Conséquences pathologiques des expansions CTG sur le système nerveux central d'un modèle murin de la dystrophie myotonique de Steinert (approches moléculaires, protéomiques et cellulaires)

La dystrophie myotonique de type I (DM1) constitue la plus fréquente des pathologies musculaires héréditaires chez l adulte. Bien qu initialement considérée comme une maladie musculaire, la DM1 présente une atteinte neurologique très handicapante. Cette maladie autosomique dominante résulte de l expansion anormale d un triplet CTG dans la partie 3 UTR du gène DMPK. Un effet trans du transcrit DMPK muté entraine une dérégulation de l épissage alternatif dans de nombreux tissus. Cependant, les mécanismes pathologiques de la DM1 dans le cerveau restent encore peu compris. Afin de disséquer ce mécanisme, notre laboratoire a créé des souris transgéniques exprimant le transcrit DMPK avec de larges expansions CUG dans de nombreux tissus. Ces souris nommées DMSXL, recréent d importants aspects pathologiques de la DM1, comme des anomalies du comportement et électrophysiologiques du cerveau. Elles représentent donc un excellent outil pour explorer l effet pathologique de la mutation dans le SNC. En m appuyant sur ce modèle, j ai exploré dans un premier temps l effet trans des ARNs toxiques et l ampleur de la splicéopathie dans le SNC. De façon intéressante, certains défauts d épissage sont régions spécifiques, et ne montrent pas d aggravation avec l âge des souris DMSXL. Mes résultats démontrent que les ARNs mutés sont capables de déréguler l épissage alternatif dans l ensemble du SNC. La région du cervelet a aussi montré des anomalies de l épissage dans les souris DMSXL, qui, en plus, présentent des perturbations cognitives dépendantes de cette région cérébrale. Le cervelet des souris DMSXL présente aussi des déficits électrophysiologiques suggérant une dysfonction cérébelleuse et plus précisément une dysfonction des cellules de Purkinje. Dans la recherche des populations cellulaires les plus affectées dans le cervelet, j ai démontré la présence de signes de la toxicité de l ARN plus marqués dans la glie de Bergman, entourant les cellules de Purkinje. Pour trouver les voies moléculaires perturbées dans le cervelet, et disséquer le mécanisme derrière les anomalies observées, j ai réalisé une approche protéomique globale et trouvé une sévère baisse de l expression du transporteur glial de glutamate GLT1/EAAT2, suggérant une dysfonction du cervelet, en conséquence d un possible métabolisme anormal du glutamate. L analyse protéomique globale du cerveau des souris DM1 a aussi identifié des différences d expression et des modifications post-traductionnelles de protéines impliquées dans la signalisation du calcium. L étude du métabolisme des ARNm dans la DM1 a mis en évidence la dérégulation de l épissage de gènes impliqués dans le métabolisme du calcium, soutenant l hypothèse d une dysfonction calcique dans le SNC. Pour étudier les conséquences de la mutation sur les variations calciques cellulaires, j ai caractérisé un modèle cellulaire astrocytaire de la DM1. Ce modèle m a permis de démontrer une localisation anormale du récepteur GRIN1/NMDAR1, ainsi qu une réponse calcique anormale dans les astrocytes primaires porteurs des amplifications CTG. Malgré les avancés thérapeutiques dans le muscle, on ne sait pas à quel point les stratégies en cours de développement sont efficaces dans le SNC. Pour étudier ce problème, j ai utilisé le modèle astrocytaire de la DM1 afin de valider in cellulo une stratégie thérapeutique qui vise à rétablir une activité normale du facteur d épissage MBNL1 endogène. Mes travaux de thèse ont permis d avancer dans la compréhension de la neuropathologie de la DM1. Ils ont mis en évidence pour la première fois une dysfonction du cervelet, ainsi que la possible dérégulation de la voix calcique dans le SNC. Mes résultats ont donc contribué à mieux comprendre le mécanisme de la DM1 dans le SNC, pour, à long terme, développer des approches thérapeutiques ciblant des évènements moléculaires précis.Myotonic dystrophy type 1 (DM1) is the most frequent inherited muscular disorder in adults. Although traditionally regarded as a muscle disease, DM1 presents debilitating neurological manifestations. DM1 is an autosomic dominant disease caused by the abnormal expansion of a CTG triplet within the 3 UTR of the DMPK gene. Many molecular aspects of the DM1 are mediated by a trans effect of the expanded DMPK transcripts, whose accumulation leads to splicing deregulation in many tissues. Despite recent progress in the understanding of DM1 pathogenesis in muscle and central nervous system (CNS), the detailed molecular disease mechanism operating in the brain is still poorly understood. In order to investigate the pathophysiology, our laboratory has generated DMSXL transgenic mice expressing DMPK transcripts containing large CUG expansions in many tissues. DMSXL mice mimic important features of the DM1, notably in the CNS, showing behaviour as well as electrophysiological abnormalities. Therefore, this mouse line represents an excellent tool to investigate the toxic effects of the mutation in the CNS. Taking advantages of this transgenic model, I have first explored the trans effect of the toxic RNA and the extent of DM1-associated spliceopathy in the CNS. Interestingly, some splicing defects were region-specific, and their severity did not increase with the age of the DMSXL mice. My data demonstrate that CUG-containing RNAs have a wide deleterious effect and deregulate alternative splicing in many areas of the CNS. In addition to splicing abnormalities in cerebellum, DMSXL mice also displayed deficits in cerebellum-dependant motor coordination. Plus, DMSXL cerebellum showed electrophysiological abnormalities, suggesting cerebellar dysfunction and more precisely Purkinje cell dysfunction. In the search for the cellular populations showing the greatest susceptibility to RNA toxicity in the cerebellum, I have found extensive foci accumulation as well as pronounced splicing defects in the Bergman glia, surrounding Purkinje cells, in DMSXL and DM1 patients cerebellum. In order to identify molecular pathways and mechanisms behind the behaviour and electrophysiological abnormalities detected, I have performed a global proteomics approach and found a severe decrease in the expression of a glial glutamate transporter GLT1/EAAT2, suggesting that DM1 causes cerebellum dysfunction, through abnormal glutamate metabolism. Global proteomic analysis of DMSXL cerebellum also identified expression and post-translational changes of several proteins involved in calcium signalling. Missplicing of different transcripts involved in calcium metabolism reinforces the idea of calcium dysfunction in the neuropathogenesis of the DM1. To study the effects of toxic RNA on calcium homeostasis and flux, I have established and characterised a brain cell model of DM1. DMSXL primary astrocyte cultures allowed me to show the mislocalisation of the glutamate receptor GRIN1/NMDAR1, as well as abnormal calcium responses to stimulation. Despite recent therapeutic advances in muscle, we do not know the CNS efficiency of the therapeutic strategies currently being developed. To address this problem, I have used the DM1 astrocyte cell model to validate in cellulo a therapeutic strategy aiming to restore the activity of the endogenous splicing factor MBNL1. My thesis work provided a significant step in the understanding of the DM1 pathology in the CNS. My results revealed for the first time signs of cerebellum dysfunction in DM1, as well as signs of calcium homeostasis deregulation in the SNC. My work contributed to better understand the pathological mechanisms of DM1, the brain pathways and cell types most susceptible to toxic RNA. In the long term, my data will contribute to the rational development of therapeutic strategies targeting precise and deleterious molecular events.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Development of a novel polyamide-based agent to inhibit EVI1 function

The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME. Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor

Conséquences pathologiques des expansions CTG sur le système nerveux central d’un modèle murin de la dystrophie myotonique de Steinert : approches moléculaires, protéomiques et cellulaires

Myotonic dystrophy type 1 (DM1) is the most frequent inherited muscular disorder in adults. Although traditionally regarded as a muscle disease, DM1 presents debilitating neurological manifestations. DM1 is an autosomic dominant disease caused by the abnormal expansion of a CTG triplet within the 3’UTR of the DMPK gene. Many molecular aspects of the DM1 are mediated by a trans effect of the expanded DMPK transcripts, whose accumulation leads to splicing deregulation in many tissues. Despite recent progress in the understanding of DM1 pathogenesis in muscle and central nervous system (CNS), the detailed molecular disease mechanism operating in the brain is still poorly understood. In order to investigate the pathophysiology, our laboratory has generated DMSXL transgenic mice expressing DMPK transcripts containing large CUG expansions in many tissues. DMSXL mice mimic important features of the DM1, notably in the CNS, showing behaviour as well as electrophysiological abnormalities. Therefore, this mouse line represents an excellent tool to investigate the toxic effects of the mutation in the CNS. Taking advantages of this transgenic model, I have first explored the trans effect of the toxic RNA and the extent of DM1-associated spliceopathy in the CNS. Interestingly, some splicing defects were region-specific, and their severity did not increase with the age of the DMSXL mice. My data demonstrate that CUG-containing RNAs have a wide deleterious effect and deregulate alternative splicing in many areas of the CNS. In addition to splicing abnormalities in cerebellum, DMSXL mice also displayed deficits in cerebellum-dependant motor coordination. Plus, DMSXL cerebellum showed electrophysiological abnormalities, suggesting cerebellar dysfunction and more precisely Purkinje cell dysfunction. In the search for the cellular populations showing the greatest susceptibility to RNA toxicity in the cerebellum, I have found extensive foci accumulation as well as pronounced splicing defects in the Bergman glia, surrounding Purkinje cells, in DMSXL and DM1 patients cerebellum. In order to identify molecular pathways and mechanisms behind the behaviour and electrophysiological abnormalities detected, I have performed a global proteomics approach and found a severe decrease in the expression of a glial glutamate transporter GLT1/EAAT2, suggesting that DM1 causes cerebellum dysfunction, through abnormal glutamate metabolism. Global proteomic analysis of DMSXL cerebellum also identified expression and post-translational changes of several proteins involved in calcium signalling. Missplicing of different transcripts involved in calcium metabolism reinforces the idea of calcium dysfunction in the neuropathogenesis of the DM1. To study the effects of toxic RNA on calcium homeostasis and flux, I have established and characterised a brain cell model of DM1. DMSXL primary astrocyte cultures allowed me to show the mislocalisation of the glutamate receptor GRIN1/NMDAR1, as well as abnormal calcium responses to stimulation. Despite recent therapeutic advances in muscle, we do not know the CNS efficiency of the therapeutic strategies currently being developed. To address this problem, I have used the DM1 astrocyte cell model to validate in cellulo a therapeutic strategy aiming to restore the activity of the endogenous splicing factor MBNL1. My thesis work provided a significant step in the understanding of the DM1 pathology in the CNS. My results revealed for the first time signs of cerebellum dysfunction in DM1, as well as signs of calcium homeostasis deregulation in the SNC. My work contributed to better understand the pathological mechanisms of DM1, the brain pathways and cell types most susceptible to toxic RNA. In the long term, my data will contribute to the rational development of therapeutic strategies targeting precise and deleterious molecular events.La dystrophie myotonique de type I (DM1) constitue la plus fréquente des pathologies musculaires héréditaires chez l’adulte. Bien qu’initialement considérée comme une maladie musculaire, la DM1 présente une atteinte neurologique très handicapante. Cette maladie autosomique dominante résulte de l’expansion anormale d’un triplet CTG dans la partie 3’UTR du gène DMPK. Un effet trans du transcrit DMPK muté entraine une dérégulation de l‘épissage alternatif dans de nombreux tissus. Cependant, les mécanismes pathologiques de la DM1 dans le cerveau restent encore peu compris. Afin de disséquer ce mécanisme, notre laboratoire a créé des souris transgéniques exprimant le transcrit DMPK avec de larges expansions CUG dans de nombreux tissus. Ces souris nommées DMSXL, recréent d’importants aspects pathologiques de la DM1, comme des anomalies du comportement et électrophysiologiques du cerveau. Elles représentent donc un excellent outil pour explorer l’effet pathologique de la mutation dans le SNC. En m’appuyant sur ce modèle, j’ai exploré dans un premier temps l’effet trans des ARNs toxiques et l’ampleur de la splicéopathie dans le SNC. De façon intéressante, certains défauts d’épissage sont régions spécifiques, et ne montrent pas d’aggravation avec l’âge des souris DMSXL. Mes résultats démontrent que les ARNs mutés sont capables de déréguler l’épissage alternatif dans l’ensemble du SNC. La région du cervelet a aussi montré des anomalies de l’épissage dans les souris DMSXL, qui, en plus, présentent des perturbations cognitives dépendantes de cette région cérébrale. Le cervelet des souris DMSXL présente aussi des déficits électrophysiologiques suggérant une dysfonction cérébelleuse et plus précisément une dysfonction des cellules de Purkinje. Dans la recherche des populations cellulaires les plus affectées dans le cervelet, j’ai démontré la présence de signes de la toxicité de l’ARN plus marqués dans la glie de Bergman, entourant les cellules de Purkinje. Pour trouver les voies moléculaires perturbées dans le cervelet, et disséquer le mécanisme derrière les anomalies observées, j’ai réalisé une approche protéomique globale et trouvé une sévère baisse de l’expression du transporteur glial de glutamate GLT1/EAAT2, suggérant une dysfonction du cervelet, en conséquence d’un possible métabolisme anormal du glutamate. L’analyse protéomique globale du cerveau des souris DM1 a aussi identifié des différences d’expression et des modifications post-traductionnelles de protéines impliquées dans la signalisation du calcium. L’étude du métabolisme des ARNm dans la DM1 a mis en évidence la dérégulation de l’épissage de gènes impliqués dans le métabolisme du calcium, soutenant l’hypothèse d’une dysfonction calcique dans le SNC. Pour étudier les conséquences de la mutation sur les variations calciques cellulaires, j’ai caractérisé un modèle cellulaire astrocytaire de la DM1. Ce modèle m’a permis de démontrer une localisation anormale du récepteur GRIN1/NMDAR1, ainsi qu’une réponse calcique anormale dans les astrocytes primaires porteurs des amplifications CTG. Malgré les avancés thérapeutiques dans le muscle, on ne sait pas à quel point les stratégies en cours de développement sont efficaces dans le SNC. Pour étudier ce problème, j’ai utilisé le modèle astrocytaire de la DM1 afin de valider in cellulo une stratégie thérapeutique qui vise à rétablir une activité normale du facteur d’épissage MBNL1 endogène. Mes travaux de thèse ont permis d’avancer dans la compréhension de la neuropathologie de la DM1. Ils ont mis en évidence pour la première fois une dysfonction du cervelet, ainsi que la possible dérégulation de la voix calcique dans le SNC. Mes résultats ont donc contribué à mieux comprendre le mécanisme de la DM1 dans le SNC, pour, à long terme, développer des approches thérapeutiques ciblant des évènements moléculaires précis

Pathological consequences of CTG expansions on the central nervous system of a mouse model of the myotonic dystrophy of Steinert : molecular, proteomics and cellular approaches

La dystrophie myotonique de type I (DM1) constitue la plus fréquente des pathologies musculaires héréditaires chez l’adulte. Bien qu’initialement considérée comme une maladie musculaire, la DM1 présente une atteinte neurologique très handicapante. Cette maladie autosomique dominante résulte de l’expansion anormale d’un triplet CTG dans la partie 3’UTR du gène DMPK. Un effet trans du transcrit DMPK muté entraine une dérégulation de l‘épissage alternatif dans de nombreux tissus. Cependant, les mécanismes pathologiques de la DM1 dans le cerveau restent encore peu compris. Afin de disséquer ce mécanisme, notre laboratoire a créé des souris transgéniques exprimant le transcrit DMPK avec de larges expansions CUG dans de nombreux tissus. Ces souris nommées DMSXL, recréent d’importants aspects pathologiques de la DM1, comme des anomalies du comportement et électrophysiologiques du cerveau. Elles représentent donc un excellent outil pour explorer l’effet pathologique de la mutation dans le SNC. En m’appuyant sur ce modèle, j’ai exploré dans un premier temps l’effet trans des ARNs toxiques et l’ampleur de la splicéopathie dans le SNC. De façon intéressante, certains défauts d’épissage sont régions spécifiques, et ne montrent pas d’aggravation avec l’âge des souris DMSXL. Mes résultats démontrent que les ARNs mutés sont capables de déréguler l’épissage alternatif dans l’ensemble du SNC. La région du cervelet a aussi montré des anomalies de l’épissage dans les souris DMSXL, qui, en plus, présentent des perturbations cognitives dépendantes de cette région cérébrale. Le cervelet des souris DMSXL présente aussi des déficits électrophysiologiques suggérant une dysfonction cérébelleuse et plus précisément une dysfonction des cellules de Purkinje. Dans la recherche des populations cellulaires les plus affectées dans le cervelet, j’ai démontré la présence de signes de la toxicité de l’ARN plus marqués dans la glie de Bergman, entourant les cellules de Purkinje. Pour trouver les voies moléculaires perturbées dans le cervelet, et disséquer le mécanisme derrière les anomalies observées, j’ai réalisé une approche protéomique globale et trouvé une sévère baisse de l’expression du transporteur glial de glutamate GLT1/EAAT2, suggérant une dysfonction du cervelet, en conséquence d’un possible métabolisme anormal du glutamate. L’analyse protéomique globale du cerveau des souris DM1 a aussi identifié des différences d’expression et des modifications post-traductionnelles de protéines impliquées dans la signalisation du calcium. L’étude du métabolisme des ARNm dans la DM1 a mis en évidence la dérégulation de l’épissage de gènes impliqués dans le métabolisme du calcium, soutenant l’hypothèse d’une dysfonction calcique dans le SNC. Pour étudier les conséquences de la mutation sur les variations calciques cellulaires, j’ai caractérisé un modèle cellulaire astrocytaire de la DM1. Ce modèle m’a permis de démontrer une localisation anormale du récepteur GRIN1/NMDAR1, ainsi qu’une réponse calcique anormale dans les astrocytes primaires porteurs des amplifications CTG. Malgré les avancés thérapeutiques dans le muscle, on ne sait pas à quel point les stratégies en cours de développement sont efficaces dans le SNC. Pour étudier ce problème, j’ai utilisé le modèle astrocytaire de la DM1 afin de valider in cellulo une stratégie thérapeutique qui vise à rétablir une activité normale du facteur d’épissage MBNL1 endogène. Mes travaux de thèse ont permis d’avancer dans la compréhension de la neuropathologie de la DM1. Ils ont mis en évidence pour la première fois une dysfonction du cervelet, ainsi que la possible dérégulation de la voix calcique dans le SNC. Mes résultats ont donc contribué à mieux comprendre le mécanisme de la DM1 dans le SNC, pour, à long terme, développer des approches thérapeutiques ciblant des évènements moléculaires précis.Myotonic dystrophy type 1 (DM1) is the most frequent inherited muscular disorder in adults. Although traditionally regarded as a muscle disease, DM1 presents debilitating neurological manifestations. DM1 is an autosomic dominant disease caused by the abnormal expansion of a CTG triplet within the 3’UTR of the DMPK gene. Many molecular aspects of the DM1 are mediated by a trans effect of the expanded DMPK transcripts, whose accumulation leads to splicing deregulation in many tissues. Despite recent progress in the understanding of DM1 pathogenesis in muscle and central nervous system (CNS), the detailed molecular disease mechanism operating in the brain is still poorly understood. In order to investigate the pathophysiology, our laboratory has generated DMSXL transgenic mice expressing DMPK transcripts containing large CUG expansions in many tissues. DMSXL mice mimic important features of the DM1, notably in the CNS, showing behaviour as well as electrophysiological abnormalities. Therefore, this mouse line represents an excellent tool to investigate the toxic effects of the mutation in the CNS. Taking advantages of this transgenic model, I have first explored the trans effect of the toxic RNA and the extent of DM1-associated spliceopathy in the CNS. Interestingly, some splicing defects were region-specific, and their severity did not increase with the age of the DMSXL mice. My data demonstrate that CUG-containing RNAs have a wide deleterious effect and deregulate alternative splicing in many areas of the CNS. In addition to splicing abnormalities in cerebellum, DMSXL mice also displayed deficits in cerebellum-dependant motor coordination. Plus, DMSXL cerebellum showed electrophysiological abnormalities, suggesting cerebellar dysfunction and more precisely Purkinje cell dysfunction. In the search for the cellular populations showing the greatest susceptibility to RNA toxicity in the cerebellum, I have found extensive foci accumulation as well as pronounced splicing defects in the Bergman glia, surrounding Purkinje cells, in DMSXL and DM1 patients cerebellum. In order to identify molecular pathways and mechanisms behind the behaviour and electrophysiological abnormalities detected, I have performed a global proteomics approach and found a severe decrease in the expression of a glial glutamate transporter GLT1/EAAT2, suggesting that DM1 causes cerebellum dysfunction, through abnormal glutamate metabolism. Global proteomic analysis of DMSXL cerebellum also identified expression and post-translational changes of several proteins involved in calcium signalling. Missplicing of different transcripts involved in calcium metabolism reinforces the idea of calcium dysfunction in the neuropathogenesis of the DM1. To study the effects of toxic RNA on calcium homeostasis and flux, I have established and characterised a brain cell model of DM1. DMSXL primary astrocyte cultures allowed me to show the mislocalisation of the glutamate receptor GRIN1/NMDAR1, as well as abnormal calcium responses to stimulation. Despite recent therapeutic advances in muscle, we do not know the CNS efficiency of the therapeutic strategies currently being developed. To address this problem, I have used the DM1 astrocyte cell model to validate in cellulo a therapeutic strategy aiming to restore the activity of the endogenous splicing factor MBNL1. My thesis work provided a significant step in the understanding of the DM1 pathology in the CNS. My results revealed for the first time signs of cerebellum dysfunction in DM1, as well as signs of calcium homeostasis deregulation in the SNC. My work contributed to better understand the pathological mechanisms of DM1, the brain pathways and cell types most susceptible to toxic RNA. In the long term, my data will contribute to the rational development of therapeutic strategies targeting precise and deleterious molecular events

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics

International audienceWe present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional. Platelets are small anucleate cells that circulate in blood and are responsible for the arrest of bleeding. Platelets are formed by fragmentation of larger cells called megakaryocytes (MKs). Thrombocytopenia (insufficient platelet count) is a major condition, often requiring platelet transfusions. High collection costs, donor shortage, immuno-genicity, risk of contamination and storage issues represent the main limits of this therapeutic approach. People have tried to make artificial platelets but these objects seem promising for drug targeting rather than for therap

Toxic RNA affects astrocyte adhesion, spreading and migration in myotonic dystrophy, and impacts neuritogenesis through abnormal glial-neuronal interactions

International audienceMyotonic dystrophy type 1 (DM1) is a multisystemic condition that affects many tissues, as well as age groups. Compelling clinical evidence clearly demonstrates the impairment of the central nervous system (CNS), through cognitive/attention deficits, executive dysfunction, prevalent hypersomnia, behavioral changes and intellectual disability in the most severe cases. The neurological manifestations are highly debilitating and distressing for patients and their relatives, and there is no cure for this devastating condition.DM1 is caused by the abnormal expansion of a non-coding trinucleotide CTG repeats. Expanded CUG transcripts accumulate in toxic RNA aggregates or foci in the cell nucleus, perturbing the activity of key RNA-binding proteins and deregulating the splicing and general RNA metabolism of downstream targets. However, important gaps exist in our understanding of the disease mechanisms in the brain. In particular, we do not know the cell types primarily affected or the molecular pathways mostly dysregulated by the repeat expansion in the CNS. Using a transgenic mouse model of DM1 we found preferential accumulation of toxic RNA foci and missplicing in cortical astrocytes relative to neurons, pointing to glia pathology. We then used our DM1 mice as a source of primary neurons and astrocytes to resolve cell type-specific phenotypes and their associated molecular abnormalities. DM1 primary astrocytes show greater RNA foci accumulation and missplicing, relative to neurons, in association with defective cell growth, adhesion, cell spreading, polarization and migration. In contrast, the growth profile of DM1 primary neurons remained unaltered, but late neurite arborization was significantly impaired. Interestingly, defects in neuritogenesis were aggravated by the presence of DM1 mouse astrocytes in co-culture systems. To dissect the molecular mechanisms behind astrocyte dysfunction we performed global proteomics and transcriptomics approaches on homogenous astrocyte cultures. In line with the cell phenotypes described, we found relevant splicing and expression changes in critical regulators of cytoskeletal dynamics and cell adhesion.In conclusion, the DM1 repeat expansion has a deleterious impact on glia cell biology, which may in turn affect neuronal physiology through defective glial-neuronal crosstalk. Our results provide new insight into the cellular and molecular mechanisms of DM1 brain disease

RNA toxicity in myotonic dystrophy causes pronounced spliceopathy in astrocytes, in association with defective cell adhesion and morphology, erratic migration and impaired polarization

International audienceMyotonic dystrophy type 1 (DM1) is a severe multisystemic condition. The impairment of the central nervous system (CNS) is demonstrated by cognitive and attention deficits, executive dysfunction, prevalent hypersomnia, behavioral changes, as well as intellectual disability in the most severe cases. DM1 is caused by the abnormal expansion of a non-coding trinucleotide CTG repeat. Expanded CUG transcripts accumulate in toxic RNA aggregates (or foci) in the cell nucleus, which perturb primarily the regulation of alternative splicing. Important gaps still exist in our understanding of the disease mechanisms in the brain: we do not know the cell types and the molecular pathways most predominantly affected, and how they contribute to the onset of the debilitating neurological manifestations of DM1.Using a transgenic mouse model of DM1 we found preferential accumulation of toxic RNA foci and missplicing in cortical astrocytes, relative to neurons, pointing to glia cell pathology. We used our DM1 mice as a source of primary neurons and astrocytes to resolve cell type-specific disease mechanisms by RNA sequencing of homogenous cell cultures. DM1 mouse astrocytes confirmed greater RNA foci accumulation and showed critical missplicing of transcripts that regulate cell adhesion, cytoskeleton dynamics and cell morphogenesis. Astrocyte spliceopathy translated into defective cell adhesion, reduced spreading and erratic migration in culture, as well as decreased astrocyte ramification and aberrant reorientation in DM1 mouse brains. We confirmed the abnormal splicing of relevant transcripts in brain tissue from DM1 patients, and the defective spreading of human glia cells expressing toxic CUG RNA in culture.In conclusion, we have shown the CTG repeat expansion has a deleterious impact on glia cell biology, which may impair the glial-neuronal crosstalk and synaptic function in DM1 brains, contributing to cognitive and behavioural deficits. Our results provide new insight into the cellular and molecular mechanisms of DM1 brain disease

RNA toxicity in myotonic dystrophy causes pronounced spliceopathy in astrocytes, in association with defective cell adhesion and morphology, erratic migration and impaired polarization

Myotonic dystrophy type 1 (DM1) is a severe multisystemic condition. The impairment of the central nervous system (CNS) is demonstrated by cognitive and attention deficits, executive dysfunction, prevalent hypersomnia, behavioral changes, as well as intellectual disability in the most severe cases. DM1 is caused by the abnormal expansion of a non-coding trinucleotide CTG repeat. Expanded CUG transcripts accumulate in toxic RNA aggregates (or foci) in the cell nucleus, which perturb primarily the regulation of alternative splicing. Important gaps still exist in our understanding of the disease mechanisms in the brain: we do not know the cell types and the molecular pathways most predominantly affected, and how they contribute to the onset of the debilitating neurological manifestations of DM1.Using a transgenic mouse model of DM1 we found preferential accumulation of toxic RNA foci and missplicing in cortical astrocytes, relative to neurons, pointing to glia cell pathology. We used our DM1 mice as a source of primary neurons and astrocytes to resolve cell type-specific disease mechanisms by RNA sequencing of homogenous cell cultures. DM1 mouse astrocytes confirmed greater RNA foci accumulation and showed critical missplicing of transcripts that regulate cell adhesion, cytoskeleton dynamics and cell morphogenesis. Astrocyte spliceopathy translated into defective cell adhesion, reduced spreading and erratic migration in culture, as well as decreased astrocyte ramification and aberrant reorientation in DM1 mouse brains. We confirmed the abnormal splicing of relevant transcripts in brain tissue from DM1 patients, and the defective spreading of human glia cells expressing toxic CUG RNA in culture.In conclusion, we have shown the CTG repeat expansion has a deleterious impact on glia cell biology, which may impair the glial-neuronal crosstalk and synaptic function in DM1 brains, contributing to cognitive and behavioural deficits. Our results provide new insight into the cellular and molecular mechanisms of DM1 brain disease