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J C Martinou

Publications and source records attributed to J C Martinou.

At least 73 records · Page 4Linked to original sources
PubMed1995

Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.

Adenovirus E1A Proteins↗
PubMed1995

Bcl-2 overexpression prevents motoneuron cell body loss but not axonal degeneration in a mouse model of a neurodegenerative disease.

Bcl-2 and its analogs protect different classes of neurons from apoptosis in several experimental situations. These proteins may therefore provide a means for treatment of neurodegenerative diseases. We examined the effects of Bcl-2 overexpression in a genetic mouse model with motor neuron disease (progressive motor neuronopathy/pmn). Pmn/pmn mice lose motoneurons and myelinated axons, and die at 6 weeks of age. When these mice were crossed with transgenic mice that overexpress human Bcl-2, there was a rescue of the facial motoneurons with a concomitant restoration of their normal soma size and expression of choline acetyltransferase. However, Bcl-2 overexpression did not prevent degeneration of myelinated axons in the facial and phrenic motor nerves and it did not increase the life span of the animals. Since Bcl-2 acts strictly on neuronal cell body survival without compensating for nerve degeneration in pmn/pmn/bcl-2 mice, this proto-oncogene would not in itself be sufficient for treatment of neurodegenerative diseases where axonal impairment is a major component.

Animals↗
PubMed1994

Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death.

In vitro, the overexpression of the bcl-2 protooncogene in cultured neurons has been shown to prevent apoptosis induced by neurotrophic factor deprivation. We have generated transgenic mice overexpressing the Bcl-2 protein in neurons, including motoneurons of the facial nucleus. We have tested whether Bcl-2 could protect these motoneurons from experimentally induced cell death in new born mice. To address this question, we performed unilateral lesion of the facial nerve of wild-type and transgenic 2-day-old mice. In wild-type mice, the lesioned nerve and the corresponding motoneuron cell bodies in the facial nucleus underwent rapid degeneration. In contrast, in transgenic mice, facial motoneurons survived axotomy. Not only their cell bodies but also their axons were protected up to the lesion site. These results demonstrate that in vivo Bcl-2 protects neonatal motoneurons from degeneration after axonal injury. A better understanding of the mechanisms by which Bcl-2 prevents neuronal cell death in vivo could lead to the development of strategies for the treatment of motoneuron degenerative diseases.

Animals↗
PubMed1994

Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia.

Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.

Animals↗
PubMed1994

Multiple promoters of human choline acetyltransferase and aromatic L-amino acid decarboxylase genes.

The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative noncoding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5' flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5' flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.

Animals↗
PubMed1994

The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Amino Acid Sequence↗
PubMed1992

Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene.

Approximately half of the neurons produced during embryogenesis normally die before adulthood. Although target-derived neurotrophic factors are known to be major determinants of programmed cell death--apoptosis--the molecular mechanisms by which trophic factors interfere with cell death regulation are largely unknown. Overexpression of the bcl-2 proto-oncogene in cultured sympathetic neurons has now been shown to prevent apoptosis normally induced by deprivation of nerve growth factor. This finding, together with the previous demonstration of bcl-2 expression in the nervous system, suggests that the Bcl-2 protein may be a major mediator of the effects of neurotrophic factors on neuronal survival.

Animals↗
PubMed1992

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.

Animals↗
PubMed1991

Acetylcholine receptor-inducing activity stimulates expression of the epsilon-subunit gene of the muscle acetylcholine receptor.

Motor neurons regulate the transcription of acetylcholine receptor subunit genes in postsynaptic muscle fibers both through muscle electrical activity produced by motor neuron acetylcholine release and by mechanisms independent of such transmitter release. Factors secreted by the motor neuron may mediate activity-independent regulation, including the postnatal switch from alpha 2 beta gamma delta (embryonic type) to alpha 2 beta epsilon delta (adult type) receptors. We have investigated the effect of putative trophic factors, agents affecting second-messenger systems, and muscle activity on the levels of acetylcholine receptor subunit mRNAs in primary mouse muscle cultures. We found that ARIA (acetylcholine receptor-inducing activity), a 42-kDa glycoprotein purified on the basis of its ability to increase the synthesis of acetylcholine receptors in chick myotubes, increases epsilon-subunit mRNA levels up to 10-fold. In addition, ARIA stimulated alpha-, gamma-, and delta-subunit mRNA levels 2-fold but had no effect on the expression of the beta-subunit gene. These effects of ARIA were independent of muscle activity, and they were not mimicked by calcitonin gene-related peptide nor by thyroxine, forskolin, phorbol 12-myristate 13-acetate, the calcium ionophore A23187, basic fibroblast growth factor, or transforming growth factor beta. Based on these data, we suggest that ARIA may act at the mammalian neuromuscular junction to induce adult-type acetylcholine receptors.

Acetylcholine↗
PubMed1991

Selective expression of an acetylcholine receptor-lacZ transgene in synaptic nuclei of adult muscle fibers.

Acetylcholine receptors (AChRs) are highly concentrated in the postsynaptic membrane at the neuromuscular junction. To investigate mechanisms that lead to the formation or maintenance of this synaptic specialization, we generated transgenic mice in which regulatory elements from the AChR alpha or epsilon-subunit genes are linked to a gene for a reporter protein that is targeted to the nucleus (nlacZ). Both transgenes were selectively expressed and developmentally regulated in muscle; nuclei in both extrafusal (ordinary) and intrafusal (spindle) muscle fibers were labeled. Within individual muscle fibers from epsilon-nlacZ mice, nuclei near synaptic sites were nlacZ-positive, whereas extrasynaptic nuclei were nlacZ-negative. In contrast, nlacZ was expressed in both synaptic and extrasynaptic nuclei when under the control of regulatory elements from the AChR alpha-subunit gene; however, synaptic nuclei were somewhat more intensely stained than extrasynaptic nuclei in a minority of muscle fibers from these mice. Together, our results provide direct evidence for molecular differences between synaptic and extrasynaptic nuclei within a single cytoplasm, and suggest that the motor nerve regulates synapse formation by selectively affecting transcription in synaptic nuclei.

Animals↗
PubMed1991

Nerve-dependent modulation of acetylcholine receptor epsilon-subunit gene expression.

The relative abundance of mRNAs encoding the gamma- and epsilon-subunits of acetylcholine receptor changes in opposite directions during mouse development. Both gamma- and epsilon-mRNAs are expressed early in muscle development in vivo, and in aneural embryonic muscle and myogenic cell lines in vitro, though gamma-mRNA is at least 20-fold more abundant than epsilon-mRNA in these circumstances. While during normal development, gamma-mRNA decreases to an undetectable level by postnatal day 12, epsilon-mRNA first increases 10-fold between day 2 and day 12-15, then decreases to the level characteristic of adult muscle. We have found that the transition form gamma- to epsilon-mRNA is influenced by the levels of thyroid hormones. Indeed, high and low levels of thyroid hormones, respectively, accelerated and delayed the switch between gamma- and epsilon-mRNAs. Neither the dramatic postnatal rise in epsilon-mRNA nor its sensitivity to thyroid hormones was observed in denervated newborn animals. By contrast, denervation was without effect on epsilon-mRNA expression in adult muscle. These results suggest that, although not required for the initial activation of the epsilon-gene nor its maintenance in adult muscle, the nerve plays a major role in the perinatal regulation of epsilon-gene transcription.

Aging↗
PubMed1990

Embryonic motoneurons grafted into the adult CNS can differentiate and migrate.

Embryonic mouse motoneurons were labelled by retrograde transport with a fluorescent dye (diI), partially purified on a density gradient and grafted into adult mouse CNS for a maximum of 10 weeks. In both the spinal cord and striatum, a small number of fluorescently labelled motoneurons were found to survive as judged by their round, bright appearance and also to differentiate as assessed by their ability to extend neurites. The motoneurons travelled long distances in the spinal cord (2 mm) and in the brain (4 mm) in both white and grey matter.

Animals↗
PubMed1990

Transforming growth factor beta 1 is a potent survival factor for rat embryo motoneurons in culture.

We have studied the effects of transforming growth factor beta 1 (TGF beta 1) on the survival of embryonic motoneurons in culture. For this purpose, E14 rat embryo motoneurons were purified to more than 90% homogeneity by cell sorting and cultured at low density on monolayers of cortex astrocytes. Subnanomolar concentrations of TGF beta 1 (40-500 pM) increased the survival of motoneurons 2-fold after 9-11 days in culture. The increase in choline acetyltransferase (ChAT) activity per culture caused by TGF beta 1 was attributable to its effects on survival. Comparable results were found with motoneurons cultured on lysed astrocytes, suggesting that the effects of the factor are not mediated by non-neuronal cells, but that motoneurons are a target for TGF beta 1.

Adrenergic Fibers↗
PubMed1989

Influence of the culture substratum on the expression of choline acetyltransferase activity in purified motoneurons from rat embryos.

Motoneurons from E14 rat embryos have been retrogradely labelled with a carbocyanine derivative and purified approximately 20-fold by density gradient centrifugation. For certain experiments, motoneurons have been purified to near homogeneity by cell sorting. These neurons were then cultured at low density on various substrata. Laminin stimulated neurite outgrowth of the identified motoneurons and stimulated choline acetyltransferase (ChAT) development by 2-fold; these cultures could not be maintained for more than 4 days. Motoneurons could be cultured for at least 7 days on monolayers of both living and lysed astrocytes from newborn cortex. The cells survived equally well on both substrata but neurite outgrowth was less elaborated on lysed astrocytes on which motoneurons displayed a mono- or bipolar, rather than a multipolar, morphology. Furthermore, ChAT developed at a 2-fold higher level on lysed, rather than living, newborn astrocytes. Such a difference was not observed using astrocytes from E14 rat embryos. In the latter case, both living and lysed astrocytes supported ChAT development at the same rate as lysed newborn astrocytes. Consequently, living astrocytes from newborn rat cortex are relatively less permissive for ChAT expression. On the other hand, both living and lysed astrocytes from newborn cortex induced ChAT expression by sympathetic neurons to the same extent. We propose that, in addition to retrograde factors from muscle origin, cell surface molecules expressed by embryonic astrocytes stimulate ChAT expression by motoneurons. Such molecules would be present in newborn astrocytes in an intracellular form and could be unmasked by cell lysis. Different cell surface molecules may induce ChAT activity in sympathetic neurons.

Animals↗
PubMed1989

Characterization of two factors enhancing choline acetyltransferase activity in cultures of purified rat motoneurons.

Motoneurons from E14 rat embryos have been retrogradely labeled with the carbocyanine derivative dil and purified 12-fold by centrifugation on a density gradient made of Nycodenz, as assessed by the increase in CAT activity per cell and in the percentage of dil-labeled cells. A 20- to 36-fold purification was achieved by the microdissection of the labeled lumbar motor columns followed by density-gradient centrifugation. Motoneurons were then purified to near homogeneity by fluorescence-activated cell sorting. In certain experiments, motoneurons were purified by a novel cell-sorting procedure that only uses light-scatter parameters and does not necessitate retrograde labeling. In cultures of motoneurons enriched by centrifugation, muscle-conditioned media (CM) had only a marginal effect on the overall neuronal survival but increased up to 10-fold the number of labeled motoneurons surviving after 7 d in culture. CAT activity per culture was quantitatively stimulated to the same extent, suggesting that the increase in CAT activity caused by CM mostly reflected better survival of motoneurons. In cultures of motoneurons purified by cell sorting, CM increased both survival and CAT expression per motoneuron. We have partially purified 2 factors from CM that stimulate CAT activity per motoneuron 2- to 3-fold without affecting their survival. One of these factors is probably identical to the factor involved in the cholinergic/noradrenergic choice of sympathetic neurons (Fukada, 1985).

Animals↗
PubMed1985

[Evaluation of the performance of an automatic coagulation tester: the Electra 700].

The Electra 700, an automatic coagulation tester recently available in France, was investigated for its performance on routine tests: Quick's test, APTT, thrombin clotting time test, assay of fibrinogen and prothrombin complex factors. Reproducibility was exceptionally good for Quick's test (CV less than 1%), excellent for the other tests (CV less than 3%), acceptability of the various commercial reagents very good, and standard linearity excellent. There was close correlation with manually obtained (electromagnetic B.M.) or semiautomated (KC 10) results (0.9948 to 0.9345). The apparatus is entirely automatic once specimens have been loaded, and it is particularly adapted to dealing with long series of specimens (60 samples). Its original loading system makes it possible to carry out two types of test concurrently, as well as to insert emergency tests. Economically, the cost of disposables and "reagent loss" is satisfactory; however, all tests are carried out relatively slowly, though this is compensated for by the fact that the reactions are completely automatic.

Blood Coagulation Tests↗