Gilbert Baillat

Early adhesion induces interaction of FAK and Fyn in lipid domains and activates raft-dependent Akt signaling in SW480 colon cancer cells

Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.

Evidence for a new type of opioid binding site in the brain of the frog Rana ridibunda

The crude membrane fraction from the brain of the frog Rana ridibunda was shown to contain 0.7-0.8 pmol/mg protein for a site with high (KD = 0.1 nM) and about 3.2 pmol/mg protein for a site with lower (KD = 10-15 nM) affinity for the opiate agonist [3H]etorphine and for the opiate antagonist [3H]diprenorphine. In addition to its very high affinity for the two tritiated oripavine derivatives, the high affinity site displayed (i) a considerably reduced ability to bind the agonist but not the antagonist in the presence of Na+ ions and (ii) pronounced stereospecificity. These properties are all typical of an opioid receptor site. The lower affinity site, which was about four times as abundant as the other exhibited none of the aforementioned characteristics and is therefore probably not opioid in nature. Detailed testing of the potency of various unlabelled opioid ligands to inhibit the binding of [3H]etorphine at the high affinity site showed that the latter consists of a mixture of several types of opioid sites, including a major type with an apparent binding profile clearly different from those of mammalian brain mu, delta- and kappa-opioid sites. In particular, this major type of site, which accounted for about 70% of the opioid binding in frog brain membranes, bound mu ([D-Ala2,MePhe4,Glyol5]enkephalin), delta ([D-Thr2,Leu5]enkephalyl-Thr) and kappa (U50,488) selective ligands with much lower affinity than did mu-, delta- and kappa-opioid receptor sites, respectively.

Antiretroviral therapy does not block the secretion of the human immunodeficiency virus tat protein.

Tat is a viral protein secreted from HIV infected cells and extra cellular Tat is suspected to prevent destruction of HIV infected cells from cells of the cellular immunity. The effect of anti retroviral therapy (ART) on Tat secretion has never been investigated. In this study, we tested for antibody reactivity against Tat variants representative of the main HIV subtypes in HIV positive patients receiving ART with undetectable viral loads ( < 40 copies/mL) over the course of one year with a blood sampling every three months. For each of theses five blood sampling, an average of 50 % of patients had Anti-Tat IgG, it turned out that 86% of patients could recognize Tat at least in one blood sampling during the course of the study. Amazingly, anti-Tat IgG appeared and/or disappeared in 66 % of patients. Only 20% had anti-Tat IgG remaining persistently while 14% were consistently without anti Tat IgG in the five blood sampling. No significant correlation was found between anti-Tat IgG and CD4+ T cell, CD8+ T cell and B cell counts revealing the incapacity of these anti Tat IgG to neutralize extra cellular Tat. Interestingly the absence and then the appearance of anti-Tat IgG in patients suggest the presence of HIV infected cells in the blood that may constitute a significant reservoir of HIV infected cells. As a conclusion antiretroviral therapy does not block the secretion of Tat and may explain why HIV infected cells can survive in spite of an effective ART treatment.

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.

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.

Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique

Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.

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

BackgroundPathological Golgi fragmentation represents a constant pre-clinical feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) but its molecular mechanisms remain hitherto unclear.ResultsHere, we show that the severe Golgi fragmentation in transgenic mutant SOD1G85R and SOD1G93A 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.ConclusionsWe conclude that Stathmin-triggered microtubule destabilization mediates Golgi fragmentation in mutant SOD1-linked ALS and potentially also in related motor neuron diseases.

Identification of a Highly Conserved Surface on Tat Variants

Background: The Tat OYI vaccine might reduce HIV-1-infected cells due to neutralizing antibodies targeting extracellular Tat. Results: MIMOOX is a peptide designed to mimic a three-dimensional epitope of Tat OYI-inducing neutralizing antibodies against Tat variants. Conclusion: There is a highly conserved surface on Tat variants that the Tat OYI vaccine helps to recognize. Significance: The Tat OYI vaccine could be an alternative to antiretroviral therapy. Extracellular Tat is suspected to protect HIV-1-infected cells from cellular immunity. Seropositive patients are unable to produce neutralizing antibodies against Tat, and Tat is still secreted under antiviral treatment. In mice, the Tat OYI vaccine candidate generates neutralizing antibodies such as the mAb 7G12. A peptide called MIMOOX was designed from fragments of Tat OYI identified as the possible binding site for mAb 7G12. MIMOOX was chemically synthesized, and its structure was stabilized with a disulfide bridge. Circular dichroism spectra showed that MIMOOX had mainly β turns but no α helix as Tat OYI. MIMOOX was recognized by mAb 7G12 in ELISA only in reduced conditions. Moreover, a competitive recognition assay with mAb 7G12 between MIMOOX and Tat variants showed that MIMOOX mimics a highly conserved surface in Tat variants. Rat immunizations with MIMOOX induce antibodies recognizing Tat variants from the main HIV-1 subtypes and confirm the Tat OYI vaccine approach.

HIV-1 Infected Patients have Antibodies Recognizing Folded Tat

Tat is a regulatory viral protein known as transactivator of HIV-1 genes but Tat is also secreted in the blood from HIV-1 infected cells. Extra cellular Tat can cross cellular membranes to trigger apoptosis and might explain the incapacity of the cellular immunity to eliminate HIV-1 infected cells. There is a controversy regarding Tat structure with studies suggesting that Tat would be a naturally unfolded protein. Here, we show that synthetic Tat variants need to be folded to have a transactivation activity in a cellular assay but this folding is unstable regarding the buffers and/or pH used as solvent. We show also that the recognition of a Tat variant versus peptides, covering its sequence, was different. Using an indirect ELISA method with 40 sera from volunteer HIV-1 infected patients, we show that Tat was recognized by 19 human sera either exclusively (n=8) or with Tat peptides (n=11). Dot Blot showed that unfolded Tat was no longer detectable by sera of the first group (n=8) compared to folded Tat. As a conclusion, this study suggests that Tat could be a naturally folded protein in the blood of HIV infected patients.