Stéphane Gaillard

Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin‐binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein–protein association domains, among which a putative caveolin‐binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970–19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca2+‐dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234–243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co‐immunoprecipitate with caveolin‐1; (2) all are pulled down by glutathione‐S‐transferase (GST)–caveolin‐1; (3) a fragment of recombinant striatin containing the putative caveolin‐binding domain binds GST–caveolin‐1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663–673].

Identification of living oligodendrocyte developmental stages by fractal analysis of cell morphology

The Mandelbrots fractal dimension (D), a measure of shape complexity, has been used to quantify the complex morphology of living cells. Previous studies on glial cells have shown that as cells increase in morphological complexity, their “D” value increases, suggesting that “D” could be used to estimate their stage of differentiation. In the present study the box‐counting method was used to calculate the “D” values of rat cerebellar oligodendrocytes during their differentiation in primary culture. These values were correlated with the immunoreactivity of cells to antigenic markers commonly used for assessing their stages of differentiation: A2B5, O4 and anti‐galactocerebroside (Gal‐C). Our results show that changes of the fractal dimension during differentiation follow the well known pattern of markers expression by these cells. These results demonstrate that A2B5‐, O4‐, and Gal‐C‐expressing oligodendrocytes can be confidently estimated from their respective fractal dimension values. Based on this immunocytochemical calibration, the calculation of “D” allows an easy and fast determination of the developmental stage of living (unstained) oligodendrocytes before the study of their physiological characteristics. Using this method we precisely identified living oligodendrocyte progenitors and early pro‐oligodendrocytes expressing voltage‐activated sodium currents that is a common characteristic of these two immature developmental stages (Sontheimer et al. [ 1989b ] Neuron 2:1135–1145). J. Neurosci. Res. 65:439–445, 2001.

Morphological and functional characterization of cholinergic interneurons in the dorsal horn of the mouse spinal cord

Endogenous acetylcholine is an important modulator of sensory processing, especially at the spinal level, where nociceptive (pain‐related) stimuli enter the central nervous system and are integrated before being relayed to the brain. To decipher the organization of the local cholinergic circuitry in the spinal dorsal horn, we used transgenic mice expressing enchanced green fluorescent protein specifically in cholinergic neurons (ChAT::EGFP) and characterized the morphology, neurochemistry, and firing properties of the sparse population of cholinergic interneurons in this area. Three‐dimensional reconstruction of lamina III ChAT::EGFP neurons based either on their intrinsic fluorescence or on intracellular labeling in live tissue demonstrated that these neurons have long and thin processes that grow preferentially in the dorsal direction. Their dendrites and axon are highly elongated in the rostrocaudal direction, beyond the limits of a single spinal segment. These unique morphological features suggest that dorsal horn cholinergic interneurons are the main contributors to the plexus of cholinergic processes located in lamina IIi, just dorsal to their cell bodies. In addition, immunostainings demonstrated that dorsal horn cholinergic interneurons in the mouse are γ‐aminobutyric acidergic and express nitric oxide synthase, as in rats. Finally, electrophysiological recordings from these neurons in spinal cord slices demonstrate that two‐thirds of them have a repetitive spiking pattern with frequent rebound spikes following hyperpolarization. Altogether our results indicate that, although they are rare, the morphological and functional features of cholinergic neurons enable them to collect segmental information in superficial layers of the dorsal horn and to modulate it over several segments. J. Comp. Neurol. 519:3139–3158, 2011.

Hepatocyte Growth Factor-Met Signaling Is Required for Runx1 Extinction and Peptidergic Differentiation in Primary Nociceptive Neurons

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.

Voltage-dependent Na+-HCO3− cotransporter and Na+/H+ exchanger are involved in intracellular pH regulation of cultured mature rat cerebellar oligodendrocytes

Intracellular pH (pHi) was measured at 37°C in mature rat cerebellar oligodendrocytes dissociated in culture by using the pH‐sensitive probe BCECF. Cells were identified by anti‐galactocerebroside antibody. The mean steady‐state pHi was 7.02 in the absence of CO2/bicarbonate (Hepes‐buffered solution) at an external pH of 7.40 and 7.04 in 5% CO2/25 mM bicarbonate‐buffered solution at the same external pH; this value was modified neither by the removal of external chloride nor by the addition of the chloride‐coupled transport blocker DIDS. In both external solutions steady‐state pHi values were strongly dependent on external pH. In Hepes‐buffered solution pHi recovery following an acid load required external Na+ and was completely inhibited by amiloride, indicating the presence of a Na+/H+ exchanger. In CO2/bicarbonate‐buffered solution amiloride partially reduced the pHi recovery rate, indicating the presence of a bicarbonate‐dependent pHi regulating mechanism. Membrane depolarization induced by increasing external K+ concentration elicited an alkalinization only in the presence of external Na+ and bicarbonate. Analysis of the calculated HCO3 fluxes with respect to membrane potential indicated that these fluxes were mediated by a Na+‐HCO3 cotransport with a stoichiometry of 1:3. These results demonstrate that a Na+/H+ exchanger and a Na+ HCO3 cotransporter are involved in pHi regulation of mature oligodendrocytes. GLIA 19:74–84, 1997.

Intracellular pH changes during oligodendrocyte differentiation in primary culture

We have studied the characteristics of pHi regulation at different stages of rat oligodendrocyte differentiation in primary culture. pHi was measured at 37°C using the pH‐sensitive fluorescent probe BCECF. In immature oligodendrocyte progenitor (OLP), three distinct ionic mechanisms were involved in pHi regulation: (i) a sodium‐independent Cl−/HCO  −3 exchanger, (ii) a Na+/H+ exchanger and (iii) a voltage‐dependent Na+‐HCO  −3 cotransporter. The two latter mechanisms were also detected in more differentiated pro‐oligodendrocytes and in mature oligodendrocytes whereas the Cl−/HCO  −3 exchanger was not active in these two later stages of differentiation. The presence of this Cl−/HCO  −3 exchanger (that acts as a chronic acidifying mechanism) only in immature OLP maintains in these cells a steady‐state pHi value significantly lower than values measured in more differentiated cells. The possible involvement of this pHi change in triggering cell differentiation is discussed. J. Neurosci. Res. 59:731–739, 2000

Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination

Epsilon toxin (ET) is produced by Clostridium perfringens types B and D and causes severe neurological disorders in animals. ET has been observed binding to white matter, suggesting that it may target oligodendrocytes. In primary cultures containing oligodendrocytes and astrocytes, we found that ET (10−9 M and 10−7 M) binds to oligodendrocytes, but not to astrocytes. ET induces an increase in extracellular glutamate, and produces oscillations of intracellular Ca2+ concentration in oligodendrocytes. These effects occurred without any change in the transmembrane resistance of oligodendrocytes, underlining that ET acts through a pore‐independent mechanism. Pharmacological investigations revealed that the Ca2+ oscillations are caused by the ET‐induced rise in extracellular glutamate concentration. Indeed, the blockade of metabotropic glutamate receptors type 1 (mGluR1) prevented ET‐induced Ca2+ signals. Activation of the N‐methyl‐D‐aspartate receptor (NMDA‐R) is also involved, but to a lesser extent. Oligodendrocytes are responsible for myelinating neuronal axons. Using organotypic cultures of cerebellar slices, we found that ET induced the demyelination of Purkinje cell axons within 24 h. As this effect was suppressed by antagonizing mGluR1 and NMDA‐R, demyelination is therefore caused by the initial ET‐induced rise in extracellular glutamate concentration. This study reveals the novel possibility that ET can act on oligodendrocytes, thereby causing demyelination. Moreover, it suggests that for certain cell types such as oligodendrocytes, ET can act without forming pores, namely through the activation of an undefined receptor‐mediated pathway.

stac1 and stac2 genes define discrete and distinct subsets of dorsal root ganglia neurons

Deciphering the precise in vivo function of a particular neuronal subpopulation is one of the most challenging issues in neurobiology. Dorsal root ganglia (DRG) neurons represent a powerful model system to address this fundamental question. These neurons display many morphological, anatomical and few molecular characteristics. With the aim of expanding the molecular description of the primary sensory neurons, we used Affimetrix microarrays to compare global gene expression profiles of DRG of wild type and trkA(trkC/trkC) knock-in mice at birth and identified several hundred potential markers of nociceptive neurons and few markers of proprioceptive neurons. Here, we describe the identification of two members of a family of putative adapter proteins STAC1 and STAC2. We found STAC1 and STAC2 being expressed in a mutually exclusive fashion in adult DRG neurons. STAC1 mainly marks peptidergic nociceptive neurons while STAC2 is expressed in a subset of nonpeptidergic nociceptors, in all trkB+ neurons and in a subpopulation of proprioceptive neurons. Our expression data demonstrate that STAC proteins identify four categories of primary sensory neurons; one class of peptidergic neurons, a subset of nonpeptidergic neurons, all TrkB+neurons and a subset of proprioceptive neurons. Genetic marking of STACs-expressing sensory neurons will lend significant advance into our understanding of DRG neuronal functional diversity.

Intracellular acidification mediates the proliferative response of PC12 cells induced by potassium ferricyanide and involves MAP kinase activation

Potassium ferricyanide is known to elicit cell growth and mitogenesis in various cells by stimulating a transplasma membrane electron‐transport system. When serum‐starved PC12 cells were treated with potassium ferricyanide, stimulation of mitogenesis was evidenced by enhanced DNA synthesis, as well as by increased cell numbers. Intracellular pH (pHi) of PC12 cells was measured at 37°C by microfluorimetric analysis of 2′,7′‐bis‐(2‐carboxyethyl)‐5(and‐6)‐carboxyfluorescein (BCECF). The resting pHi of unstimulated cells was 7.52 (external pH 7.40). Addition of potassium ferricyanide (100 μM) decreased pHi by about 0.25 pH units. Lowering pHi to a similar extent, either by decreasing external pH (pHo) or by adding a weak acid, also elicited a mitogenic response, indicating that intracellular acidification by itself has growth factor‐mimicking, mitogenic effects. Nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) triggered proliferation without changes in pHi. The mitogenic treatments eliciting intracellular acidification did not activate protein kinase C (PKC) but stimulated the p42/p44 mitogen‐activated protein (MAP) kinase. Our results indicate that 2 distinct mitogenic pathways are active in PC12 cells: the first is independent of pHi and involves activation of the PKC pathway and the second requires a permissive pHi value around 7.25 and involves activation of the p42/p44 MAP kinase pathway.

Expression and distribution of phocein and members of the striatin family in neurones of rat peripheral ganglia

Phocein and members of the striatin family (striatin, SG2NA and zinedin) are intracellular proteins, mainly expressed in neurones of the mammalian central nervous system where they are thought to be involved in vesicular traffic and Ca2+ signalling. Here, we have investigated whether these proteins are also present in the peripheral nervous system, by analysing their expression and distribution within sensory neurones of the vagal (nodose and jugular) ganglia, the petrosal ganglion, the dorsal root ganglion, and also in the sympathetic neurones of the superior cervical ganglion. RT-PCR experiments showed that mRNAs of phocein, striatin, SG2NA and zinedin are present in all studied peripheral ganglia. Immunocytochemical detections demonstrate that phocein, striatin and SG2NA are expressed in neurones of vagal, petrosal and dorsal root ganglia. Immunoblotting experiments confirm these data and in addition demonstrate that: (1) the proteins phocein, striatin and SG2NA are also present in the superior cervical ganglion and (2) zinedin is detected in all studied ganglia. The distribution appears to differ: immunoreactivity for striatin and SG2NA is found only in soma of sensory neurons, whereas immunoreactivity for phocein is observed in both soma and processes. Our study thus demonstrates that phocein and the members of the striatin family are expressed not only in central nervous system but also in the peripheral nervous system and, in particular, in afferent sensory neurones.