weight‐drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24‐h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell‐permeable calpain inhibitor E‐64‐d (1 mg/kg). Following 24‐h treatment, a 5‐cm‐long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68‐kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E‐64‐d‐treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E‐64‐d providing neuroprotective benefit.

Upregulation of calpain correlates with increased neurodegeneration in acute experimental auto-immune encephalomyelitis.

Although calpain up‐regulation is well established in experimental auto‐immune encephalomyelitis (EAE), a link between increased calpain expression and activity and neurodegeneration has not been examined. Therefore, spinal cord tissue from Lewis rats with EAE was examined to test the hypothesis that increased calpain expression in neurons would correlate with increased cell death and axonal damage in a time‐dependent manner following EAE induction. We found that increased calpain expression in EAE corresponded to increased TUNEL‐positive neurons and to increased expression of dephosphorylated neurofilament protein, markers of cell death and axonal degeneration, respectively. An increase in internucleosomal DNA fragmentation in EAE spinal cord suggested that cell death was, at least partially, due to apoptosis. Axonal damage was further demonstrated in EAE spinal cord compared with control via morphological analysis, revealing granular degeneration of filament and microtubule integrity, loss of myelin, and mitochondrial damage. Calcium (Ca2+) influx, which is required for calpain activation, was also increased in EAE spinal cord. From these findings, we conclude that increases in Ca2+‐induced calpain activity may play a crucial role in neurodegeneration in acute EAE.