Marguerite Lucas

Expression of two neuronal markers, growth-associated protein 43 and neuron-specific enolase, in rat glial cells

Abstract Recent studies have revealed that proteins such as growth-associated protein 43 (GAP-43) and neuron-specific enolase (NSE), believed for many years to be expressed exclusively in neurons, are also present in glial cells under some circumstances. Here we present an overview of these observations. GAP-43 is expressed both in vitro and in vivo transiently in immature rat oligodendroglial cells of the central nervous system, in Schwann cell precursors, and in non-myelin-forming Schwann cells of the peripheral nervous system. GAP-43 mRNA is also present in oligodendroglial cells and Schwann cells, indicating that GAP-43 is synthesized in these cells. GAP-43 is also expressed in type 2 astrocytes (stellate-shaped astrocytes) and in some reactive astrocytes but not in type 1 astrocytes (flat protoplasmic astrocytes). These results suggest that GAP-43 plays a more general role in neural plasticity during development of the central and peripheral nervous systems. NSE enzymatic activity and protein and mRNA have been detected in rat cultured oligodendrocytes at levels comparable to those of cultured neurons. NSE expression increases during the differentiation of oligodendrocyte precursors into oligodendrocytes. In vivo, NSE protein is expressed in differentiating oligodendrocytes and is repressed in fully mature adult cells. The upregulation of NSE in differentiating oligodendrocytes coincides with the formation of large amounts of membrane structures and of protoplasmic processes. Similarly, NSE becomes detectable in glial neoplasms and reactive glial cells at the time when these cells undergo morphological changes. The expression of the glycolytic isozyme NSE in these cells, which do not normally contain it, could reflect a response to higher energy demands. This expression may also be related to the neurotrophic and neuroprotective properties demonstrated for this enolase isoform. NSE activity and protein and mRNA have also been found in cultured rat type 1-like astrocytes but at much lower levels than in neurons and oligodendrocytes. Thus GAP-43 and NSE should be used with caution as neuron-specific markers in studies of normal and pathological neural development.

Activation of the gene encoding the glycolytic enzyme β-enolase during early myogenesis precedes an increased expression during fetal muscle development

We define the spatial and temporal patterns of expression of the gene encoding the glycolytic enzyme, beta-enolase, during mouse ontogenesis. Transcripts were detected by in situ hybridization using 35S labelled cRNA probes. The beta-enolase gene is expressed only in striated muscles. It is first detected in the embryo, in the cardiac tube and in newly formed myotomes. In the muscle masses of the limb, beta gene expression occurs at a low level in primary fibers, and subsequently greatly increases at a time which corresponds to the onset of innervation and secondary fiber formation. Later in development, it becomes undetectable in slow-twitch fibers. Our results demonstrate the multistep regulation of the beta-enolase gene. The regulation of this muscle-specific gene in somites is discussed in terms of the myogenic sequences of the MyoD family shown to be present when it is activated.

Fibre‐type distribution and subcellular localisation of α and β enolase in mouse striated muscle

Enolase is a dimeric glycolytic enzyme exhibiting tissue specific isoforms. During ontogenesis, a transition occurs from the embryonic αα towards the specific αβ, and ββ isoforms in striated muscle. Immunocytochemical analyses on transverse sections of adult mouse gastrocnemius muscle, allowed us to compare the expression of α and β subunits to that of myosin heavy chain (MHC) isoforms. Levels of β immunoreactivity followed the order IIB > IIX > IIA > I. This gradient parallels the ATPase activity associated to MHC isoforms, indicating that the expression of β enolase in myofibres is finely regulated as a function of energetic requirements. By contrast, variations in α immunolabelling intensity appeared independent of fibre types.

Expression of the neuron-specific enolase gene by rat oligodendroglial cells during their differentiation

Abstract: We have examined the regulation of neuron‐specific γ‐enolase gene (NSE) expression in oligodendrocytes at various steps of their differentiation/maturation. We have demonstrated for the first time that NSE is expressed in oligodendroglial cells in vitro and in vivo, and only at a certain stage of differentiation. A heterogeneity of the γ subunit was observed in cultured oligodendrocytes and the same one was found in adult rat brain. The level of γ mRNA increased when precursor cells differentiated into oligodendrocytes. By contrast, no significant change in α‐enolase gene expression was observed. High NSE (γγ and αγ) enolase activity was detected in cultured oligodendrocytes. Treatment with basic fibroblast growth factor, which stimulates the proliferation of oligodendrocyte precursor cells and reversibly blocks their differentiation, resulted in lower αγ‐ and γγ‐enolase activities in these cells, but it enhanced αα‐enolase activity slightly. These data indicate that γ‐enolase gene expression is associated with the differentiation of the oligodendrocytes and that it is repressed in adult fully mature cells.

Photo affinity labelling of β-adrenergic receptors of C6 glioma cells: Presence of a nucleophilic group in the receptor☆

Abstract (±) 1-[1-( p -Nitrophenoxy), 2-methyl, 2-propylamino], 3-(α-naphtyloxy), 2-propanol (PNP) was synthesized and found to be a potent photoaffinity label for β-adrenergic receptors of C 6 glioma cells. In the dark, PNP displaced all the [ 3 H]DHA binding sites on C 6 glioma cell membranes ( K D = 5.5 × 10 −8 M). Upon photolysis on isolated C 6 glioma cell membranes: (a) PNP reduced in a dose-dependent manner maximally stimulated β-adrenergic sensitive adenylate cyclase. After extensive washing of the membranes, the maximal β-adrenergic stimulation was reduced without change in the apparent affinity for isoproterenol. A 62% decrease in activity was obtained with 10 −5 M PNP without any change in basal and NaF stimulated adenylate cyclase activities, (b) PNP also irreversibly reduced in a dose-dependent manner the total number of [ 3 H]DHA binding sites without changing the affinity of the remaining sites. The effects of PNP on adenylate cyclase and [ 3 H]DHA binding were suppressed in the presence of (−)alprenolol. Upon photolysis on intact C 6 glioma cells, PNP inactivated β-adrenergic receptors coupled with adenylate cyclase without any change in basal, NaF and Gpp(NH)p stimulated adenylate cyclase activities. These results indicate that PNP photolabelling occurred on the β-adrenergic receptors. Furthermore, as PNP was shown to react with model nucleophiles upon photolysis, this labelling implies the presence of a nucleophilic group in the β-adrenergic receptor.

Irreversible blockade of β-adrenergic receptors with a bromoacetyl derivative of pindolol

SummaryA potent irreversible β-adrenergic derivative of pindolol possessing a chemically reactive group (Br-AAM-pindolol) was synthesized. This compound devoid of agonist properties, competed for all (3H)-dihydroalprenolol (3H-DHA) binding sites in C6 glioma cell and rat cerebellum membranes. Pretreatment of C6 glioma cell membranes with Br-AAM-pindolol and subsequent washing resulted in a time- and dose-dependent blockade of β-adrenergic receptors. A 50% blockade was achieved in the presence of 1.6 nM Br-AAM-pindolol.This blockade occurs specifically at the β-adrenergic receptor level, as: 1) it induced a decrease of maximal isoproterenol stimulated adenylate cyclase activity with no modification of basal and sodium fluoride stimulated activity and 2) decreases of (3H)-DHA binding and stimulation of adenylate cyclase activity by the agonist were suppressed in the presence of isoproterenol, a β-adrenergic agonist. Furthermore, Br-AAM-pindolol treatment did not affect (3H)-diazepam binding in C6 glioma cell membranes.Pretreatment of C6 glioma cells with Br-AAM-pindolol also reduced the response of adenylate cyclase to isoproterenol and the number of β-adrenergic receptors. The blockade of β-adrenergic receptors of C6 glioma cells by Br-AAM-pindolol was non-competitive, whereas the blockade obtained with AM-pindolol, a derivative of pindolol devoid of alkylating properties, was competitive.The irreversible blockade of β-adrenergic receptors by Br-AAM-pindolol in rat erythrocyte membranes was substantiated by the demonstration that no recovery of β-adrenergic receptors occurred during long term incubation of the membranes (48 h) following Br-AAM-pindolol treatment and subsequent washing.Double reciprocal plotting of equiactive isoproterenol concentrations in dose-response curves of adenylate cyclase from membranes of control and Br-AAM-pindolol treated C6 glioma cells permitted calculation of the dissociation constant for isoproterenol from its binding sites (1.5±0.2×10−7 M, n=11). This is very close to the dissociation constant of the agonist derived from binding experiments (1.7±0.5×10−7 M, n=13).These results suggest that Br-AAM-pindolol is a potent irreversible β-adrenergic antagonist and may be useful for pharmacological and physiological studies of σ-adrenergic receptors.

A comparative study of the distribution of α- and γ-enolase subunits in cultured rat neural cells and fibroblasts

we report the presence and distribution of α (ubiquitous) and γ (neuron‐specific) subunits of the dimeric glycolytic enzyme enolase (2‐phospho‐D‐glycerate hydrolase) in cultured neural cells. The γγ enolase is found in vivo at high levels only in neurons and neuroendocrine cells. Neuronal cells in culture also contain relatively high levels of αγ and γγ enolase. Here we show, by enzymatic and immunological techniques, that the γ subunit also is expressed in cultured rat astrocytes and meningeal fibroblasts and, as we previously reported, in oligodendrocytes. Both neuron‐specific isoforms αγ and γγ are expressed in all these cells, but the αα isoform accounts for the major part of total enolase activity. The sum of αγ and γγ enolase activities increases with time in culture, i.e. maturation processes, reaching the highest level in oligodendrocytes (40% of total enolase activity) and 15 and 10% of total enzymatic activity in astrocytes and fibroblasts, respectively. The γ enolase transcripts were found not only in cultured neuronal cells but also in cultured oligodendrocytes, astrocytes, and meningeal fibroblasts. Our data indicate that neuron‐specific enolase should be used with caution as a specific marker for neuronal cell differentiation.

Direct interaction of LSD with central "beta"-adrenergic receptors.

Abstract (+)-lysergic acid diethylamide (LSD) has been found to interact with central β-adrenergic receptors, both in the cerebral cortex of the rat and in cultured C 6 glioma cells. LSD inhibited the binding of 3 H-dihydroalprenolol ( 3 H-DHA) with an apparent inhibition constant of 10- 7 M in the cerebral cortex and 10- 6 M in C 6 glioma cells. The displacement of 3 H-DHA binding by LSD was found to be competitive in the cortex, with a dissociation constant of 1.9 × 10- 7 M, compared to 1.4 × 10- 8 M for (−) alprenolol and 5.6 × 10- 8 M for (−) isoproterenol. BOL, an analogue of LSD without hallucinogenic properties, showed the same affinity as LSD for the cortical β-adrenergic receptor. However, several dopamine and serotonin agonists and antagonists were without effect at 10- 6 M. The stimulation of adenylate cyclase by isoproterenol was inhibited by LSD with an apparent inhibition constant of 1.6 × 10- 7 M in homogenates of cerebral cortex and 5 × 10- 6 M in the C 6 glioma cell system. The results suggest that central β-adrenergic receptors represent one of the several sites of action of LSD.

Developmental Expression of Alpha- and Gamma-Enolase Subunits and mRNA Sequences in the Mouse Brain

Nonneuronal alpha alpha- and neuron-specific alpha gamma- and gamma gamma-enolase activities were measured in the mouse brain during development. The corresponding mRNA sequences were quantified directly by hybridization with cDNA probes. The variations in alpha- and gamma-monomer levels inferred from the enzymatic activities were very similar to those of their respective mRNAs. We conclude that monomer levels are primarily controlled by the amounts of their mRNAs during mouse brain development.

Influence de l'acide glyoxylique sur les propriétés des mitochondries isolées

Glyoxalate is an effector of oxidative phosphorylation in isolated mitochondria : it slows down State 3 but does not affect State 4 respiration. This report presents the findings of our study on the mechanism of action of glyoxalate ; these findings are listed below. The inhibition of Stage 3 respiration by glyoxalate does not set in immediately, can be reversed in part by the addition of an uncoupling agent or a dithiol, is non-competitive against succinate and can be demonstrated with substrates requiring the involvement of other membrane transport systems. Glyoxalate prevents the increased oxygen uptake stimulated by 2,4-DNP or Sr++. Glyoxalate also inhibits phosphate transport and this inhibition can account for most of the effect observed. The inhibition of State 3 respiration is paralleled by a decrease in the mitochondrial accumulation of succinate : this decrease could arise from a direct effect of glyoxalate on dicarboxylic acid transport or could be the result of an inhibiton of the phosphate transport system, which is connected with the former. The decrease in the respiratory rate of uncoupled mitochondria placed in a phosphate free medium demonstrates that the effector acts directly at the substrate transport or/and electron transfer level. Phosphate, by delaying the respiratory inhibiton due to glyoxalate, has a protecting effect on mitochondrial functions. Glyoxalate is thus acting at several mitochondrial sites. It acts presumably by forming hemimercaptals, blocking sulfhydryl groups. Its effects can be accounted for by the unfolding of such (hemicercaptal) groups under the influence of ADP, Pi, uncoupling or others agents which bring about conformational changes in the internal mitochondrial membrane.