Apoptosis. Key to the mitochondrial gate.
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Apoptosis↗
Biomedical subjects
J C Martinou
Publications and source records attributed to J C Martinou.
The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event.
During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.
Amino Acid Chloromethyl Ketones↗
Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.
Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.
Animals↗
Oxidative stress is attenuated in mice overexpressing BCL-2.
The protooncogene Bcl-2 inhibits apoptosis in neural cells, which may involve mitochondrial stabilization and decreased generation of reactive oxygen species. Using in vivo microdialysis we found that following administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) there was a significant increase in the conversion of 4-hydroxybenzoic acid (4-HBA) to 3,4-dihydroxybenzoic acid (3,4-DHBA) in control mice, but not in Bcl-2 overexpressing mice. Striatal lesions were observed in littermate control mice, whereas, lesions were minimal or absent in Bcl-2 overexpressing mice. This shows that Bcl-2 overexpression in vivo attenuates the generation of reactive oxygen species.
Animals↗
Overproduction of Cu/Zn-superoxide dismutase or Bcl-2 prevents the brain mitochondrial respiratory dysfunction induced by glutathione depletion.
Recent work has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Low concentrations of the powerful antioxidant glutathione (GSH) and impaired brain energy metabolism, particularly in the substantia nigra, are key features of Parkinson's disease (PD). The main goal of this study was to better characterize the deleterious effects of brain GSH depletion on mitochondrial function. We depleted GSH in the brains of newborn wild-type (WT) and transgenic (Tg) mice overproducing either human Cu/Zn-superoxide dismutase (h-CuZnSOD) or human Bcl2 (h-Bcl-2), by subcutaneous injection of l-buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. GSH was 97% depleted in brain homogenates and 90% depleted in brain mitochondria for both WT and Tg mice. This depletion of brain GSH led to a decrease in the activity of the GSH-dependent antioxidant enzyme glutathione peroxidase, both in WT and in Tg animals. BSO treatment decreased the activities of respiratory complexes I, II, and IV in the brain homogenates of WT mice. BSO-treated h-CuZnSOD or h-Bcl-2 Tg mice had no respiratory chain deficiencies. Thus, brain GSH depletion leads to the impairment of mitochondrial respiratory chain activity. The protection of mitochondrial respiratory function by overproduction of Bcl-2 may result from a decrease in the generation of reactive oxygen species (ROS) or lipid peroxidation. The protection of mitochondria by overproduction of CuZnSOD is consistent with the involvement of superoxide or superoxide-derived ROS in the mitochondrial dysfunction caused by brain GSH depletion. This study demonstrates that the antioxidant balance is critical for maintenance of brain mitochondrial function, and its disruption may contribute to the pathogenesis of PD.
Animals↗
Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid beta-peptide neurotoxicity.
The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid beta-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer's disease victims. In this study, we have compared the toxicity of amyloid beta-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper-zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper-zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid beta-peptide(25-35) [the minimal cytotoxic fragment of amyloid beta-peptide(1-42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid beta-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper-zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid beta-peptide(25-35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid beta-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid beta-peptide(25-35) and amyloid beta-peptide(1-42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer's disease.
Amyloid beta-Peptides↗
Hu-Bcl-2 transgenic mice with supernumerary neurons exhibit timing impairment in a complex motor task.
Programmed neuronal cell death is common during development, and is thought to be important in the elimination of errors in axonal projection, cell position and sculpting of neuronal circuits. However, the potential importance of programmed cell death for complex behaviour in the adult animal has never been addressed. We studied motor abilities in a strain of transgenic mice with neuronal overexpression of the human Bcl-2 protein, which have supernumerary neurons due to reduced developmental cell death. Our results show that these mice have a clear deficiency in fine timing of motor coordination without impairment of basic motor functions. This is the first indication that altered developmental cell death and the consequent neuronal surplus can impair complex behaviour in the adult animal.
Animals↗
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing Bcl-2.
The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases mitochondrial calcium-buffering capacity. The neurotoxicity of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyride (MPTP) is thought to involve both mitochondrial dysfunction and free radical generation. We therefore investigated MPTP neurotoxicity in both Bcl-2 overexpressing mice and littermate controls. MPTP-induced depletion of dopamine and loss of [3H]mazindol binding were significantly attenuated in Bcl-2 overexpressing mice. Protection was more profound with an acute dosing regimen than with daily MPTP administration over 5 d. 1-Methyl-4-phenylpyridinium (MPP+) levels after MPTP administration were similar in Bcl-2 overexpressing mice and littermates. Bcl-2 blocked MPP+-induced activation of caspases. MPTP-induced increases in free 3-nitrotyrosine levels were blocked in Bcl-2 overexpressing mice. These results indicate that Bcl-2 overexpression protects against MPTP neurotoxicity by mechanisms that may involve both antioxidant activity and inhibition of apoptotic pathways.
1-Methyl-4-phenylpyridinium↗
Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions.
Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.
Animals↗
Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons.
We show that the pro-apoptotic proteins Bax and Bak trigger apoptosis when over-expressed in sympathetic neurons cultured in the presence of NGF. This effect can be blocked with z-VAD-fmk, a peptide inhibitor of caspases, but not with anti-apoptotic chemical compounds such as antioxidants or proteasome inhibitors. These results demonstrate that in sympathetic neurons Bax and Bak are sufficient to induce apoptosis in the absence of any other apparent cell death stimulus and that their effect is mediated by caspases but does not require reactive oxygen species nor activity of the proteasome.
Acetylcysteine↗
Postnatal expression of Hu-bcl-2 gene in Lurcher mutant mice fails to rescue Purkinje cells but protects inferior olivary neurons from target-related cell death.
The Lurcher mutant has been extensively studied as a model for cell-autonomous and target-related cell death, yet there are still many unknowns concerning the mechanisms of neuronal degeneration in this mutant. As a key regulator of apoptosis, a bcl-2 transgene has been overexpressed in the heterozygous Lurcher mutant to investigate the effects of BCL-2 on two types of in vivo neuronal cell loss in Lurcher: cell-autonomous Purkinje cell degeneration and target-related olivary neuron death. Six adult +/Lc mutants expressing a human bcl-2 transgene (Hu-bcl-2) were generated by crossing +/Lc mutants with NSE71 Hu-bcl-2 transgenic mice. Analysis of these brains showed that bcl-2 overexpression did not prevent +/Lc Purkinje cell degeneration, but it did rescue most olivary neurons from target-related cell death. Although the number of olivary neurons was equivalent to wild-type numbers, the inferior olive nucleus was significantly shorter in its rostrocaudal extent, suggesting that olivary neurons are atrophied. We propose that Lurcher gene action causes Purkinje cell degeneration independently of a BCL-2-mediated pathway. Furthermore, although bcl-2 overexpression rescues olivary neurons from target-related cell death, it does not prevent the atrophy associated with the loss of target-related trophic support.
Animals↗
Purification and biochemical properties of soluble recombinant human Bax.
Bax is a member of the Bcl-2 protein family with proapoptotic properties. The proteins of this family contain three highly conserved regions termed BH1, BH2, and BH3 as well as a hydrophobic COOH-terminal domain, which is responsible for the membrane attachment of the proteins. We have expressed human Bax truncated of the 20 amino acid COOH-terminal hydrophobic domain to obtain large amounts of soluble protein suitable for biochemical and structural studies. The truncated protein was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The GST-Bax fusion protein was bound to glutathione-Sepharose, and Bax was released by thrombin cleavage and further purified by sequential chromatography on heparin-Sepharose and DEAE-Sepharose. The purified protein was present in solution as a heptamer and multimers of the heptamer complex. Limited tryptic digestion cleaved the protein in the region preceding the BH3 domain and produced a specific stable protein fragment of 15 kDa. Phosphorylation has been proposed as a possible regulatory mechanism of the bcl-2 proteins. The Bax protein was an in vitro substrate for specific serine/threonine protein kinases.
Amino Acid Sequence↗
Overexpression of a Hu-bcl-2 transgene in Lurcher mutant mice delays Purkinje cell death.
Cerebellar Purkinje cells in the heterozygous Lurcher mutant undergo cell autonomous degeneration beginning in the second week of postnatal development and becoming almost total around 30-45 days. The Lurcher mutation was recently identified as gain-of-function defect in the delta 2 glutamate receptor causing a constitutive current leak, suggesting that +/Lc Purkinje cells die by an excitotoxic mechanism. In previous studies we have shown that overexpression of bcl-2, a key regulator of cell death, in the heterozygous Lurcher mutant does not prevent +/Lc Purkinje cell death. To investigate further the mechanisms of +/Lc Purkinje cell death, we have crossed +/Lc mutants with a second line of Hu-bcl-2 transgenics (NSE73a) that shows an earlier onset of transgene expression and higher expression levels. Analysis of eight +/Lc-NSE73a mutants (4 at 2 months and 4 at 5-6 months) showed that Hu-bcl-2 overexpression delayed, but ultimately could not prevent +/Lc Purkinje cell death.
Animals↗
Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia.
In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.
Amino Acid Chloromethyl Ketones↗
Death-signalling cascade in mouse cerebellar granule neurons.
Molecular mechanisms of neuronal cell death are still largely unknown. In the present study, the signal transduction pathway of cell death in cerebellar granule neurons was examined by employing various death-preventative agents. When death was induced by the depletion of serum and a depolarizing level of potassium, transient increase in active c-Jun, mitochondrial membrane potential (deltapsi) loss, activation of caspase-3 (-like) proteases, and nuclear condensation and fragmentation were observed. The protein synthesis inhibitor cycloheximide blocked all these phenomena, whereas RNA synthesis inhibitor actinomycin-D, survival factor such as insulin-like growth factor-1, brain-derived neurotrophic factor, high K+ (25 mM) and overproduced antiapoptotic protein Bcl-2, prevented deltapsi, loss, caspase activation, and nuclear change, but not an increase in active c-Jun. The caspase inhibitor z-Asp-CH2-DCB (carbobenzoxy-L-aspartyl-alpha-[(2,6-dichlorobenzoyl) oxy]methane) only inhibited activation of caspases and nuclear change. These results suggest that the death signal in cerebellar granule neurons is sequentially transduced in the order of c-Jun activation, de novo RNA synthesis, mitochondrial deltapsi loss, activation of caspase-3 (-like) proteases and nuclear change.
Animals↗
Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice.
BACKGROUND AND PURPOSE: Reports suggesting the involvement of apoptosis in ischemic neuronal damage have been accumulating, and protection against apoptotic death by BCL-2 has been shown in many types of cells. Overexpression of BCL-2 has been shown to reduce infarct size after focal ischemia. The purpose of the present study was to assess whether BCL-2 exerted its effect on selective neuronal vulnerability after transient global ischemia. METHODS: Transgenic mice overexpressing BCL-2 in neurons and their littermates were subjected to transient forebrain ischemia for 12 minutes, and the hippocampus was examined 7 days later with conventional histology, immunohistochemistry, and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of fragmented DNA. RESULTS: Although both types of mice showed a similar degree of ischemic insult, transgenic mice showed a lesser degree of neuronal death together with DNA fragmentation in the hippocampus than their littermates. CONCLUSIONS: Overexpression of BCL-2 in neurons mitigates selective neuronal vulnerability in the hippocampus of transgenic mice after transient global ischemia.
Animals↗
Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.
We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.
Amino Acid Sequence↗
Fear decrease in transgenic mice overexpressing bcl-2 in neurons.
Neuronal destruction in the amygdala, hypothalamus and cerebellum provokes a diminution in anxiety and neophobia. In transgenic mice that express the human bcl-2 gene under the control of neuron specific enolase promotor (Hu-bcl-2), BCL-2 overexpression reduces the naturally occurring neuronal death, producing an increase of the number of neurons and brain size. Since BCL-2 over-expression has been observed in different parts of the brain and especially in the amygdaloid nuclei, the hypothalamus and the cerebellum, we studied the fear-related behavior of these transgenic mice. Hu-bcl-2 transgenic mice showed a decrease in anxiety and neophobia, indicating that, for this particular behavior, supernumerary neurons elicit the same modification as that observed after neuronal destruction.
Animals↗