Calpain inhibitors prevent nitric oxide-triggered excitotoxic apoptosis.

The pathogenesis of some neurodegenerative disorders has been linked to excitotoxicity, excess generation of nitric oxide (NO) and apoptosis. Here, we used a model of NO-triggered neuronal apoptosis that was strictly dependent on autocrine NMDA receptor (NMDA-R) activation and intracellular Ca2+ increase. We investigated the efficiency and potentially beneficial effects of calpain inhibition. Three calpain inhibitors that prevented intracellular fodrin proteolysis also blocked apoptotic features such as decrease in mitochondrial membrane potential, chromatin breakdown, and subsequent death of cerebellar granule neurons exposed to NO donors (S-nitroso-L-glutathione, S-nitroso-N-acetyl-d,l-penicillamine, and diethylamino-diazenolate-2-oxide). Since inhibitors did not interfere with NMDA-R activation, we suggest that block of calpains blunts NO-triggered neuronal apoptosis by stopping the cascade downstream of primary autocrine excitotoxic events.

Apoptosis in caspase-inhibited neurons.

BACKGROUND: There is growing evidence of apoptosis in neurodegenerative disease. However, it is still unclear whether the pathological manifestations observed in slow neurodegenerative diseases are due to neuronal loss or whether they are related to independent degenerative events in the axodendritic network. It also remains elusive whether a single, caspase-based executing system involving caspases is responsible for neuronal loss by apoptosis. MATERIALS AND METHODS: Long-term exposure to the microtubule-disassembling agent, colchicine, was used to disrupt the axodendritic network and eventually trigger caspase-3-mediated apoptosis in cultures of cerebellar granule cells. For this model, we investigated the role of Bcl-2 and caspases in neurite degeneration and death of neuronal somata. RESULTS: Early degeneration of the axodendritic network occurred by a Bcl-2 and caspase-independent mechanism. Conversely, apoptosis of the cell body was delayed by Bcl-2 and initially blocked by caspase inhibition. However, when caspase activity was entirely blocked by zVAD-fmk, colchicine-exposed neurons still underwent delayed cell death characterized by cytochrome c release, chromatin condensation to irregularly shaped clumps, DNA-fragmentation, and exposure of phosphatidylserine. Inhibitors of the proteasome reduced these caspase-independent apoptotic-like features of the neuronal soma. CONCLUSION: Our data suggest that Bcl-2-dependent and caspase-mediated death programs account only partially for neurodegenerative changes in injured neurons. Blockage of the caspase execution machinery may only temporarily rescue damaged neurons and classical apoptotic features can still appear in caspase-inhibited neurons.

Cascade of caspase activation in potassium-deprived cerebellar granule neurons: targets for treatment with peptide and protein inhibitors of apoptosis.

Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.

Energy requirement for caspase activation and neuronal cell death.

Recent work has shown that execution of the apoptotic program involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can be found also when cells are treated with inhibitors of caspases as the cell permeable tripeptide, Z-Val-Ala-Asp-fluoro-methyl-ketone (Z-VAD-fmk), or analogous compounds. This has posed the question as to whether apoptosis may occur in a caspase independent way, and whether caspase inhibitors may then be used to treat diseases characterised by an excess apoptosis. It is also becoming clear, that ATP depletion during the early phases of apoptosis can preclude caspase activation, and consequently switch execution of cell death towards necrosis. In vivo, a block or partial inhibition of the typical apoptotic demise may have profound implications, as persistence of damaged but "undead" cells within the nervous system, followed by delayed lysis may favour neuroinflammatory reactions. In this review, we discuss some recent findings, which suggest that cells may use diverging execution pathways, with different implications in neuropathology and therapy.

Execution of apoptosis: converging or diverging pathways?

There is increasing evidence that apoptosis and necrosis represent only two of several possible ways for cells to die. These two types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus, and often local metabolic conditions and the intensity of the same initial insult decide the prevalence of either apoptosis or necrosis. Recent work has shown that execution of the apoptotic programme involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can be observed also in cells where caspases are inhibited by cell-permeable tripeptides, such as z-VaD-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), or analogous compounds. This has posed the question as to whether apoptosis may or may not occur in a caspase independent way, and whether caspase inhibitors may be effective in the treatment of disease. Also relevant is the understanding that low intracellular energy levels during apoptosis can preclude caspase activation, and consequently decide the occurrence and mode of demise in damaged cells. In vivo, incomplete execution of damaged cells by apoptosis may have profound implications, as their persistence within a tissue, followed by delayed lysis, may elicit delayed pro-inflammatory reactions. In this minireview, we discuss some recent findings suggesting that cells may use diverging execution pathways, with different implications in pathology and therapy.

ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation.

BACKGROUND: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. MATERIALS AND METHODS: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, 1-methyl-4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. RESULTS: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. CONCLUSIONS: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.

CD95-Mediated murine hepatic apoptosis requires an intact glutathione status.

Agonistic engagement of the cytokine receptor CD95 in mice leads to activation of hepatic caspases, followed by massive hepatocyte apoptosis, acute liver failure, and death. This mechanism of cell death is thought to be associated with several human liver disorders. Because hepatic glutathione represents the major defense against toxic liver injury, we investigated its role in CD95-mediated liver failure, which represents a model for hyperinflammatory organ destruction. As a tool for modulating the liver glutathione status of mice in vivo, we used the GSH transferase substrate, phorone, which rapidly depleted hepatic glutathione in a dose-dependent manner. When GSH was depleted, CD95-initiated hepatic caspase-3-like activity and DNA fragmentation were completely blocked, and animals were protected from liver injury dose-dependently as assessed by histological examination and determination of liver enzymes in plasma. Conversely, repletion of hepatic glutathione by treatment with the permeable glutathione monoethylester restored susceptibility of GSH-depleted mice toward CD95-mediated liver injury. In contrast, the antioxidants, GSH, N-acetyl cysteine, alpha-tocopherol, butyl-hydroxytoluene, and catalase failed to do so. Animals treated once with phorone survived for more than 3 months after an otherwise lethal injection of the activating anti-CD95 antibody. We investigated the thiol sensitivity of recombinant caspase-3 in vitro and observed that its activity was dependent on the presence of a reducing agent such as GSH, while GSSG attenuated proteolytic activity. Based on our finding that CD95-mediated hepatocyte apoptosis requires an intact intracellular glutathione status, we propose that the activation of apoptosis-executing caspases is controlled by reduced glutathione.

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+.

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.

1-Methyl-4-phenylpyridinium induces autocrine excitotoxicity, protease activation, and neuronal apoptosis.

The pathogenesis of several neurodegenerative diseases may involve indirect excitotoxic mechanisms, where glutamate receptor overstimulation is a secondary consequence of initial functional defects of neurons (e.g., impairment of mitochondrial energy generation). The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and other mitochondrial inhibitors (e.g., rotenone or 3-nitropropionic acid) elicited apoptosis in cerebellar granule cell cultures via stimulation of autocrine excitotoxicity. Cell death, increase in intracellular Ca2+ concentration, release of cytochrome c, and all biochemical and morphological signs of apoptosis were prevented by blockade of the N-methyl-D-aspartate receptor with noncompetitive, glycine-site or glutamate-site inhibitors. In addition, MPP+-induced apoptosis was reduced by high Mg2+ concentrations in the medium or by inhibiting exocytosis with clostridial neurotoxins. Two classes of cysteine proteases were involved in the execution of cell death: caspases and calpains. Inhibitors of either class of proteases prevented cell death, cleavage of intracellular proteins (i.e., fodrin), and the appearance of typical features of apoptosis such as phosphatidylserine translocation or DNA fragmentation. However, protease inhibitors did not interfere with the initial intracellular Ca2+ concentration increase. We suggest that MPP+ as well as other mitochondrial inhibitors trigger indirect excitotoxic processes, which lead to Ca2+ overload, protease activation, and subsequent neuronal apoptosis.

Apoptosis in the absence of poly-(ADP-ribose) polymerase.

Cleavage of poly-(ADP-ribose) polymerase is a process occurring early during the execution phase of apoptosis. Although in many experimental systems PARP cleavage indicates a point of no return, the significance of this proteolytic step for apoptosis remains unclear. Here we compare the susceptibility of cells from wild-type mice and PARP-/- mice to several inducers of apoptosis. Neither the susceptibility of hepatocytes towards CD95 or TNF-mediated apoptosis nor the activation of PARP-cleaving caspases was modified in PARP-/- liver cells. Thymocytes with either genotype exhibited similar sensitivity to treatments with ceramide, dexamethasone, or etoposide. The sensitivity of primary neurons towards apoptosis induced by staurosporine, colchicine, potassium withdrawal, peroxynitrite, or the neurotoxin MPP+ was also unaltered. These data suggest that neither activation nor cleavage of PARP has a causal role in apoptotic cell death of primary, non-transformed cells.

Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide.

BACKGROUND: Excitotoxicity and excess generation of nitric oxide (NO) are believed to be fundamental mechanisms in many acute and chronic neurodegenerative disorders. Disturbance of Ca2+ homeostasis and protein nitration/nitrosylation are key features in such conditions. Recently, a family of proteases collectively known as caspases has been implicated as common executor of a variety of death signals. In addition, overactivation of poly-(ADP-ribose) polymerase (PARP) has been observed in neuronal excitotoxicity. We therefore designed this study to investigate whether triggering of caspase activity and/or activation of PARP played a role in cerebellar granule cell (CGC) apoptosis elicited by peroxynitrite (ONOO-) or NO donors. MATERIALS AND METHODS: CGC from wild-type or PARP -/- mice were exposed to various nitric oxide donors. Caspase activation and its implications for membrane alterations, Ca2+ homeostasis, intracellular proteolysis, chromatin degradation, and cell death were investigated. RESULTS: CGC exposed to NO donors undergo apoptosis, which is mediated by excess synaptic release of excitotoxic mediators. This excitotoxic mechanism differs from direct NO toxicity in some other neuronal populations and does not involve PARP activation. Inhibition of caspases with different peptide substrates prevented cell death and the related features, including intracellular proteolysis, chromatin breakdown, and translocation of phosphatidylserine to the outer surface of the cell membrane. Increased Ca2+ influx following N-methyl-D-aspartate (NMDA) receptor (NMDA-R) activation was not inhibited by caspase inhibitors. CONCLUSIONS: In CGC, NO donors elicit apoptosis by a mechanism involving excitotoxic mediators, Ca2+ overload, and subsequent activation of caspases.