calpain and 3-nitropropionic-acid

Excitotoxicity due to excessive activation of glutamate receptors is a primary mediator of cell death in acute and chronic neurological disorders, and NMDA-type glutamate receptors (NMDARs) are thought to be involved. NMDARs assemble from heteromeric combinations of GluN1, GluN2 and GluN3 subunits, yielding a variety of receptor subtypes that differ in biophysical properties, signaling, and synaptic targeting. Inclusion of inhibitory GluN3 subunits reduces Ca2+ influx via NMDAR channels and alters their synaptic targeting, thus modifying the two hallmarks of NMDARs that are critical for their roles on neuronal death and survival. Here we evaluated the neuroprotective potential of GluN3A subunits by analyzing the susceptibility to striatal excitotoxic damage of transgenic mice overexpressing GluN3A. We found that mild GluN3A overexpression protected susceptible striatal neurons from lesions induced by the neurotoxin 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex II/succinate dehydrogenase. GluN3A-mediated neuroprotection was dose-dependent, and correlated with the levels of transgenic GluN3A expressed by two different mice strains. Neuroprotection was associated with a potent reduction of the activation of calpain, a Ca2+-dependent protease, which was measured as a decrease in 3-NP-induced fodrin and STEP cleavage in GluN3A transgenic mice relative to controls. We further show that transgenic GluN3A subunits incorporate into extrasynaptic compartments in mouse striatum, suggesting that reductions of toxic calpain activation might be linked to inhibition by GluN3A of pathological extrasynaptic NMDAR activity.

Topics: Animals; Blotting, Western; Calpain; Convulsants; Corpus Striatum; Enzyme Activation; Immunohistochemistry; Immunoprecipitation; Mice; Mice, Transgenic; Nitro Compounds; Propionates; Protein Subunits; Receptors, N-Methyl-D-Aspartate

8-Oxoguanine (8-oxoG), a common DNA lesion caused by reactive oxygen species, is associated with carcinogenesis and neurodegeneration. Although the mechanism by which 8-oxoG causes carcinogenesis is well understood, the mechanism by which it causes neurodegeneration is unknown. Here, we report that neurodegeneration is triggered by MUTYH-mediated excision repair of 8-oxoG-paired adenine. Mutant mice lacking 8-oxo-2'-deoxyguanosine triphosphate-depleting (8-oxo-dGTP-depleting) MTH1 and/or 8-oxoG-excising OGG1 exhibited severe striatal neurodegeneration, whereas mutant mice lacking MUTYH or OGG1/MUTYH were resistant to neurodegeneration under conditions of oxidative stress. These results indicate that OGG1 and MTH1 are protective, while MUTYH promotes neurodegeneration. We observed that 8-oxoG accumulated in the mitochondrial DNA of neurons and caused calpain-dependent neuronal loss, while delayed nuclear accumulation of 8-oxoG in microglia resulted in PARP-dependent activation of apoptosis-inducing factor and exacerbated microgliosis. These results revealed that neurodegeneration is a complex process caused by 8-oxoG accumulation in the genomes of neurons and microglia. Different signaling pathways were triggered by the accumulation of single-strand breaks in each type of DNA generated during base excision repair initiated by MUTYH, suggesting that suppression of MUTYH may protect the brain under conditions of oxidative stress.

Topics: Animals; Apoptosis Inducing Factor; Benzamides; Calpain; Cell Nucleus; Corpus Striatum; Dipeptides; DNA Breaks, Single-Stranded; DNA Glycosylases; DNA Repair; DNA, Mitochondrial; Guanine; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Mitochondria; Motor Activity; Neurodegenerative Diseases; Nitro Compounds; Oxidative Stress; Phosphoric Monoester Hydrolases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Propionates

Glycogen synthase kinase-3beta (GSK-3beta) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain-mediated cleavage. Calpain mediates in cell death induced by 3-nitropropionic acid (3-NP), but GSK-3beta regulation has not been demonstrated. Here we studied changes in total GSK-3beta protein levels and GSK-3beta phosphorylation at Ser-9 in this model. The 3-NP treatment induced GSK-3beta truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3-NP-induced neuronal loss, inhibition of GSK-3beta by SB-415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3-NP-induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross-talk between calpain and GSK-3beta in the 3-NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3-NP-induced neuronal loss.

Topics: Amino Acid Chloromethyl Ketones; Aminophenols; Animals; Calpain; Caspases; Cell Survival; Cells, Cultured; Convulsants; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Male; Maleimides; Mice; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Nitro Compounds; Propionates; Purines; Rats; Roscovitine; Signal Transduction; Time Factors

In this study we tested whether phosphodiesterase 5 (PDE5) inhibitors, sildenafil and vardenafil, would afford protection against 3-nitropropionic acid (3NP), which produces striatal lesions that closely mimic some of the neuropathological features of Huntington's Disease (HD). The neurotoxin was given over 5 days by constant systemic infusion using osmotic minipumps. Animals treated with PDE5 inhibitors (sildenafil or vardenafil) showed improved neurologic scores, reduced the loss of striatal DARPP-32 protein levels and lesion volumes, and decreased calpain activation produced by 3NP. This protective effect was independent of changes in 3NP-induced succinate dehydrogenase inhibition. Furthermore, striatal p-CREB levels along with the expression of BDNF were significantly increased in sildenafil-treated rats. In summary, PDE5 inhibitors protected against 3NP-induced striatal degeneration by reducing calpain activation and by promoting survival pathways. These data encourage further evaluation of PDE5 inhibitors in transgenic mouse models of HD.

Topics: Analysis of Variance; Animals; Blotting, Western; Brain-Derived Neurotrophic Factor; Calpain; Corpus Striatum; Cyclic AMP Response Element-Binding Protein; Dopamine and cAMP-Regulated Phosphoprotein 32; Male; Motor Activity; Neurons; Neurotoxicity Syndromes; Neurotoxins; Nitro Compounds; Phosphodiesterase Inhibitors; Piperazines; Propionates; Purines; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Sildenafil Citrate; Succinate Dehydrogenase; Sulfones

Lithium reduced striatal neurodegeneration induced in rats by 3-nitropropionic acid inhibiting calpain activation. Lithium prevented an increase in cdk5 activity, as shown by the levels of the co-activator p35. Myocite enhancer factor 2 (MEF2), a downstream substrate for cdk5 with pro-survival activity, showed increased phosphorylation. In primary cultures of neurons treated with 3-NP, lithium also reduced protease activity mediated by calpain, cdk5 activation and cellular death. These observations indicate that lithium has a neuroprotective effect. Lithium treatment also reduced the intracellular increase in calcium induced by 3-NP. The finding that lithium mediates the modulation of the calpain/cdk5 pathway further supports its use in the treatment of neurodegenerative diseases.

Topics: Animals; Calcium; Calpain; Cell Survival; Cells, Cultured; Cyclin-Dependent Kinase 5; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Gene Expression Regulation; Hippocampus; Huntington Disease; Lithium Chloride; Male; Mice; Neurons; Neuroprotective Agents; Nitro Compounds; Propionates; Rats; Rats, Sprague-Dawley; Signal Transduction; Succinate Dehydrogenase

3-Nitropropionic acid (NPA) produces degeneration of striatum and some neurological disturbances characteristic of Huntington's disease in rodents and primates. We have shown that the flavonoid kaempferol largely reduced striatal damage induced by cerebral ischaemia-reperfusion in rats (Lopez-Sanchez et al. 2007). In this work, we report that intraperitoneal (i.p.) administration of kaempferol affords an efficient protection against NPA-induced neurodegeneration in Wistar rats. We studied the effects of daily i.p. injections of 7, 14 and 21 mg of kaempferol/kg body weight during the NPA-treatment (25 mg/kg body weight/12 h i.p., for 5 days) on the neurological deficits, degeneration of rat striatum and oxidative stress markers. Intraperitoneal injections of 14-21 mg of kaempferol/kg body weight largely attenuated motor deficit and delayed mortality. The higher dose of kaempferol prevented the appearance of NPA-induced striatal lesions up to the end of treatment, as revealed by haematoxylin-eosin and TUNEL staining, and also NPA-induced oxidative stress, because it blocked the fall of reduced glutathione and the increase of protein nitrotyrosines in NPA-treated rats. It was found that striatal degeneration was associated with calpains activation and a large inactivation of creatine kinase, which were also prevented when the higher doses of kaempferol were administered.

Topics: Animals; Calpain; Caspases; Convulsants; Corpus Striatum; Creatine Kinase; Disease Models, Animal; Huntington Disease; Kaempferols; Male; Nerve Degeneration; Neuroprotective Agents; Nitro Compounds; Oxidative Stress; Propionates; Rats; Rats, Wistar; Reactive Nitrogen Species; Reactive Oxygen Species

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.

Topics: Animals; Calcium Signaling; Calpain; Cell Death; Cerebral Cortex; Convulsants; Corpus Striatum; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Inhibitors; G1 Phase; Huntington Disease; Nitro Compounds; Organ Specificity; Phosphorylation; Propionates; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Retinoblastoma Protein; RNA, Small Interfering; Succinate Dehydrogenase

3-Nitropropionic acid (3-NP) is a neurotoxin that inhibits mitochondrial complex II and is used in an experimental model of Huntington's disease. Treatment of rats with 3-NP 30mgkg(-1) i.p. once a day for 5 days induced an increase in calpain activation in rat striatum, measured by the formation of 145kDa fragment of alpha-spectrin breakdown and by an increase in enzymatic calpain activity. In this neurotoxic model, Western blot studies revealed that calpain activity increase was followed by changes in cyclin-dependent kinase 5 (cdk5) and its activator p25. Our results indicated, after 10 days of treatment with 3-NP, a decrease in myocyte enhancer factor phosphorylation, a neuronal prosurvival factor. Thus, a decrease in its expression indicates a new potential mechanism of neuronal cell death mediated by the neurotoxin 3-NP. Accordingly, in our study we demonstrated in rat striatum the activation of the calpain/cdk5/p25 pathway in the 3-NP model. Previous studies have linked the deregulation of cdk5 with neurodegenerative diseases, such as Alzheimer's and Parkinson's. We suggest that calpain/cdk5 activation could also be a common pathway activated in other neurodegenerative diseases, which is liable to be targeted.

Topics: Analysis of Variance; Animals; Calpain; Convulsants; Corpus Striatum; Cyclin-Dependent Kinase 5; Enzyme Activation; Male; Nitro Compounds; Propionates; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors

According to the "indirect" excitotoxicity hypothesis, mitochondrial defects increase Ca2+ entry into neurons by rendering NMDA-R hypersensitive to glutamate. We tested this hypothesis by investigating in the rat striatum and cultured striatal cells how partial mitochondrial complex II inhibition produced by 3-nitropropionic acid (3NP) modifies the toxicity of the NMDA-R agonist quinolinate (QA). We showed that nontoxic 3NP treatment, leading to partial inhibition of complex II activity, greatly exacerbated striatal degeneration produced by slightly toxic QA treatment through an "all-or-nothing" process. The potentiation of QA-induced cell death by 3NP was associated with increased calpain activity and massive calpain-mediated cleavage of several postsynaptic proteins, suggesting major neuronal Ca2+ deregulation in the striatum. However, Ca2+ anomalies probably do not result from NMDA-R hypersensitivity. Indeed, brain imaging experiments using [(18)F]fluorodeoxyglucose indirectly showed that 3NP did not increase QA-induced ionic perturbations at the striatal glutamatergic synapses in vivo. Consistent with this, the exacerbation of QA toxicity by 3NP was not related to an increase in the QA-induced entry of 45Ca2+ into striatal neurons. The present results demonstrate that the potentiation of NMDA-R-mediated excitotoxicity by mitochondrial defects involves primarily intracellular Ca2+ deregulation, in the absence of NMDA-R hypersensitivity.

Topics: Animals; Calcium Signaling; Calpain; Cells, Cultured; Corpus Striatum; Male; Mitochondria; Neurons; Nitro Compounds; Propionates; Quinolinic Acid; Rats; Rats, Inbred Lew; Receptors, N-Methyl-D-Aspartate

Huntington's disease has an increase in the activated calpain, which is enhanced by the NMDA receptor activation. We investigated the neuroprotective effect of memantine in 3-nitropropionic acid (3NP)-induced striatal degeneration model. Either memantine (20 mg/kg/day) or PBS was intraperitoneally administered for five days with 3NP continuous infusion. In the memantine-treated group, the striatal lesion volume, the number of TUNEL+ cells, and Fluoro-Jade C+ degenerating neurons were all decreased. Memantine increased Bcl-xl and decreased Bax level. Memantine also exerted an inhibitory effect on the micro-calpain level and decreased the huntingtin proteolytic fragments. Those rats treated with memantine showed less degree of weight loss at 5 days. Subsequently, memantine was found to have neuroprotective effects and save striatal cells with decreasing calpain levels in the 3NP model of Huntington's disease.

Topics: Animals; Apoptosis Regulatory Proteins; Calpain; Cell Death; Corpus Striatum; Disease Models, Animal; Down-Regulation; Excitatory Amino Acid Antagonists; Huntingtin Protein; Huntington Disease; Male; Memantine; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Neurotoxins; Nitro Compounds; Nuclear Proteins; Peptide Fragments; Propionates; Rats; Rats, Sprague-Dawley

Minocycline has been shown to be neuroprotective in various models of neurodegenerative diseases. However, its potential in Huntington's disease (HD) models characterized by calpain-dependent degeneration and inflammation has not been investigated. Here, we have tested minocycline in phenotypic models of HD using 3-nitropropionic acid (3NP) intoxication and quinolinic acid (QA) injections. In the 3NP rat model, where the development of striatal lesions involves calpain, we found that minocycline was not protective, although it attenuated the development of inflammation induced after the onset of striatal degeneration. The lack of minocycline activity on calpain-dependent cell death was also confirmed in vitro using primary striatal cells. Conversely, we found that minocycline reduced lesions and inflammation induced by QA. In cultured cells, minocycline protected against mutated huntingtin and staurosporine, stimulations known to promote caspase-dependent cell death. Altogether, these data suggested that, in HD, minocycline may counteract the development of caspase-dependent neurodegeneration, inflammation, but not calpain-dependent neuronal death.

Topics: Animals; Calpain; Caspases; Cell Death; Cells, Cultured; Corpus Striatum; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalitis; Glutamic Acid; Huntingtin Protein; Huntington Disease; Male; Minocycline; Nerve Degeneration; Nerve Tissue Proteins; Neuroprotective Agents; Nitro Compounds; Nuclear Proteins; Phenotype; Propionates; Quinolinic Acid; Rats; Rats, Inbred Lew; Rats, Wistar; Staurosporine

The contribution of calpains and caspases to cell death has been widely studied using pharmacological inhibitors. Among them, the caspase inhibitor N-benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone (zVAD) has been used as a specific caspase inhibitor in nearly 1000 published studies. However, several studies showed that zVAD also behaves as a calpain inhibitor in peripheral cells. The effects of zVAD as a calpain inhibitor have never been assessed in neurodegeneration models. We examined here whether zVAD could reduce neurodegeneration in Huntington's disease models using the mitochondrial inhibitor 3-nitropropionic acid (3NP). In these models, 3NP toxicity has been shown to require calpain activation. In rats, intra-cerebro-ventricular infusion of zVAD significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain and calpain-dependent cleavage of fodrin. zVAD (100 microM) also blocked 3NP-induced death of cultured striatal neurons. In vitro, zVAD inhibited purified mu-calpain with high affinity (IC50=10 nM). The present data demonstrate that zVAD protects neurons against 3NP through calpain inhibition. This suggests that, in certain models of neuronal death where zVAD showed protective effects, caspases but also calpains may be involved.

Topics: Animals; Calpain; Carrier Proteins; Cell Death; Huntington Disease; Immunohistochemistry; Injections, Intraventricular; Male; Microfilament Proteins; Neostriatum; Nerve Degeneration; Neuroprotective Agents; Nitro Compounds; Oligopeptides; Propionates; Protease Inhibitors; Rats; Rats, Inbred Lew; Rats, Wistar

Molecular machinery involved in apoptosis plays a role in neuronal death in neurodegenerative disorders such as Parkinson's disease (PD) and Huntington's disease (HD). Several caspase inhibitors, such as the well-known peptidyl inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (zVADfmk), can protect neurons from apoptotic death caused by mitochondrial toxins. However, the poor penetrability of zVADfmk into brain and toxicity limits its use therapeutically. In the present study, a novel peptidyl broad-spectrum caspase inhibitor, Q-VD-OPH, which offers improvements in potency, stability, and toxicity over zVADfmk, showed significant protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3NP), and malonate toxicities. Q-VD-OPH significantly reduced dopamine depletion in striatum produced by MPTP administration and prevented MPTP-induced loss of dopaminergic neurons in the substantia nigra. It significantly reduced the size of striatal lesions produced by intrastriatal malonate injections and systemic administration of 3NP. Western blots performed on tissues from the midbrain following administration of MPTP or the striatum in 3NP-treated animals showed increases of the active forms of caspase-9 and caspase-8, as well as the caspase-8-mediated proapoptotic protein Bid, which were inhibited Q-VD-OPH treatment. These findings suggest that systematically active broad-spectrum caspase inhibitors maybe useful in the treatment of neurodegenerative diseases such as PD and HD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Chloromethyl Ketones; Animals; BH3 Interacting Domain Death Agonist Protein; Brain; Calpain; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Corpus Striatum; Dopamine Agents; Enzyme Inhibitors; Male; Malonates; Mesencephalon; Mice; Neurotoxins; Nitro Compounds; Propionates; Quinolines; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley

Striatal cell death in Huntington's Disease (HD) may involve mitochondrial defects, NMDA-mediated excitotoxicity, and activation of death effector proteases such as caspases and calpain. However, the precise contribution of mitochondrial defects in the activation of these proteases in HD is unknown. Here, we addressed this question by studying the mechanism of striatal cell death in rat models of HD using the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP). The neurotoxin was either given by intraperitoneal injections (acute model) or over 5 d by constant systemic infusion using osmotic pumps (chronic model) to produce either transient or sustained mitochondrial deficits. Caspase-9 activation preceded neurodegeneration in both cases. However, caspase-8 and caspase-3 were activated in the acute model, but not in the chronic model, showing that 3-NP does not require activation of these caspases to produce striatal degeneration. In contrast, activation of calpain was specifically detected in the striatum in both models and this was associated with a calpain-dependent cleavage of huntingtin. Finally, in the chronic model, which mimics a steady blockade of complex II activity reminiscent of HD, selective calpain inhibition prevented the abnormal calpain-dependent processing of huntingtin, reduced the size of the striatal lesions, and almost completely abolished the 3-NP-induced DNA fragmentation in striatal cells. The present results demonstrate that calpain is a predominant effector of striatal cell death associated with mitochondrial defects in vivo. This suggests that calpain may play an important role in HD pathogenesis and could be a potential therapeutic target to slow disease progression.

Topics: Acute Disease; Animals; Calpain; Caspases; Cell Death; Chronic Disease; Corpus Striatum; Disease Models, Animal; DNA Fragmentation; Drug Administration Routes; Electron Transport Complex II; Enzyme Inhibitors; Huntingtin Protein; Huntington Disease; Male; Mitochondria; Multienzyme Complexes; Nerve Tissue Proteins; Neuroprotective Agents; Nitro Compounds; Nuclear Proteins; Oxidoreductases; Propionates; Rats; Rats, Inbred Lew; Succinate Dehydrogenase

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, has been used to model features of neurodegenerative disorders including Huntington disease, as well as acute neuronal insults such as cerebral ischemia. 3NP induces rapid necrosis and delayed apoptosis in primary cultures of rat hippocampal neurons. Low levels of extracellular glutamate shift the cell death mechanism to necrosis, whereas antagonism of NMDA receptors results in predominately apoptotic death. In the present study, the involvement of cysteine proteases in the morphologic and biochemical alterations accompanying 3NP-induced neuron death was investigated. Immunoblots of spectrin breakdown products indicated Ca(2+)-dependent cysteine protease (calpain) activation within the 8 hours of 3NP administration, whereas caspase-3 activation was not evident until 16 to 48 hours after treatment. The NMDA receptor antagonist MK-801 (dizocilpine) decreased 3NP-induced calpain activity, but did not alter caspase-3 activity. Similar to MK-801, calpain inhibitors (Z-Val-Phe.H and Z-Leu-Phe-CONHEt) shifted the cell death morphology towards apoptosis and delayed, but did not prevent, the 3NP-induced cell death. Together, the results indicate that following 3NP administration, increased calpain activity precedes caspase-3 activation, contributes to the necrotic morphology, and facilitates and accelerates the cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspases; Cell Death; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Drug Synergism; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Immunoblotting; Immunohistochemistry; Necrosis; Neurons; Neurotoxins; Nitro Compounds; Oligopeptides; Propionates; Rats; Spectrin; Time Factors

The role of caspases and calpains in neurodegeneration remains unclear. In this study, we focused on these proteases in a rat model of Huntington's disease using the mitochondrial toxin 3-nitropropionic acid (3NP). Results showed that 3NP-induced death of striatal neurons was preceded by cytochrome c redistribution, transient caspase-9 processing, and activation of calpain, whereas levels of the active/processed form of caspase-3 remained low and were even reduced as compared with control animals. We evidenced here that this decrease in active caspase-3 levels could be attributed to calpain activation. Several observations supported this conclusion. 1) Pharmacological blockade of calpain in 3NP-treated rats increased the levels of endogenous processed caspase-9 and caspase-3. 2) Cell-free extracts prepared from the striatum of 3NP-treated rats degraded in vitro the p34 and p20 subunits of active recombinant caspase-9 and caspase-3, respectively. 3) This degradation of p34 and p20 could be mimicked by purified mu-calpain and was prevented by calpain inhibitors. 4) mu-Calpain produced a loss of the DEVDase (Asp-Glu-Val-Asp) activity of active caspase-3. 5) Western blot analysis and experiments with 35S-radiolabeled caspase-3 showed that mu-calpain cleaved the p20 subunit of active caspase-3 near its catalytic site. 6) mu-Calpain activity was selectively inhibited (IC50 of 100 mum) by a 12 amino acid peptide corresponding to the C terminus of p20. Our results showed that calpain can down-regulate the caspase-9/caspase-3 cell death pathway during neurodegeneration due to chronic mitochondrial defects in vivo and that this effect may involve, at least in part, direct cleavage of the caspase-3 p20 subunit.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Brain; Calpain; Caspase 3; Caspase 9; Caspases; Cell-Free System; Cytosol; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Inhibitory Concentration 50; Male; Models, Biological; Molecular Sequence Data; Neurodegenerative Diseases; Neurons; Nitro Compounds; Propionates; Protein Binding; Protein Structure, Tertiary; Rats; Rats, Inbred Lew; Recombinant Proteins; Sequence Homology, Amino Acid; Time Factors

Huntington's disease (HD) is caused by a polyglutamine expansion in the amino-terminal region of huntingtin. Mutant huntingtin is proteolytically cleaved by caspases, generating amino-terminal aggregates that are toxic for cells. The addition of calpains to total brain homogenates also leads to cleavage of wild-type huntingtin, indicating that proteolysis of mutant and wild-type huntingtin may play a role in HD. Here we report that endogenous wild-type huntingtin is promptly cleaved by calpains in primary neurons. Exposure of primary neurons to glutamate or 3-nitropropionic acid increases intracellular calcium concentration, leading to loss of intact full-length wild-type huntingtin. This cleavage could be prevented by calcium chelators and calpain inhibitors. Degradation of wild-type huntingtin by calcium-dependent proteases thus occurs in HD neurons, leading to loss of wild-type huntingtin neuroprotective activity.

Topics: Animals; Blotting, Western; Brain; Calcimycin; Calcium; Calpain; Cell-Free System; Cells, Cultured; Densitometry; Glutamic Acid; Huntingtin Protein; Ionophores; Nerve Tissue Proteins; Neurons; Nitro Compounds; Nuclear Proteins; Propionates; Protein Binding; Rats; Rats, Sprague-Dawley; Time Factors

Oxidative stress has been implicated as a pathogenic mediator of neuronal perikarya cell death. Axons and oligodendrocytes, components of white matter, could also be vulnerable to oxidative damage. An experimental model of oxidative stress was induced by systemic injection of 3-nitropropionic acid (3-NPA). Animals received an i.p. injection of 10, 15, 20 or 30 mg/kg 3-NPA or vehicle and were killed 24 h later. 3-NPA produced a concentration-dependent increase in axonal pathology within the striatum reflected by the amount of beta-APP and SNAP-25 accumulation. Axonal damage was anatomically coincident with the neuronal lesion. There was no neuronal or axonal damage in the subcortical white matter or cerebral cortex in any of the animals treated with 3-NPA. Manganese superoxide dismutase (Mn-SOD) immunoreactivity was present in the vehicle and all 3-NPA treated groups. The amount of Mn-SOD cellular staining was concentration-dependently increased within the striatum supporting a role for oxidative stress in the mechanism of 3-NPA neurotoxicity. Oligodendrocyte-like cells within the subcortical white matter were immunopositive for calpain-mediated spectrin breakdown products and increased in a concentration-dependent manner. Therefore in this experimental model, mitochondrial inhibition may lead to the initiation of oxidative stress and calpain activation, which could mediate cytoskeletal breakdown in axons and oligodendrocytes suggesting an interaction between at least two pathogenic mechanisms.

Topics: Amyloid beta-Protein Precursor; Animals; Axons; Brain Diseases; Calpain; Cytoskeleton; Free Radicals; Immunohistochemistry; Male; Mitochondria; Neuroglia; Neurotoxins; Nitro Compounds; Oxidative Stress; Propionates; Rats; Rats, Sprague-Dawley; Superoxide Dismutase