Caractérisation du gène Spatial et identification de sa fonction dans les cellules hautement polarisées

Le gène Spatial est exprimé par des cellules hautement polarisées : les cellules épithéliales du thymus, neuronales du système nerveux central et germinales du testicule. La différenciation de ces cellules est accompagnée d'une polarisation de la distribution de Spatial, dans des structures microtubulaires hautement organisées telles que la manchette, le flagelle et la dendrite.Mon projet a porté sur l’identification de la fonction du gène Spatial au niveau du thymus et du cerveau murins. De plus, j’ai été impliquée dans la caractérisation du gène Spatial chez l’homme et l’évaluation de son impact sur la spermatogenèse et l’infertilité humaine.Nos résultats montrent qu’au niveau du thymus, Spatial est détecté tout au long de l’organogenèse thymique jusqu’aux stades adultes. Son profil d’expression correspond à un « promiscuous gene » impliqué dans l’acquisition de la tolérance des lymphocytes T. Au niveau du système nerveux central, Spatial présente une distribution somatodendritique dans les cultures de neurones hippocampiques et son expression est fortement détectée lors de la poussée dendritique. Nous avons montré que le transport de Spatial du corps cellulaire vers les dendrites est dépendant de la kinésine Kif17. De plus, Spatial semble être impliqué dans la formation des dendrites via la voie de signalisation stimulée par le Nerve Growth Factor.Chez l’homme, le gène H-Spatial est fortement exprimé au niveau du testicule et son expression est spécifique de la spermiogenèse, étape de différenciation des spermatides rondes en spermatozoïdes. Chez les patients infertiles asthéno- et/ou tératozoospermiques, présentant des anomalies de mobilité et de forme des spermatozoïdes, le niveau d’ARNm d’H-Spatial est fortement réduit. Ces résultats suggèrent qu’H-Spatial est un marqueur potentiel de l’infertilité masculine.J’ai également participé à la validation des dendrimères PAMAM (poly-amidoamine) comme vecteurs efficaces pour le transfert de siRNA et d’ADN in vitro sur différents lignées cellulaires et in vivo sur des thymus murins. Ce système pourrait être un moyen thérapeutique pour traiter des immunodéficiences liées aux lymphocytes T.Spatial gene is expressed in highly polarized cells such as, thymic epithelial cells, testicular germ cells and neuronal cells of the central nervous system. The differentiation of these cells is accompanied by the polarized distribution of Spatial in highly organized microtubule structures such as the manchette, the flagellum and dendrites. This project aims to identify the function of Spatial gene in the thymus and in the brain. Moreover, we characterize Spatial gene in humans and evaluate its impact on spermatogenesis and human infertility.Our results showed that, in the thymus, Spatial is detected throughout the thymic development, until adulthood. Its expression profile corresponds to a ‘promiscuous gene’, implicated in the acquisition of T lymphocyte tolerance.At the level of the central nervous system, Spatial showed a somatodendritic distribution in hippocampal neuron cultures. Moreover, its expression was highly detected during dendritic growth. We have also shown that the transport of Spatial from the cell body to the dendrites is dependent on the kinesin Kif17. In addition, our results suggest that Spatial seems to be implemented in dendrite formation by the Nerve Growth Factor mediated signaling pathway.In the humans, H-Spatial is highly expressed in the testis and its expression is specific to spermiogenesis: the phase of differentiation of round spermatids to spermatozoids. In infertile astheno and/or teratozoospermic patients with sperm shape and mobility anomalies, H-Spatial levels were drastically reduced. These results propose H-Spatial as a potential marker for human male infertility.Finally, we have equally participated in the validation of PAMAM (poly-amidoamine) dendrimers as efficient vectors for the transfer of DNA and siRNA in vitro, in different cell lines; and in vivo, in murine thymi. This system could serve as a new therapeutic model for treating diseases linked to T lymphocytes

Spatial gene's (Tbata) implication in neurite outgrowth and dendrite patterning in hippocampal neurons

The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based kinesin motor transport to deliver essential cargo into axons and dendrites. In developing neurons, kinesin trafficking is essential for delivering organelles and molecules that are crucial for elongation and guidance of the growing axonal and dendritic termini. In mature neurons, kinesin cargo delivery is essential for neuron dynamic physiological functions which are critical in brain development. In this work, we followed Spatial (Tbata) gene expression during primary hippocampal neuron development and showed that it is highly expressed during dendrite formation. Spatial protein exhibits a somatodendritic distribution and we show that the kinesin motor Kif17, among other dendrite specific kinesins, is crucial for Spatial localization to dendrites of hippocampal neurons. Furthermore, Spatial down regulation in primary hippocampal cells revealed a role for Spatial in maintaining neurons' polarity by ensuring proper neurite outgrowth. This polarity is specified by intrinsic and extracellular signals that allow neurons to determine axon and dendrite fate during development. Neurotrophic factors, such as the Nerve Growth Factor (NGF), are candidate extracellular polarity-regulating cues which are proposed to accelerate neuronal polarization by enhancing dendrite growth. Here, we show that NGF treatment increases Spatial expression in hippocampal neurons. Altogether, these data suggest that Spatial, in response to NGF and through its transport by Kif17, is crucial for neuronal polarization and can be a key regulator of neurite outgrowth. © 2013.This work was supported by the Inserm, the Provence-Alpes-Côte d'Azur region and the “Association Francaise contre les Myopathies” (AFM)Peer Reviewe

Sperm mRNAs and microRNAs as candidate markers for the impact of toxicants on human spermatogenesis: an application to tobacco smoking

International audienceSpermatozoa contain a complex population of RNAs including messenger RNAs (mRNAs) and small RNAs such as microRNAs (miRNA). It has been reported that these RNAs can be used to understand the mechanisms by which toxicological exposure affects spermatogenesis. The aim of our study was to compare mRNA and miRNA profiles in spermatozoa from eight smokers and eight non-smokers, and search for potential relationships between mRNA and miRNA variation. All men were selected based on their answers to a standard toxic exposure questionnaire, and sperm parameters. Using mRNA and miRNA microarrays, we showed that mRNAs from 15 genes were differentially represented between smokers and non-smokers (p \textless 0.01): five had higher levels and 10 lower levels in the smokers. For the microRNAs, 23 were differentially represented: 16 were higher and seven lower in the smokers (0.004 \textless= p \textless 0.01). Quantitative RT-PCR confirmed the lower levels in smokers compared to non-smokers for hsa-miR-296-5p, hsa-miR-3940, and hsa-miR-520d-3p. Moreover, we observed an inverse relationship between the levels of microRNAs and six potential target mRNAs (B3GAT3, HNRNPL, OASL, ODZ3, CNGB1, and PKD2). Our results indicate that alterations in the level of a small number of microRNAs in response to smoking may contribute to changes in mRNA expression in smokers. We conclude that large-scale analysis of spermatozoa RNAs can be used to help understand the mechanisms by which human spermatogenesis responds to toxic substances including those in tobacco smoke

Structurally Flexible Triethanolamine Core PAMAM Dendrimers Are Effective Nanovectors for DNA Transfection in Vitro and in Vivo to the Mouse Thymus

With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy

Divergent transcription is associated with promoters of transcriptional regulators.

International audienceBACKGROUND: Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. RESULTS: We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. CONCLUSIONS: We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription