Optogenetic acidification of synaptic vesicles and lysosomes

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes

V-ATPase is a pH sensor controlling vesicular membrane fusion

International audienc

Alternative splicing controls neuronal expression of v-ATPase subunit a1 and sorting to nerve terminals.

Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1-a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression

Thymic myoid cells express high levels of muscle genes

International audienceTo explore the possible contribution of thymic myoid cells in tolerance induction mechanisms, we quantified by real-time RT-PCR, the expression of 12 muscle genes (the five subunits of acetylcholine receptor, Musk, rapsyn, utrophin, ErbB2, ErbB3, troponin T, and MCK) in a human thymic myoid cell line (MITC), compared to thymic epithelial cells (TEC) and thymocytes. Although expression of all the genes analyzed was detected in TEC and thymocytes, the level of expression in these cells was much lower than in MITC, except for -AChR, utrophin and ErbB3 genes. Since myoid cells express high level of most muscle genes and are consistently found in the thymic medulla, they may contribute to the mechanisms involved in the induction and maintenance of immune tolerance

The molecular basis of p21-activated kinase-associated neurodevelopmental disorders: From genotype to phenotype

International audienceAlthough the identification of numerous genes involved in neurodevelopmental disorders (NDDs) has reshaped our understanding of their etiology, there are still major obstacles in the way of developing therapeutic solutions for intellectual disability (ID) and other NDDs. These include extensive clinical and genetic heterogeneity, rarity of recurrent pathogenic variants, and comorbidity with other psychiatric traits. Moreover, a large intragenic mutational landscape is at play in some NDDs, leading to a broad range of clinical symptoms. Such diversity of symptoms is due to the different effects DNA variations have on protein functions and their impacts on downstream biological processes. The type of functional alterations, such as loss or gain of function, and interference with signaling pathways, has yet to be correlated with clinical symptoms for most genes. This review aims at discussing our current understanding of how the molecular changes of group I p21-activated kinases (PAK1, 2 and 3), which are essential actors of brain development and function; contribute to a broad clinical spectrum of NDDs. Identifying differences in PAK structure, regulation and spatio-temporal expression may help understanding the specific functions of each group I PAK. Deciphering how each variation type affects these parameters will help uncover the mechanisms underlying mutation pathogenicity. This is a prerequisite for the development of personalized therapeutic approache

La V-ATPase : un senseur de pH contrôlant la fusion membranaire [= V-ATPase is a pH sensor controlling vesicular membrane fusion].

news2014 Jun-Jul2014 07 11importe

Effects of Cytokines on Acetylcholine Receptor Expression: Implications for Myasthenia Gravis

International audienceMyasthenia gravis is an autoimmune disease associated with thymic pathologies, including hyperplasia. In this study, we investigated the processes that may lead to thymic overexpression of the triggering Ag, the acetylcholine receptor (AChR). Using microarray technology, we found that IFN-regulated genes are more highly expressed in these pathological thymic tissues compared with age- and sex-matched normal thymus controls. Therefore, we investigated whether proinflammatory cytokines could locally modify AChR expression in myoid and thymic epithelial cells. We found that AChR transcripts are up-regulated by IFN-gamma, and even more so by IFN-gamma and TNF-alpha, as assessed by real-time RT-PCR, with the alpha-AChR subunit being the most sensitive to this regulation. The expression of AChR protein was increased at the cytoplasmic level in thymic epithelial cells and at the membrane in myoid cells. To examine whether IFN-gamma could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-gamma gene knock-out mice, and found a significant decrease in AChR transcript levels in the thymus but not in the muscle, compared with wild-type mice. However, up-regulation of AChR protein expression was found in the muscles of animals with myasthenic symptoms treated with TNF-alpha. Altogether, these results indicate that proinflammatory cytokines influence the expression of AChR in vitro and in vivo. Because proinflammatory cytokine activity is evidenced in the thymus of myasthenia gravis patients, it could influence AChR expression and thereby contribute to the initiation of the autoimmune anti-AChR response

A new life for an old pump: V-ATPase and neurotransmitter release.

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca(2+) ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca(2+)-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase

Ocular and generalized myasthenia gravis induced by human acetylcholine receptor γ subunit immunization

Introduction: HLA-DQ8 transgenic mice develop ocular myasthenia gravis (oMG), which then progresses to generalized MG (gMG) when immunized with the human acetylcholine receptor (H-AChR) a subunit. Because the fetal AChR ? subunit is expressed in adult extraocular muscles, we anticipated that ? subunit immunization would generate an immune response to mouse AChR and induce MG in mice. Results: H-AChR ? subunit immunization in HLA-DQ8 mice induced an autoimmune response to mouse AChR and led to the destruction of AChR in the neuromuscular junction (NMJ) by anti-AChR antibody and complement activation, and it triggered upregulation of AChR gene transcription. Conclusion: Our findings indicate that oMG may be induced by immunity to the AChR ? subunit. Muscle Nerve, 201

Description clinique et analyse de corrélation phénotype/génotype dans une cohorte de patients atteints d’une déficience intellectuelle liée à des mutations du gène PAK3

International audienceLa prévalence de la déficience intellectuelle (DI) est estimée entre 2 à 3% de la population générale. Chez les individus de sexe masculin 5 à 10% des DI seraient liées à l’X. On connait actuellement 146 gènes de DI sur le chromosome X. Le premier variant pathogène retrouvé dans le gène PAK3 (P21-Activated Kinase 3) est un variant faux-sens identifié par Allen et al. en 1998 dans une famille de patients atteints d’une DI liée à l’X. Les patients, décrits comme peu dysmorphiques, avaient une microcéphalie et des troubles du comportement, hyperactivité dans l’enfance et hétéroagressivité à l’âge adulte. Notre étude multicentrique rétrospective avait pour objectif de décrire les caractéristiques cliniques des patients ayant une mutation dans le gène PAK3 puis de faire une analyse de corrélation phénotype/génotype. Elle a porté sur dix patients recrutés en France et quarante-cinq patients de la littérature. Nous avons retrouvé une DI non spécifique associée à une hypotonie dans 92% des cas, un retard de langage dans 91%, une microcéphalie dans 46% et des réflexes vifs dans 32% des cas. Les troubles du comportement présents chez 97% des patients sont au-devant du tableau clinique avec une auto- ou une hétéroagressivité dans 58% des cas et une psychose dans 26% des cas. Sur le plan morphologique, il n’existe pas de caractères spécifiques, mais des oreilles longues sont retrouvées chez 60% des patients et un palais creux chez 71%. Enfin, une agénésie du corps calleux est retrouvée dans 42% des cas mais peu de patients ont eu une IRM cérébrale. Au total, 14 patients issus de 4 familles ont une mutation nulle et 40 patients issus de 12 familles ont une mutation faux-sens. Nous n’avons pas mis en évidence de corrélation génotype-phénotype significative, notamment les patients ayant une mutation nulle n’ont pas une atteinte clinique plus sévère que les autres. Une nouvelle étude avec un plus grand nombre de patients et davantage de données d’imagerie cérébrale serait intéressante pour évaluer la fréquence de l’agénésie du corps calleux et définir si elle est liée à certains types de mutations