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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.

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., (Copyright 1999 Elsevier Science B.V.)

Quantitative measurements of the time course of calcium levels in experimental spinal cord trauma have been made. The injury was produced in rats by dropping a 10 g weight from 30 cm upon exposed dura-invested spinal cord. Lumbar sections of traumatized spinal cord and internal controls from remote cervical cord were excised and analyzed for calcium using atomic absorption spectroscopy. Total calcium levels in the lesioned cord were significantly elevated over control values within 45 min post-trauma (P less than 0.005), with maximal increase at 8 h. The increased levels of calcium in the lesion tissue confirm the previous morphologic finding of calcium deposits within axons in the lesion.

The distribution of calcium-activated neutral proteinase (CANP) activity was examined in the subcellular fractions of quaking and control mouse brain. The CANP activity was determined in purified myelin, cytosol and pellet (P2, consisting of nuclei, mitochondria and microsomes) fractions using [14C]azocasein as substrate. The enzyme activity in quaking brain was 1.3-fold greater than control. Fifty-seven percent of the control brain activity was in purified myelin compared to only 7% in quaking myelin. The specific activity of the control purified myelin was 4-fold greater than homogenate while that of the quaking was two-fold greater. In contrast, 51% of the quaking brain activity was present in cytosol compared to only 18% in the control. Triton X-100 greatly increased the control brain activity (10-fold) while the quaking brain activity was increased by only 1.2-fold. The total calcium content in the quaking brain was greatly elevated (6-fold) compared to control. Approximately 30% of the brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was in quaking myelin while 77% of the CNPase activity in control brain was in myelin. These results suggest that in quaking brain much of the CANP is not incorporated into the myelin membrane and remains cytosolic.

Long-Evans rats were undernourished from birth by removing the mother from the sucling rats for part of each day; the undernutrition was continued after weaning till 60 days of age by restricting the daily food intake. Brain development was monitored by histologic and selected biochemical analyses coordinated with an ultrastructural morphometric analysis of the pyramidal tract of 30-day-old rats. Brain and body growth were already reduced after 10 days of undernutrition. At 20 days of age, the peak of myelination in the controls, the body and brain weights of the undernourished rats, compared to the controls, were reduced by 25 and 60%, respectively, and by the end of the study (60 days), the brain and body weights were reduced by 30 and 70%, respectively. Morphometric analysis indicated that the proportion of myelinated axons was significantly reduced in starved (34%) relative to control (43%) animals. Fiber analysis revealed that not only were the myelin sheaths thinner in the undernourished rats, but that the sheath was disproportionately reduced relative to the axon diameter. Chemical analysis on a whole brain basis demonstrated a greater than 60% deficit in the relatively myelin-specific galactolipids. (Whole brain analysis included regions more severely affected than the morphometrically analyzed pyramidal tract.) We also obtained evidence for both a delay in the initiation and a general retardation of myelinogenesis. The promyelinating fibers (axons with one or two non-compacted myelin lamellae) still constituted 4.5% of the myelinated fibers in the undernourished animals at 30 days but had declined to 0.3% in the controls. Analysis of the fabtty acids of cerebroside and sulfatide (lipids enriched in myelin) demonstrated in the undernourished group a pattern characteristic of a younger animal. Thus, the effect of undernutrition on the developing rat appears to be one of inhibited and somewhat retarded myelination. These effects were most likely due to a reduction in the number of myelinating glia formed and the restricted capacity of those which form to generate myeline lamellae.

The defect in myelinogenesis present in the Quaking mutant mouse was investigated using a double radioisotope technique for comparing the incorporation of amino acid into myelin proteins of normal and mutant mice. Quaking mice and littermate controls received intracranial injections of 150 muCi [3H]glycine and 25 muCi of [14C]glycine respectively. After 2 h their brains were combined and jointly processed to obtain subcellular fractions. The 3H/14C ratio for the myelin subfraction was 1.88 as compared to a 3H/14C ratio of 3.0 for the other subfractions, indicating a 40% decrease in glycine incorporation into myelin of Quaking mice. Myelin proteins were separated by discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and the 3H/14C ratios determined in each gel slice. In contrast to the microsomal subfractions which gave a 3H/14C ratio of 2.6 across the gel, the 3H/14C ratio of myelin showed large variations with values ranging from 0.54 for proteolipid protein to 2.0 for some of the high molecular weight proteins. During development, the Quaking mutant exhibited a preferential depression in glycine incorporation into proteolipid protein in 18-day-old mice, while in older animals (32-54 days) the fast migrating basic protein, as well as the proteolipid protein, was labeled to a significantly lesser extent.

The incorporation of radioactive label into the myelin basic protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. four Quaking mice (32 days) and 4 littermate controls were injected intracranially with 150 muCi [2-3H]glycine and 25 muCi of [2-14C]glycine, respectively. One hour later, the 8 mice were sacrificed and their brains combined for common homogenization. The 3H/14C ratios of the small and large basic protiens in whole brain were 3.44 and 2.48 respectively, while the 3H/14C ratios for these proteins in myelin were 0.79 and 1.00, respectively. In the same experiment, the microsomal fraction had a 3H/14C ratio of 2.98 which is the expected ratio for normal incorporation. The results indicate that the synthesis of basic protein in whole brain of Quaking mouse proceeds at a normal rate, but specifically, the incorporation of basic protein into myelin is depressed suggesting a defect at the step of assembly of myelin components into a final membrane product.