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J C Martinou

Publications and source records attributed to J C Martinou.

At least 55 records · Page 3Linked to original sources
PubMed1997

Bcl-2 promotes regeneration of severed axons in mammalian CNS.

Most neurons of the mammalian central nervous system (CNS) lose the ability to regenerate severed axons in vivo after a certain point in development. At least part of this loss in regenerative potential is intrinsic to neurons. Although embryonic retinal ganglion cells (RGCs) can grow axons into tectum of any age, most RGCs from older animals fail to extend axons into CNS tissue derived from donors of any age, including the embryonic tectum. Here we report that the proto-oncogene bcl-2 plays a key role in this developmental change by promoting the growth and regeneration of retinal axons. This effect does not seem to be an indirect consequence of its well-known anti-apoptotic activity. Another anti-apoptotic drug, ZVAD, supported neuronal survival but did not promote axon regeneration in culture. This finding could lead to new strategies for the treatment of injuries to the CNS.

Animals↗
PubMed1997

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene.

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells.

Animals↗
PubMed1997

Anti-apoptotic effect of Bcl-2 overexpression in RIN cells infected with Semliki Forest virus.

RIN cells were infected with recombinant Semliki Forest virus (SFV) particles containing the LacZ gene. X-gal staining showed 100% infectivity of the cell cultures and high-level expression of bacterial beta-galactosidase in these cells. The cytopathogenic effects of the SFV infection were studied by measuring the viability of the RIN cells. Comparisons between control RIN cells and Bcl-2 overexpressing RIN cells were done 72 h post-infection. Significant differences in viability levels were observed. The control RIN cells showed in the MTT assay a mean value of 0.156+/-0.017 compared to 0.347+/-0.057 for the RIN/Bcl-2 cells. FACS analysis of cells labelled with propidium iodide indicated that only an average of 4.5+/-0.5% of the control cells were viable 72 h post-infection, while 44.5+/-3.5% of the RIN/Bcl-2 cells were still alive. Thus, the Bcl-2 overexpression clearly protected the SFV-infected cells from undergoing apoptosis.

Journal Article↗
PubMed1997

Bcl-2 prevents activation of CPP32 cysteine protease and cleavage of poly (ADP-ribose) polymerase and U1-70 kD proteins in staurosporine-mediated apoptosis.

Members of the the Bcl-2 and ICE/ced-3 gene families have been implicated as essential components in the control of the cell death pathway. Bcl-2 overexpression can prevent programmed cell death (PCD) in different cell types. ICE/ced-3-like proteases are synthesized as pro-enzymes and are activated by limited proteolysis. When overexpressed in diverse cell types, they trigger PCD. Bcl-2 can inhibit PCD mediated by these proteases, although as yet it is not clear at what specific step in the cell death pathway the protein acts. Here, we demonstrate that CPP32/Yama/Apopain, a member of the ICE/Ced-3 gene family, is processed during staurosporine-induced apoptosis in HeLa cells and that concomitant with CPP32 activation, two other proteins, poly (ADP-ribose) polymerase (PARP) and the U1-70 K small ribonucleoprotein, also undergo proteolysis. Overexpression of Bcl-2 prevents cleavage of CPP32, PARP and U1-70 K and protects HeLa cells from PCD. These results demonstrate that Bcl-2 controls PCD, by acting upstream of CPP32/Yama/Apopain.

Journal Article↗
PubMed1997

Susceptibility of cerebellar granule neurons derived from Bcl-2-deficient and transgenic mice to cell death.

Overproduced Bcl-2 oncoprotein has been shown to suppress cell death induced by a variety of stimuli in many cell types, including neuronal cells. Because bcl-2 is expressed in the nervous system where massive cell death is observed during development, endogenous Bcl-2 is likely to be involved in regulating neuronal cell death. Here we examined the possible role of endogenous Bcl-2 in the regulation of neuronal cell survival in the central nervous system using primary cultured cerebellar granule neurons from bcl-2-deficient, wild-type and NSE-bcl-2-transgenic mice. Cerebellar granule neurons from bcl-2-deficient mice were more susceptible than those from normal littermates to death induced by reducing the K+ concentration of the medium from high (25 mM) to low (5 mM), and neurons from bcl-2-transgenic mice were least susceptible. Similar results were obtained when cell death was induced by serum withdrawal under high K+ conditions or by the presence of etoposide, A23187 or nimodipine. Consistently, bcl-2 deficiency reduced the number of cerebellar granule neurons per mouse. These results indicate that Bcl-2 impedes neuronal cell death induced by various stimuli in a dose-dependent manner, and that endogenous levels of Bcl-2 are able to regulate neuronal cell survival in the central nervous system.

Animals↗
PubMed1997

[Caenorhabditis elegans and neuronal death in mammals].

The development of the nervous system implies not only the generation of neurons, but also their death. This neuronal death can occur through several mechanisms, one of them being apoptosis. This type of cell death seems to be also implicated in some neurodegenerative diseases. This study of the nematode Caenorhabditis elegans has led to the discovery of several genes controlling apoptosis in neurons. Two of them, the pro-apoptotic ced3 and the anti-apoptotic ced9, have mammalian homologs. The mammalian homologs to Ced9 form the Bcl-2 family and can be either pro-apoptotic or anti-apoptotic. Some of them, Bcl-x, and Bax have been shown to be involved in neuronal death during development in some pathological situations. The first mammalian homolog of Ced3 to be described was the Interleukin-1b Converting Enzymes (ICE). Since then, many other homologs of the proteases Ced3 and ICE have been discovered constituting the Caspases family. These Cysteinyl Aspartate Specific Proteases are pro-apoptotic in many different systems. Several studies using viral or peptidic inhibitors of the Caspases have demonstrated their role in neuronal death in vitro. In vivo, CPP32, a member of the Caspases family, has been shown to be clearly involved in the development of the nervous system. Finally, the analysis of apoptosis in Caenorhabditis elegans has led to the discovery of two families of genes involved in the cascade of events inducing neuronal death in mammals. Indeed, the Caspases seem to be controlled by the Bcl-2 family, as Ced3 is by Ced9.

Animals↗
PubMed1996

Increased cerebellar Purkinje cell numbers in mice overexpressing a human bcl-2 transgene.

The Purkinje cell is a primary organizer in the development of the cerebellum. Purkinje cells may provide positional information cues that regulate afferent innervation, and Purkinje cell target size controls the adult number of afferent olivary neurons and granule cells. While Purkinje cells are necessary for the survival of olivary neurons and granule cells during periods of programmed cell death, little is known about the survival requirements of Purkinje cells in vivo. To determine if Purkinje cells are subject to programmed cell death during development we have analyzed Purkinje cell numbers in two lines of transgenic mice that overexpress a human gene for bcl-2 (Hu-bcl-2). Bcl-2 is a protooncogene that inhibits apoptosis in many cell types. Overexpression of bcl-2 in vitro and in vivo rescues neurons from trophic factor deprivation or naturally occurring cell death. In the mice analyzed in this study, transgene expression is driven by the neuron-specific enolase promoter that is first expressed embryonically in most regions of the brain in one line and postnatally in the second line. We have counted Purkinje cells in three adult control mice, five early overexpressing transgenics, and three late expressing transgenics. The number of Purkinje cells in the Hu-bcl-2 transgenic mice is significantly increased above control numbers, with an increase of 43% in the embryonically overexpressing line and an increase of 27% in the postnatally overexpressing line. Because bcl-2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl-2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development.

Animals↗
PubMed1996

Expression of the CTL-associated protein TIA-1 during murine embryogenesis.

TIA-1 is a T cell-associated protein that binds poly(A) in vitro and induces apoptosis in permeabilized thymocytes. It may be involved in the induction of apoptosis in target cells during lymphocyte attack. To elucidate the role of TIA-1 in mammalian development, a cDNA-encoding mouse TIA-1 was cloned. The predicted mouse TIA-1 protein contains three RNA binding domains at the amino terminus and a putative lysosomal targeting sequence at the carboxyl terminus. The mouse sequence shows 96% overall identity with the human TIA-1 homologue. During murine embryogenesis, abundant mouse TIA-1 mRNA is detectable from 12.5 days of development onward in the brain and the retina, where it is selectively expressed within neuronal cells. Transcripts are also found in the lung, kidney, and thymus. TIA-1 in the adult mouse is expressed mainly in T cells and NK cells. The expression of TIA-1 during mouse embryogenesis is endogenous to tissues in which apoptotic cell death occurs. The conservation of this RNA-binding protein throughout evolution implicates its importance in embryogenesis, and in particular neuronal development.

Amino Acid Sequence↗
PubMed1996

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.

Acetylcysteine↗
PubMed1996

p53 protein in sympathetic neurons: cytoplasmic localization and no apparent function in apoptosis.

The p53 tumour suppressor gene plays a major role in controlling cell cycle and apoptosis in many different cell types. Here we have examined the status and the potential apoptosis inducing activity of p53 in sympathetic neurons. The p53 protein is expressed in rat sympathetic neurons cultured in the presence of NGF. The protein is not upregulated when these neurons are induced to die upon NGF deprivation. Over-expression of wild-type human p53 in neurons cultured in the presence of NGF does not trigger apoptosis nor does it accelerate apoptosis when the neurons are deprived of NGF. Finally endogenous p53 expression is not necessary for neuronal cell death triggered by NGF deprivation since neurons prepared from p53 knockout mice undergo normal cell death upon NGF deprivation. Our results suggest that p53 may have an unknown function in post-mitotic neurons which is distinct from its well described roles in apoptosis or cell cycle control.

Animals↗
PubMed1996

MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.

MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases.

Aging↗
PubMed1996

ICE-like proteases execute the neuronal death program.

The past year has witnessed significant advances in our understanding of the mechanisms that kill neurons during programmed cell death. The executioners are members of a family of proteases founded by ced-3, the product of a gene that is required for programmed cell death in the nematode Caenorhabditis elegans, and by mammalian interleukin-1 beta-converting enzyme. These proteases represent interesting novel targets for the therapy of acute and chronic pathologies of the nervous system associated with neuronal death.

Amino Acid Sequence↗
PubMed1996

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

Animals↗
PubMed1996

Long-term survival of retinal ganglion cells following optic nerve section in adult bcl-2 transgenic mice.

The bcl-2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl-2 in adult transgenic mice can protect the axotomized ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice approximately 65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, < 10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl-2 protein in central neurons is a very effective strategy to ensure long-term survival in axotomized cells.

Animals↗
PubMed1995

The motoneuron degeneration in the wobbler mouse is independent of the overexpression of a Bcl2 transgene in neurons.

The wobbler mouse mutation, an autosomal recessive mutation, leads to motoneuron degeneration in early post-natal development. Transgenic mice in which neurons overexpress human bcl2 transgene have been generated: the overexpression of bcl2 reduces the neuron loss during naturally occurring and experimentally-induced cell deaths. In the present study, we generate mice co-expressing the wobbler mutant gene and the bcl2 transgene in order to determine the effects of Bcl2 overexpression on the neurodegenerative disorders of the wobbler mouse. The clinical signs of the disease (weakness, tremor, small size) as well as biochemical and histological parameters (choline acetyltransferase (ChAT) activity in muscles, gliosis in spinal cord) are similar in bcl2 positive and negative wobbler mice. These results point to the fact that the neuron-specific expression of the human bcl2 transgene does not correct the effects of the wobbler mutation.

Animals↗
PubMed1995

Function and expression of the Bcl-x gene in the developing and adult nervous system.

We have shown that overexpression of Bcl-x can rescue sympathetic neurones from nerve growth factor deprivation in vitro. We have also examined the distribution and expression of Bcl-x mRNA in the developing and adult nervous system using Northern blot and in situ hybridization. Bcl-x mRNA is widespread during development of the nervous system. In embryonic spinal cord, mRNA levels increase at the beginning of the naturally occurring cell death period, suggesting that Bcl-x may be involved in the selection of neurones during this period. In the brain, Bcl-x expression increases after birth to reach a high level in the adult brain. Neurones from the cortex, olfactory bulb, and Purkinje cells are among those expressing the highest levels of Bcl-x mRNA. The widespread expression of Bcl-x during development and in adult brain suggests of a role for Bcl-x beyond simply protecting neurones from developmental cell death.

Animals↗
PubMed1995

Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K.

A number of DNA viruses carry apoptosis-inhibiting genes which enable the virus to escape from the host response. The adenovirus E1B 19K protein can inhibit apoptosis induced by E1A, tumour-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation. The molecular basis of this inhibition remains poorly understood, but the fact that protection is seen in the absence of other viral proteins suggests that E1B 19K targets cellular proteins. We report here the identification of three cellular proteins that bind E1B 19K. One of these is a new member of the bcl-2 family, which we have called bak (for bcl-2 homologous antagonist/killer). This protein, which is expressed in a wide variety of cell types, binds to E1B 19K and to the Bcl-2 homologue Bcl-XL (ref. 17) in yeast. In addition, overexpression of bak in sympathetic neurons deprived of nerve growth factor accelerates apoptosis and blocks the protective effect of co-injected E1B 19K.

Adenovirus E1B Proteins↗