Ankyrin G Membrane Partners Drive the Establishment and Maintenance of the Axon Initial Segment

International audienceThe axon initial segment (AIS) is a highly specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple membrane proteins such as ion channels and cell adhesion molecules (CAMs) that are recruited to the AIS by the scaffold protein ankyrin G (ankG). The crucial function of ankG in the anchoring of AIS membrane components is well established, but a reciprocal role of membrane partners in ankG targeting and stabilization remained elusive. In rat cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners (voltage-gated sodium channels (Nav) or neurofascin-186) led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These effects were rescued by expressing a recombinant AIS-targeted Nav or by a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor (mABD). Moreover, overexpressing mABD in mature neurons led to ankG mislocalization. Altogether, these results demonstrate that a tight and precocious association of ankG with its membrane partners is a key step for the establishment and maintenance of the AIS

Conserved Genes Act as Modifiers of Invertebrate SMN Loss of Function Defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Les Protéines de la famille striatine (un sujet épineux)

Mon travail de thèse a été réalisé dans l'équipe qui a identifié la famille striatine ,une nouvelle famille de protéines d'échafaudage principalement exprimée dans les neurones où elle est concentrée dans les épines dendritiques. Chez les mammifères, cette famille de protéines comprend trois membres, la striatine, la zinédine et SG2NA, qui partagent quatre domaines d'interaction protéine-protéine identiques (un domaine de fixation à la cavéoline, une structure coiled-coil, un domaine de fixation au complexe Ca2+ /calmoduline et un domaine de répétitions WD), agencés dans le même ordre, très conservés au cours de l'évolution, et considérés comme la signature des membres de la famille striatine. Ces trois protéines interagissent avec de nombreuses protéines de signalisation comme la calmoduline, PP2A, ER a... (voir Benoist et al., 2006). Alors que la répartition de SG2NA et de la striatine dans le système nerveux central avait été déterminée (Salin et al., 1998; Castets et al., 2000; Gaillard et al., 2006), celle de la zinédine restait inconnue. Au cours de ma thèse, j'ai montré que la zinédine, à l inverse de SG2NA et de la striatine qui présentent une expression caudale et centrale respectivement, a une expression principalement rostrale. Elle est exprimée majoritairement dans les neurones de l'hippocampe, du cortex cérébral et du bulbe olfactif. Comme les autres membres de la famille striatine, la zinédine a une distribution polarisée dans le compartiment somatodendritiquedes neurones et est enrichie dans les épines dendritiques (Benoist et al., 2008). Nous avons également étudié l adressage des protéines de la famille striatine aux épines dendritiques, et montré que l adressage de ces protéines nécessite leur oligomérisation par le domaine coiled-coil (Gaillard et al., 2006). Enfin, j'ai commencé à disséquer le rôle des protéines de la famille striatine dans le trafic vésiculaire. Dans ce but, j'ai développé un test d'endocytose, fondé sur l'infection des cellules N2a par le virus de la stomatite vésiculeuse. Dans ce modèle, la surexpression des membres de la famille striatine inhibe l'endocytose dépendante de la clathrine. Cette inhibition nécessite le domaine entre les acides aminés 185 et 194 de SG2NA, à proximité du domaine de fixation de la calmoduline. Dans les neurones d'hippocampe en culture, un test d endocytose de la transferrine m'a permis de montrer que SG2NA est un régulateur de l'endocytose dépendante de la clathrine comme dans les cellules N2a (Benoist et al., en préparation). En conclusion, les protéines de la famille striatine ont un rôle déterminant dansl'endocytose dépendante de la clathrine. Ainsi, ces protéines agissent probablement comme un lien entre les processus de signalisation et d'endocytose. Nos données mettent ces protéines au centre de la physiologie des épines dendritiques.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

Caractérisation d'une nouvelle famille de protéines d'échafaudage (La famille striatine)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

The excitatory amino acid carrier 1 (EAAC1) in the rat nucleus of the solitary tract: subcellular localization suggests no major role in glutamate clearance

International audienceThe excitatory amino acid carrier 1 (EAAC1) is a sodium-dependent glutamate transporter widely found in the mammalian brain and mainly localized in the somatodendritic compartment of neurons. The present study was performed to determine whether EAAC1 is present in the rat nucleus of the solitary tract (NST, a sensory brainstem nucleus involved in visceroception) and to document its subcellular localization. Using fluorescent immunolabeling, peroxidase immunostaining and quantitative immunogold labeling, we showed that both intracellular and plasma membrane-associated pools of EAAC1 transporters existed in dendrites of NST neurons. Although plasma membrane-associated transporters were more concentrated in the vicinity of synapses, no labeling was found at the axon-dendrite interface, suggesting that EAAC1 was not (or barely) expressed in this portion of dendritic membrane. Using computer simulation, we next showed that the ability of EAAC1 to efficiently take up synaptically released glutamate was very low outside the axon-dendrite interface. These data suggest that EAAC1 transporters present on NST dendrites may play a minor role if any in glutamate clearance

Distribution of zinedin in the rat brain.

International audienceMembers of the striatin family are scaffolding proteins involved in numerous signaling pathways principally in neurons. Zinedin is the only member of this protein family for which the brain distribution has not been determined so far. Here, we have validated a specific antibody against zinedin and used this tool to study the localization of zinedin at cellular and sub-cellular levels in the rat brain. Zinedin is primarily expressed in neurons of the hippocampus, cerebral cortex, olfactory bulb and caudate putamen nucleus. Like other members of the striatin family, zinedin displays a polarized distribution in the somato-dendritic compartment of neurons and is enriched in dendritic spines. The rostral expression of zinedin as well as its compartmented distribution in dendritic spines may have important implications not only for zinedin function but also in the physiology of dendritic spines of a particular subset of neurons

Sex-related exacerbation of injury-induced mechanical hypersensitivity in GAD67 haplodeficient mice

International audienceAbstract Decreased GABA levels in injury-induced loss of spinal inhibition are still under intense interest and debate. Here, we show that GAD67 haplodeficient mice exhibited a prolonged injury-induced mechanical hypersensitivity in postoperative, inflammatory, and neuropathic pain models. In line with this, we found that loss of 1 copy of the GAD67-encoding gene Gad1 causes a significant decrease in GABA contents in spinal GABAergic neuronal profiles. Consequently, GAD67 haplodeficient males and females were unresponsive to the analgesic effect of diazepam. Remarkably, all these phenotypes were more pronounced in GAD67 haplodeficient females. These mice had significantly much lower amount of spinal GABA content, exhibited an exacerbated pain phenotype during the second phase of the formalin test, developed a longer lasting mechanical hypersensitivity in the chronic constriction injury of the sciatic nerve model, and were unresponsive to the pain relief effect of the GABA-transaminase inhibitor phenylethylidenehydrazine. Our study provides strong evidence for a role of GABA levels in the modulation of injury-induced mechanical pain and suggests a potential role of the GABAergic system in the prevalence of some painful diseases among females

CK2 accumulation at the axon initial segment depends on sodium channel Nav1

International audienceEdited by Maurice Montal Keywords: Protein kinase CK2 Nav1 Phosphorylation Phosphospecific antibody Dominant negative Axon initial segment a b s t r a c t Accumulation of voltage-gated sodium channel Nav1 at the axon initial segment (AIS), results from a direct interaction with ankyrin G. This interaction is regulated in vitro by the protein kinase CK2, which is also highly enriched at the AIS. Here, using phosphospecific antibodies and inhibition/ depletion approaches, we showed that Nav1 channels are phosphorylated in vivo in their ankyrin-binding motif. Moreover, we observed that CK2 accumulation at the AIS depends on expression of Nav1 channels, with which CK2 forms tight complexes. Thus, the CK2–Nav1 interaction is likely to initiate an important regulatory mechanism to finely control Nav1 phosphorylation and, consequently , neuronal excitability

Molecular Cloning and Characterization of Phocein, a Protein Found from the Golgi Complex to Dendritic Spines

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the ς subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA