TBCE Mutations Cause Early-Onset Progressive Encephalopathy with Distal Spinal Muscular Atrophy

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration

Translating lung function genome-wide association study (GWAS) findings: new insights for lung biology

Chronic respiratory diseases are a major cause of worldwide mortality and morbidity. Although hereditary severe deficiency of α1 antitrypsin (A1AD) has been established to cause emphysema, A1AD accounts for only ∼1% of Chronic Obstructive Pulmonary Disease (COPD) cases. Genome-wide association studies (GWAS) have been successful at detecting multiple loci harboring variants predicting the variation in lung function measures and risk of COPD. However, GWAS are incapable of distinguishing causal from noncausal variants. Several approaches can be used for functional translation of genetic findings. These approaches have the scope to identify underlying alleles and pathways that are important in lung function and COPD. Computational methods aim at effective functional variant prediction by combining experimentally generated regulatory information with associated region of the human genome. Classically, GWAS association follow-up concentrated on manipulation of a single gene. However association data has identified genetic variants in >50 loci predicting disease risk or lung function. Therefore there is a clear precedent for experiments that interrogate multiple candidate genes in parallel, which is now possible with genome editing technology. Gene expression profiling can be used for effective discovery of biological pathways underpinning gene function. This information may be used for informed decisions about cellular assays post genetic manipulation. Investigating respiratory phenotypes in human lung tissue and specific gene knockout mice is a valuable in vivo approach that can complement in vitro work. Herein, we review state-of-the-art in silico, in vivo, and in vitro approaches that may be used to accelerate functional translation of genetic findings

Caracterisation, solubilisation et purification du recepteur aux substances opioiedes de cerveaux de grenouilles "Rana ridibunda"

SIGLECNRS TD Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

MALADIES DES MOTONEURONES: Etude des mécanismes de dégénérescence dans la SLA

Egalement appelée maladie de Charcot, la SLA est une maladie sévère et fatale des motoneurones de l’adulte. Elle est caractérisée par la dégénérescence sélective et progressive des neurones moteurs de la moelle épinière, du tronc cérébral et du cortex cérébral. Elle provoque une paralysie progressive de l'ensemble de la musculature squelettique des membres, du tronc (y compris les muscles respiratoires) et de l'extrémité céphalique. Elle touche les deux sexes avec une incidence annuelle d’environ 2,5 cas / 100 000. Cette maladie apparait de manière sporadique dans 90 à 95% des cas et aucune cause n’a pu être retenue avec suffisamment de certitude jusqu’à présent. Parmi les cas ayant une cause génétique familiale (5 à 10%), plus de 25 gènes ont été impliqués tels que SOD1, Alsin/ALS2, Senataxin, VAP-B, Dynactin, Tau, FUS, TDP43. Récemment, des répétitions multiples de l’hexanucléotide GGGGCC dans le gène C9ORF72 ont été identifiées dans des cas de SLA sporadique et familiale mais les mécanismes aboutissant à la maladie restent à éclaircir.Les neurones moteurs qui dégénèrent dans la SLA montrent de nombreux changements structuraux tels que la présence d’agrégats intracellulaires, une désorganisation du cytosquelette et des anomalies des organelles intracellulaires. La fragmentation de l’appareil de Golgi survient précocement au cours de la pathologie, bien avant l’apparition d’autres anomalies pathologiques dans les neurones moteurs de patients. A l’heure actuelle, le mécanisme à l’origine de la fragmentation de l’appareil de Golgi n’est toujours pas identifié que ce soit dans des modèles murins ou chez les patients atteints de SLA. Une première étude a porté sur le rôle de la protéine chaperonne des tubulines TBCE dans la fragmentation du Golgi chez des souris pmn (progressive motor neuropathy) utilisées comme modèle de maladie du neurone. Les données obtenues montrent que TBCE maintient la structure du Golgi en ajustant la polymérisation des microtubules en impliquant ARF1, la petite GTPase connue pour contrôler la formation des vésicules COP I. Ces anomalies pathologiques compromettent le transport des constituants axonaux et synaptiques et représenterait une contribution importante à la dégénérescence et aux dysfonctionnements des motoneurones.Une deuxième étude a porté sur les mécanismes moléculaires et les conséquences cellulaires des mutations de la protéine SOD1 dans la SLA. Afin d’éclaircir les mécanismes moléculaires induit par ces mutations, notre équipe a utilisé les deux lignées murines les plus courantes : SOD1G93A et SOD1G85R. Une perte de microtubules est observée dans les neurones moteurs exprimant la SOD1 mutée ainsi des anomalies morphologiques de l’appareil de Golgi. Nous avons montré une augmentation de l’expression des Stathmine 1 et 2 dans les neurones moteurs des souris SOD mutées. Ce sont des phosphoprotéines qui, selon leur état de phosphorylation, induisent la dépolymérisation des MTs in vitro et in vivo en séquestrant les dimères de tubuline libres. Notre hypothèse est que la surexpression de protéines de la famille des Stathmines pourrait être à l’origine des anomalies golgienne et des altérations vésiculaires.J’ai également participé au développement d’une nouvelle méthode de purification par FACS de motoneurones humains issus d’IPS.Mon expérience professionnelle, développée sur 27 ans de carrière scientifique, m’apporte la capacité d’assumer des responsabilités plus importantes, comme diriger un étudiant en thèse. Dans ce but, je désire donc obtenir le diplôme d’Habilitation à Diriger les Recherches au sein de l’Université d’Aix-Marseille

Golgi fragmentation in pmn mice is due to a defective ARF1/TBCE cross-talk that coordinates COPI vesicle formation and tubulin polymerization

Golgi fragmentation is an early hallmark of many neurodegenerative diseases but its pathophysiological relevance and molecular mechanisms are unclear. We here demonstrate severe and progressive Golgi fragmentation in motor neurons of progressive motor neuronopathy (pmn) mice due to loss of the Golgi-localized tubulin-binding cofactor E (TBCE). Loss of TBCE in mutant pmn and TBCE-depleted motor neuron cultures causes defects in Golgi-derived microtubules, as expected, but surprisingly also reduced levels of COPI subunits, decreased recruitment of tethering factors p115/GM130 and impaired Golgi SNARE-mediated vesicle fusion. Conversely, ARF1, which stimulates COPI vesicle formation, enhances the recruitment of TBCE to the Golgi, increases polymerization of Golgi-derived microtubules and rescues TBCE-linked Golgi fragmentation. These data indicate an ARF1/TBCE-mediated cross-talk that coordinates COPI formation and tubulin polymerization at the Golgi. We conclude that interruption of this cross-talk causes Golgi fragmentation in pmn mice and hypothesize that similar mechanisms operate in human amyotrophic lateral sclerosis and spinal muscular atrophy

Stathmin 1/2-triggered microtubule loss mediates Golgi fragmentation in mutant SOD1 motor neurons

International audienceBackground: Pathological Golgi fragmentation represents a constant pre-clinical feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) but its molecular mechanisms remain hitherto unclear.Results: Here, we show that the severe Golgi fragmentation in transgenic mutant SOD1 G85R and SOD1 G93A mouse motor neurons is associated with defective polymerization of Golgi-derived microtubules, loss of the COPI coat subunit β-COP, cytoplasmic dispersion of the Golgi tether GM130, strong accumulation of the ER-Golgi v-SNAREs GS15 and GS28 as well as tubular/vesicular Golgi fragmentation. Data mining, transcriptomic and protein analyses demonstrate that both SOD1 mutants cause early presymptomatic and rapidly progressive up-regulation of the microtubule-destabilizing proteins Stathmins 1 and 2. Remarkably, mutant SOD1-triggered Golgi fragmentation and Golgi SNARE accumulation are recapitulated by Stathmin 1/2 overexpression but completely rescued by Stathmin 1/2 knockdown or the microtubule-stabilizing drug Taxol.Conclusions: We conclude that Stathmin-triggered microtubule destabilization mediates Golgi fragmentation in mutant SOD1-linked ALS and potentially also in related motor neuron diseases

A monoclonal antibody directed against a conformational epitope of the HIV-1 trans-activator (Tat) protein neutralizes cross-clade.

International audienceThe identification of a neutralizing monoclonal antibody (mAb) against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication, but also as an extracellular toxin able to disrupt the immune system. Previously, we showed that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi, and selected a mAb, named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common 3D epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing that Tat is as an unfolded protein. MAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity

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