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.

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

BACKGROUND: 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 ß-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.

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.

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.

Identification of a highly conserved surface on Tat variants.

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 ß 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.

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

The identification of a neutralizing 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. We showed previously 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 three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat 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.

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.

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.

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.

Immunogold localization of phocein in dendritic spines.

Phocein, a widely expressed intracellular protein involved in clathrin- and dynamin-dependent membrane dynamics, has been shown to interact with members of the striatin family of proteins, striatin, SG2NA, and zinedin. Immunogold labeling was performed to assess subcellular localization of phocein in neurons of the rodent cerebellar cortex and hippocampal Ammon's horn. Most of the phocein-bound gold particles were located within dendritic thorns and spines of the cerebellar Purkinje cells and hippocampal pyramidal neurons, as observed previously for striatin in striatal neurons. The postsynaptic profiles containing phocein were engaged in asymmetric synapses with the main types of afferents in the cerebellum and in the hippocampus. In the cerebellum, phocein-bound immunogold particle numbers ranged from 1-20 in approximately 50% of the Purkinje cell spines. In these spines most of the immunogold particles were found in the neuroplasm ( approximately 70%) and on nonsynaptic plasma membrane domains and related structures such as endocytic-like profiles ( approximately 18%). As soon as the first postnatal week, phocein was detected in the Purkinje cell somatic and dendritic thorns making asymmetric synapses with climbing fibers. During the following weeks the protein was located in the dendritic spines, as observed in the adult molecular layer. Finally, double immunogold labeling revealed a distribution of phocein and SG2NA suggesting that the two proteins could interact in the Purkinje cell spines. The early postnatal expression of phocein, a protein involved in membrane dynamics, suggests that it may have functional relevance in dendritic remodeling during development and potentially in spine plasticity during adulthood.

Bcl-xL/Bax ratio is altered by IFNgamma in TNFalpha- but not in TRAIL-induced apoptosis in colon cancer cell line.

Apoptosis is a crucial mechanism to eliminate harmful cells in which growth factors and cytokines are key regulators. In HT29-D4 cells, a model of human colon carcinoma, IFNgamma presensitization is essential to induce an apoptotic response to TNFalpha whereas it only slightly enhances TRAIL-induced apoptosis. To compare the transcriptional profiles induced by TNFalpha and TRAIL and their regulation by IFNgamma, we optimized a cDNA array analysis on targeted signaling pathways and confirmed the gene expression modulations by comparative RT-PCR. Although the two TNFSF ligands induced a same strong up-expression of pro-apoptotic Bax gene, the expression of anti-apoptotic Bcl-xL gene was more strongly up-regulated in TNFalpha- than in TRAIL-stimulated cells. Thus, TRAIL but not TNFalpha induced apoptotic mitochondrial cascade as highlighted by cytochrome c release into cytosol. IFNgamma presensitization of TRAIL-stimulated cells did not induce any change in cytochrome c release, suggesting that the increase of IFNgamma/TRAIL-induced apoptosis is independent of this pathway. In contrast, IFNgamma pretreatment prevented Bcl-xL gene up-expression in TNFalpha-stimulated cells and allowed cytochrome c release. Thus, we hypothesize that the Bcl-xL/Bax ratio can block the apoptotic response in TNFalpha-stimulated cells but allows cell death initiation when it is altered by a crosstalk between IFNgamma presensitization and TNFalpha induced signalings.

Membrane rafts segregate pro- from anti-apoptotic insulin-like growth factor-I receptor signaling in colon carcinoma cells stimulated by members of the tumor necrosis factor superfamily.

In the tumor microenvironment, autocrine/paracrine loops of insulin-like growth factors (IGFs) contribute to cancer cell survival. However, we report here that IGF-I can send contradictory signals that interfere with cell death induced by different ligands of the tumor necrosis factor (TNF) superfamily. IGF-I protected human colon carcinoma cells from TNF-alpha-induced apoptosis, but it enhanced the apoptotic response to anti-Fas antibody and TNF-related apoptosis inducing ligand stimulation. This proapoptotic effect of IGF-I, observed in several but not all tested colon cancer cell lines, was mediated via the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. Furthermore, IGF-I receptors (IGF-IR) were located in and out of membrane lipid rafts and were tyrosine autophosphorylated in response to IGF-I. However, disruption of rafts by acute cholesterol depletion shifted IGF-IR to non-raft domains, abolished the IGF-I-mediated proapoptotic effect, and inhibited the IGF-I-dependent IRS-1 and Akt recruitment into and phosphorylation/activation within lipid rafts. Replenishing cell membranes with cholesterol reversed these effects. Activation of extracellular-regulated kinase-1/2 and p38 mitogen-activated protein kinase, which convey the IGF-I anti-apoptotic effect, occurred independently of lipid rafts. Thus, we propose that segregation of IGF-IR in and out of lipid rafts may dynamically regulate the pro- and anti-apoptotic effects of IGF-I on apoptosis induced by TNF superfamily members.

Interactions of phocein with nucleoside-diphosphate kinase, Eps15, and Dynamin I.

Phocein, an intracellular protein interacting with striatin, bears a few homologies with the sigma-subunits of clathrin adaptor proteins (Baillat, G., Moqrich, A., Castets, F., Baude, A., Bailly, Y., Benmerah, A., and Monneron, A. (2001) Mol. Biol. Cell 12, 663-673). Using phocein as a bait in a yeast two-hybrid screen, we identified two novel interacting proteins, nucleoside-diphosphate kinase (NDPK) and Eps15. Immunoprecipitation and pull-down experiments involving native and/or recombinant phocein and, respectively, NDPK and Eps15, biochemically validated their interactions. NDPK and Eps15 were recently shown to be functional neighbors of dynamin. Dynamin I is shown here to directly interact with NDPK through its C-terminal proline-rich domain, whereas recombinant phocein associates with native dynamin I. Immunocytochemical studies of rat embryonic hippocampal neurons demonstrated partial co-localization of phocein and dynamin I. Phocein thus appears to be a component of the complexes involved in some steps of the vesicular traffic machinery.