Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In traumatic brain injury (TBI), neurons surviving the primary insult may succumb through poorly understood secondary mechanisms. In vitro, cortical neurons exposed to stretch injury exhibited enhanced vulnerability to NMDA, apoptotic-like DNA fragmentation, peroxynitrite (PN) formation, and cytoplasmic cytochrome c accumulation. Surprisingly, caspase-3 activity was undetectable by both immunoblotting and fluorogenic activity assays. Therefore, we hypothesized that PN directly inhibits caspases in these neurons. Consistent with this, stretch injury in cultured neurons elicited tyrosine nitration of procaspase-3, but not caspase-9 or Apaf-1, suggesting a direct interaction of PN with caspase-3. In an ex vivo system, PN inhibited the activity of caspase-3, and this inhibition was reversible with the addition of the sulfhydryl reducing agent dithiothreitol, indicating that PN inhibits caspases by cysteinyl oxidation. Moreover, in cultures, the PN donor 3-morpholinosydnonimine (SIN-1) blocked staurosporine-induced caspase-3 activation and its downstream effects including PARP-1 [poly-(ADP-ribose) polymerase-1] cleavage and phosphotidylserine inversion, suggesting that peroxynitrite can inhibit caspase-3-mediated apoptosis. To examine these mechanisms in vivo, rats were exposed to a lateral fluid percussion injury (FPI). FPI caused increased neuronal protein nitration that colocalized with TUNEL staining, indicating that PN was associated with neurodegeneration. Caspase-3 activity was inhibited in brain lysates harvested after FPI and was restored by adding dithiothreitol. Our data show that caspase-mediated apoptosis is inhibited in neurons subjected to stretch in vitro and to TBI in vivo, mostly because of cysteinyl oxidation of caspase-3 by PN. However, this is insufficient to prevent cell death, indicating that the TBI therapy may, at a minimum, require a combination of both anti-apoptotic and anti-oxidant strategies.
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PMID:Inhibition of caspase-mediated apoptosis by peroxynitrite in traumatic brain injury. 1732 11

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which excitotoxicity has been implicated as a cause for cell death. To examine neurofilament (NF) aggregate-mediated sensitization of motor neurons to NMDA excitotoxicity, we examined NMDA receptor expression and the impact of NO donors (NOC12 or NOC5) or sodium cyanide (NaCN) on calcium influx and viability in dissociated motor neurons derived from wt and hNFL+/+ (NF aggregate-forming) mice. Alterations in intracellular calcium were assayed using Oregon Green calcium dye and the extent of apoptosis using active caspase-3 immunoreactivity. Although NF aggregate-bearing neurons demonstrated increased intracellular calcium levels and enhanced cell death in response to NMDA receptor activation, this was not associated with increased NMDA receptor expression. The down-regulation of the NMDA receptor using NO donors decreased calcium influx and caspase-3 activation in aggregate-bearing neurons, but had no effect on wt cultures. The converse was observed with NaCN in which intracellular calcium levels increased significantly in wt cultures in association with increased cell death. No effect was observed in aggregate-bearing neurons. These findings suggest that the presence of NF aggregates renders motor neurons more susceptible to NMDA-mediated excitotoxicity, and that this can be reversed by NO.
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PMID:Loss of nitric oxide-mediated down-regulation of NMDA receptors in neurofilament aggregate-bearing motor neurons in vitro: implications for motor neuron disease. 1715 1

We previously demonstrated that ginsenoside Rg(3) (Rg(3)), one of the active ingredients in Panax ginseng, attenuates NMDA receptor-mediated currents and NMDA-induced neurotoxicity (Kim, S., Kim, T., Ahn, K., Park, W.K., Nah, S.Y., Rhim, H., 2004. Ginsenoside Rg(3) antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. Biochem. Biophys. Res. Commun. 323, 416-424). Accumulating evidence suggests that homocysteine (HC), a metabolite of methionine, exerts its excitotoxicity through NMDA receptor activation. In the present study, we examined the neuroprotective effects of Rg(3) on HC-induced hippocampal excitotoxicity in vitro and in vivo. Our in vitro studies using rat cultured hippocampal neurons revealed that Rg(3) treatment significantly and dose-dependently inhibited HC-induced hippocampal cell death, with an EC(50) value of 28.7+/-7.5 muM. Rg(3) treatment not only significantly reduced HC-induced DNA damage, but also dose-dependently attenuated HC-induced caspase-3 activity in vitro. Our in vivo studies revealed that intracerebroventricular (i.c.v.) pre-administration of Rg(3) significantly and dose-dependently reduced i.c.v. HC-induced hippocampal damage in rats. To examine the mechanisms underlying the in vitro and in vivo neuroprotective effects of Rg(3) against HC-induced hippocampal excitotoxicity, we examined the effect of Rg(3) on HC-induced intracellular Ca(2+) elevations in cultured hippocampal cells and found that Rg(3) treatment dose-dependently inhibited HC-induced intracellular Ca(2+) elevation, with an IC(50) value of 41.5+/-17.5 muM. In addition, Rg(3) treatment dose-dependently inhibited HC-induced currents in Xenopus oocytes expressing the NMDA receptor, with an IC(50) of 47.3+/-14.2 muM. These results collectively indicate that Rg(3)-induced neuroprotection against HC in rat hippocampus might be achieved via inhibition of HC-mediated NMDA receptor activation.
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PMID:Neuroprotective effects of ginsenoside Rg3 against homocysteine-induced excitotoxicity in rat hippocampus. 1723 31

Huntingtin-interacting protein 1 (HIP1) is an endocytic adaptor protein that plays a role in clathrin-mediated endocytosis and the ligand-induced internalization of AMPA receptors (AMPARs) (Metzler et al., 2003). In the present study, we investigated the role of HIP1 in NMDA receptor (NMDAR) function by analyzing NMDA-dependent transport and NMDA-induced excitotoxicity in neurons from HIP1-/- mice. HIP1 colocalizes with NMDARs in hippocampal and cortical neurons and affinity purifies with NMDARs by GST (glutathione S-transferase) pull down and coimmunoprecipitation. A profound decrease in NMDA-induced AMPAR internalization of 75% occurs in neurons from HIP1-/- mice compared with wild type, using a quantitative single-cell-based internalization assay. This defect in NMDA-dependent removal of surface AMPARs is in agreement with the observed defect in long-term depression induction in hippocampal brain slices of HIP1-/- mice and supports a role of HIP1 in AMPAR internalization in vivo. HIP1-/- neurons are partially protected from NMDA-induced excitotoxicity as assessed by LDH (lactate dehydrogenase) release, TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling) and caspase-3 activation assays, which points to a role of HIP1 in NMDA-induced cell death. Interestingly, phosphorylation of Akt and its substrate huntingtin (htt) decreases during NMDA-induced excitotoxicity by 48 and 31%, respectively. This decrease is significantly modulated by HIP1, resulting in 94 and 48% changes in P-Akt and P-htt levels in HIP1-/- neurons, respectively. In summary, we have shown that HIP1 influences important NMDAR functions and that both HIP1 and htt participate in NMDA-induced cell death. These findings may provide novel insights into the cellular mechanisms underlying enhanced NMDA-induced excitotoxicity in Huntington's disease.
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PMID:NMDA receptor function and NMDA receptor-dependent phosphorylation of huntingtin is altered by the endocytic protein HIP1. 1732 27

Collapsin response mediator proteins (CRMPs) are important molecules in neurite outgrowth and axonal guidance. Within the CRMP family, CRMP-2 has been implicated in several neurological diseases (Alzheimer's, epilepsy, and ischemia). Here, we investigated the integrity of CRMPs (CRMP-1, -2, -4, -5) after in vitro neurotoxin treatment and in vivo traumatic brain injury (TBI). After maitotoxin (MTX) and NMDA treatment of primary cortical neurons, a dramatic decrease of intact CRMP-1, -2 and -4 proteins were observed, accompanied by the appearance of distinct 55-kDa and 58-kDa breakdown products (BDP) for CRMP-2 and -4, respectively. Inhibition of calpain activation prevented NMDA-induced CRMP-2 proteolysis and redistribution of CRMP-2 from the neurites to the cell body, while attenuating neurite damage and neuronal cell injury. Similarly, CRMP-1, -2, and -4 were also found degraded in rat cortex and hippocampus following controlled cortical impact (CCI), an in vivo model of TBI. The appearance of the 55-kDa CRMP-2 BDP was observed to increase, in a time-dependent manner, between 24 and 48 h in the ipsilateral cortex, and by 48 hours in the hippocampus. The observed 55-kDa CRMP-2 BDP following TBI was reproduced by in vitro incubation of naive brain lysate with activated calpain-2, but not activated caspase-3. Sequence analysis revealed several possible cleavage sites near the C-terminus of CRMP-2. Collectively, this study demonstrated that CRMP-1, -2, and -4 are degraded following both acute traumatic and neurotoxic injury. Furthermore, calpain-2 was identified as the possible proteolytic mediator of CRMP-2 following excitotoxic injury and TBI, which appears to correlate well with neuronal cell injury and neurite damage. It is possible that the calpain-mediated truncation of CRMPs following TBI may be an inhibiting factor for post-injury neurite regeneration.
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PMID:Calpain-mediated collapsin response mediator protein-1, -2, and -4 proteolysis after neurotoxic and traumatic brain injury. 1740 52

Acute intoxication with large ammonia doses leads to activation of NMDA receptors in the brain, resulting in oxidative stress and disturbance of mitochondrial function. Altered mitochondrial function is a crucial step in some mechanisms of cellular apoptosis. This study assesses whether ammonia intoxication in vivo leads to induction of apoptotic markers such as permeability transition pore (PTP) formation, caspase-3, and caspase-9 activation, changes in p53 protein, or cytochrome c release. Acute ammonia intoxication did not affect caspase-9 or caspase-3 activities. The mitochondrial membrane potential also remained unaltered in non-synaptic brain mitochondria after injection of ammonia, indicating that ammonia did not induce PTP formation in brain in vivo. The nuclear level of p53 did not change, whereas its cytoplasmic level increased approximately two-fold. In agreement with the theory that translocation of the p53 from cytosol to nuclei is an essential step for induction of apoptosis we did not find apoptotic nuclei in brain of rats injected with ammonia. This supports the idea that ammonia neurotoxicity does not involve apoptosis and points to impaired p53 transfer from cytoplasm to nuclei as a possible preventer of apoptosis. We did not find any release of cytochrome c from mitochondria to cytosol after ammonia injection. Cytochrome c content was significantly reduced (30%) in brain mitochondria from rats injected with ammonia. This decrease may contribute to the reduced state 3 respiration, decreased respiratory control index, and disturbances in the mitochondrial electron transport chain in brain mitochondria from rats injected with ammonia.
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PMID:Acute ammonia neurotoxicity in vivo involves increase in cytoplasmic protein P53 without alterations in other markers of apoptosis. 1755 80

Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamate-induced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 microM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38 MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38 MAPK pathway.
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PMID:Glutamate-induced toxicity in hippocampal slices involves apoptotic features and p38 MAPK signaling. 1761 14

Glutamate, the major excitatory neurotransmitter, can cause the death of neurons by a mechanism known as excitotoxicity. This is a calcium-dependent process and activation of the NMDA receptor subtype contributes mainly to neuronal damage, due to its high permeability to calcium. Activation of calpain, a calcium-dependent cysteine protease, has been implicated in necrotic excitotoxic neuronal death. We have investigated the contribution of NMDA and non-NMDA ionotropic receptors to calpain activation and neuronal death induced by the acute administration of glutamate into the rat striatum. Calpain activity was assessed by the cleavage of the cytoskeletal protein, alpha-spectrin. Caspase-3 activity was also studied because glutamate can also lead to apoptosis. Results show no caspase-3 activity, but a strong calpain activation involving both NMDA and non-NMDA receptors. Although neuronal damage is mediated mainly by the NMDA receptor subtype, it can not be attributed solely to calpain activity.
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PMID:Contribution of NMDA and non-NMDA receptors to in vivo glutamate-induced calpain activation in the rat striatum. Relation to neuronal damage. 1827 Aug 15

The purpose of this study is to assess the neuroprotective effect of Rg1, a ginsenoside. We measured cell viability and lactate dehydrogenase (LDH) release from primary culture of rat hippocampal neurons and electrical activities in hippocampal slices of rats, before and after the neurons were deprived of oxygen and glucose. In addition, cerebral damage was evaluated with magnetic resonance imaging after middle cerebral artery was occluded transiently. Nissl staining was used for histological observation and immunohistochemistry analysis for activated caspase-3 expression of the brain. Furthermore, calcium influx was measured with laser confocal microscopy in neurons perfused with KCl (50 mM) or N-methyl-d-aspartate (NMDA, 1 mM), or deprived of oxygen and glucose. The influences of ginsenoside Rg1 on these parameters were determined simultaneously. We found that treatment of Rg1: 1) increased the neuronal viability; 2) promoted the recovery of electrical activity in hippocampal slices; 3) reduced the release of LDH, cerebral damage area, neuronal loss and expression of caspase-3; and 4) inhibited calcium influx induced by NMDA, KCl or oxygen/glucose deprivation. However, the protective effect of Rg1 was blocked by mifepristone, an antagonist of glucocorticoid receptors. Taken together, these results suggest that ginsenoside Rg1 can reduce neuronal death, including apoptotic cell death, induced by hypoxic-ischemic insults. This neuroprotective effect is probably mediated by the activation of glucocorticoid receptors, and by the inhibition of calcium influx through NMDA receptors and L-type voltage-dependent Ca2+ channels and the resultant reduction of intracellular free Ca2+.
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PMID:Ginsenoside Rg1 protects neurons from hypoxic-ischemic injury possibly by inhibiting Ca2+ influx through NMDA receptors and L-type voltage-dependent Ca2+ channels. 1843 Apr 19

Glutamate neurotoxicity is exacerbated when energy metabolism is impaired. In vitro studies show that neuronal death in these conditions is related to mitochondrial dysfunction, ATP depletion, and the loss of calcium homeostasis. We have recently observed that, in vivo, enhancement of glutamate toxicity elicited by previous mitochondrial inhibition does not involve severe ATP depletion, suggesting the involvement of other processes. Factors such as the activation of different proteases may determine the extent and type of cell death. Protease activation might be triggered by internal or external factors, such as mitochondrial damage or the activation of a particular glutamate receptor subtype. In the present study we aimed to investigate whether moderate inhibition of mitochondrial metabolism facilitates glutamate toxicity through caspase-3 or calpain activation, as well as the contribution of NMDA and non-NMDA glutamate ionotropic receptors to this activation. Rats were pre-treated with a subtoxic dose of 3-NP and 4 h later intrastriatally injected with glutamate. Results show that neither of these treatments alone (3-NP or Glu) or in combination (3-NP+Glu) activated caspase-3. Conversely, calpain activity is induced after glutamate injection both in intact and 3-NP pre-treated rats. Inhibition of calpain activity by MDL-28170 significantly prevented striatal damage. NMDA and non-NMDA receptors contributed equally to calpain activation and to the induction of neuronal death. Results suggest that enhancement of glutamate toxicity due to inhibition of mitochondrial metabolism in vivo, does not recruit caspase-dependent apoptosis but favors calpain activation through the stimulation of both subtypes of glutamate ionotropic receptors.
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PMID:Mild mitochondrial inhibition in vivo enhances glutamate-induced neuronal damage through calpain but not caspase activation: role of ionotropic glutamate receptors. 1849 18


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