Interactions neurogliales dans la déficience intellectuelle : étude du modèle oligophrénine-1

The synapse mediates the inter-neuron communication that forms the basis of all cognitive activity. Mutations in genes encoding for synaptic proteins are responsible for neurodevelopmental disorders called synaptopathies, covering a large clinical spectrum from intellectual disability (ID) to autism spectrum disorders. However it is currently established that neurons are not the only active cells at the synapse. Astrocytes play as well an essential role for its development and functioning. They maintain synaptic ionic homeostasis and are capable of secreting gliotransmitters, which can modulate synaptic activity. Oligophrenin-1 gene (OPHN1) was identified and associated with X-linked human ID. OPHN1 is a synaptic protein, which neuronal function is well known. It can directly interact with the actin-cytoskeleton and plays a role in the formation and maturation of dendritic spines. This protein is also expressed in astrocytes but its astrocytic function is still unknown. Using the Ophn1 KO mouse model, we were able to highlight in vitro the consequences induced by its deletion in astrocytes. We demonstrated that the absence of OPHN1 disturbed astrocytic migration and morphology in vitro. Since Ophn1 negatively regulates RhoA GTPase, we used an inhibitor of the RhoA/ROCK pathway to rescue the phenotype back to control. In vivo we took advantage of the cortical glial scar formation to observ astrocytic migration and morphology in KO mice. We found that Ophn1 deletion disrupted glial scar formation and that astrocytes near the wound were less ramified. Taken together, these results show that astrocytes are altered in our mouse model of X-linked ID. Moreover the development of an astrocytic conditional KO for Ophn1 will allow us to study the consequences of astrocytic loss of OPHN1 and determine the astrocytic contribution in the pathophysiology of this neurodevelopmental disease.La synapse est le lieu de communication entre les neurones à l'origine de nos capacités cognitives. Les mutations des gènes codant pour des protéines synaptiques sont responsables des maladies neurodéveloppementales appelées synaptopathies, recouvrant un large spectre de pathologies, de la déficience intellectuelle aux troubles du spectre autistique. Cependant, il est actuellement établi que les neurones ne sont pas les seuls acteurs au niveau de la synapse. Les astrocytes jouent également un rôle essentiel dans la mise en place du réseau neuronal et le fonctionnement de la synapse. Ils assurent aussi l'homéostasie ionique synaptique et sont capables de sécréter des glio-transmetteurs qui modulent l'activité synaptique. Oligophrénine-1 (OPHN1) est un gène associé à la déficience intellectuelle liée à l'X chez l'Homme. OPHN1 est une protéine synaptique dont les fonctions neuronales sont bien connues. La protéine peut directement interagir avec le cytosquelette d'actine et joue un rôle dans la formation et la maturation des épines dendritiques. Cette protéine est aussi exprimée dans les astrocytes mais sa fonction astrocytaire n'est pas connue. A l'aide d'un modèle KO de souris pour Ophn1, nous avons mis en évidence les conséquences de l'absence d'Ophn1 dans les astrocytes. Nous avons démontré que la délétion d'OPHN1 altère la migration et la morphologie des astrocytes in vitro. Sachant qu'Ophn1 est capable d'inactiver la GTPase RhoA, nous avons utilisé un inhibiteur de la voie RhoA/ROCK pour retrouver un phénotype de migration normal. In vivo nous avons choisi un modèle de cicatrisation gliale cortical afin de pouvoir observer la migration et la morphologie des astrocytes au niveau de la cicatrice. Nous avons observé que la délétion d'Ophn1 altérait la cicatrisation gliale et que les astrocytes à proximité de la cicatrice était moins ramifiés. L'ensemble de ces résultats nous permet de constater que les astrocytes sont altérés dans notre modèle murin de déficience intellectuelle liée à l'X. De plus, le KO conditionel astrocytaire mis en place nous permettra à l'avenir d'étudier les conséquences de la perte d'OPHN1 uniquement dans les astrocytes, et de comprendre la contribution astrocytaire dans la physiopathologie de cette maladie neuro-développementale

Astroglial contribution to synaptophaties : the oligophrenin1 gene model

La synapse est le lieu de communication entre les neurones à l'origine de nos capacités cognitives. Les mutations des gènes codant pour des protéines synaptiques sont responsables des maladies neurodéveloppementales appelées synaptopathies, recouvrant un large spectre de pathologies, de la déficience intellectuelle aux troubles du spectre autistique. Cependant, il est actuellement établi que les neurones ne sont pas les seuls acteurs au niveau de la synapse. Les astrocytes jouent également un rôle essentiel dans la mise en place du réseau neuronal et le fonctionnement de la synapse. Ils assurent aussi l'homéostasie ionique synaptique et sont capables de sécréter des glio-transmetteurs qui modulent l'activité synaptique. Oligophrénine-1 (OPHN1) est un gène associé à la déficience intellectuelle liée à l'X chez l'Homme. OPHN1 est une protéine synaptique dont les fonctions neuronales sont bien connues. La protéine peut directement interagir avec le cytosquelette d'actine et joue un rôle dans la formation et la maturation des épines dendritiques. Cette protéine est aussi exprimée dans les astrocytes mais sa fonction astrocytaire n'est pas connue. A l'aide d'un modèle KO de souris pour Ophn1, nous avons mis en évidence les conséquences de l'absence d'Ophn1 dans les astrocytes. Nous avons démontré que la délétion d'OPHN1 altère la migration et la morphologie des astrocytes in vitro. Sachant qu'Ophn1 est capable d'inactiver la GTPase RhoA, nous avons utilisé un inhibiteur de la voie RhoA/ROCK pour retrouver un phénotype de migration normal. In vivo nous avons choisi un modèle de cicatrisation gliale cortical afin de pouvoir observer la migration et la morphologie des astrocytes au niveau de la cicatrice. Nous avons observé que la délétion d'Ophn1 altérait la cicatrisation gliale et que les astrocytes à proximité de la cicatrice était moins ramifiés. L'ensemble de ces résultats nous permet de constater que les astrocytes sont altérés dans notre modèle murin de déficience intellectuelle liée à l'X. De plus, le KO conditionel astrocytaire mis en place nous permettra à l'avenir d'étudier les conséquences de la perte d'OPHN1 uniquement dans les astrocytes, et de comprendre la contribution astrocytaire dans la physiopathologie de cette maladie neuro-développementale.The synapse mediates the inter-neuron communication that forms the basis of all cognitive activity. Mutations in genes encoding for synaptic proteins are responsible for neurodevelopmental disorders called synaptopathies, covering a large clinical spectrum from intellectual disability (ID) to autism spectrum disorders. However it is currently established that neurons are not the only active cells at the synapse. Astrocytes play as well an essential role for its development and functioning. They maintain synaptic ionic homeostasis and are capable of secreting gliotransmitters, which can modulate synaptic activity. Oligophrenin-1 gene (OPHN1) was identified and associated with X-linked human ID. OPHN1 is a synaptic protein, which neuronal function is well known. It can directly interact with the actin-cytoskeleton and plays a role in the formation and maturation of dendritic spines. This protein is also expressed in astrocytes but its astrocytic function is still unknown. Using the Ophn1 KO mouse model, we were able to highlight in vitro the consequences induced by its deletion in astrocytes. We demonstrated that the absence of OPHN1 disturbed astrocytic migration and morphology in vitro. Since Ophn1 negatively regulates RhoA GTPase, we used an inhibitor of the RhoA/ROCK pathway to rescue the phenotype back to control. In vivo we took advantage of the cortical glial scar formation to observ astrocytic migration and morphology in KO mice. We found that Ophn1 deletion disrupted glial scar formation and that astrocytes near the wound were less ramified. Taken together, these results show that astrocytes are altered in our mouse model of X-linked ID. Moreover the development of an astrocytic conditional KO for Ophn1 will allow us to study the consequences of astrocytic loss of OPHN1 and determine the astrocytic contribution in the pathophysiology of this neurodevelopmental disease

Do Astrocytes Play a Role in Intellectual Disabilities?

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The intellectual disability protein Oligophrenin1 controls astrocyte morphology and migration: Structural and migratory alterations in astrocytes deficient for OPHN1 via the ROCK pathway

International audienceAstrocytes are involved in several aspects of neuronal development and properties which are altered in intellectual disability (ID). Oligophrenin-1 is a RhoGAP protein implicated in actin cytoskeleton regulation, and whose mutations are associated with X-linked ID. Oligophrenin-1 is expressed in neurons, where its functions have been widely reported at the synapse, as well as in glial cells. However, its roles in astrocytes are still largely unexplored. Using in vitro and in vivo models of oligophrenin1 disruption in astrocytes, we found that oligophrenin1 regulates at the molecular level the RhoA/ROCK/MLC2 pathway in astroglial cells. We also showed at the cellular level that oligophrenin1 modulates astrocyte morphology and migration both in vitro and in vivo, and is involved in glial scar formation. Altogether, these data suggest that oligophrenin1 deficiency alters not only neuronal but also astrocytic functions, which might contribute to the development of ID

Connexin 30 controls astroglial polarization during postnatal brain development

International audienceAstrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates in situ the extension and ramification of astroglial processes. However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an in vitro wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/β1 integrin/ Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the β1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. In vivo, connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development

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

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

International audienceIntroduction: Brain dysfunction in neurological diseases is frequently mediated by the impairment of neuronal and non-neuronal cells. Although DMPK gene expression is higher in cortical astrocytes than in neurons isolated from adult human and mouse brains, the contribution of astroglia to DM1 brain disease has been poorly investigated. Methods: Transgenic DMSXL mice express expanded human DMPK transcripts in multiple cell types of the brain, providing a good model to investigate the impact of RNA toxicity on astroglia.Results: DMSXL astrocytes exhibit impaired ramification and polarization in vivo, as well as defects in adhesion, spreading and migration in culture. In line with these pronounced phenotypes, DMSXL astrocytes express high levels of toxic RNA and accumulate abundant RNA foci, relative to neurons. RNA sequencing revealed MBNL-dependent RNA spliceopathy, which affects primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis. To study the impact of defective astrocytes on neurons, we used co-culture cell systems, and found that DMSXL astrocytes impair neuritogenesis.Conclusions: We demonstrate that DM1 impacts astrocyte cell biology, possibly compromising the support and regulation of synaptic function through defective neuroglia interplay

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

International audienceIntroduction: Brain dysfunction in neurological diseases is frequently mediated by the impairment of neuronal and non-neuronal cells. Although DMPK gene expression is higher in cortical astrocytes than in neurons isolated from adult human and mouse brains, the contribution of astroglia to DM1 brain disease has been poorly investigated. Methods: Transgenic DMSXL mice express expanded human DMPK transcripts in multiple cell types of the brain, providing a good model to investigate the impact of RNA toxicity on astroglia.Results: DMSXL astrocytes exhibit impaired ramification and polarization in vivo, as well as defects in adhesion, spreading and migration in culture. In line with these pronounced phenotypes, DMSXL astrocytes express high levels of toxic RNA and accumulate abundant RNA foci, relative to neurons. RNA sequencing revealed MBNL-dependent RNA spliceopathy, which affects primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis. To study the impact of defective astrocytes on neurons, we used co-culture cell systems, and found that DMSXL astrocytes impair neuritogenesis.Conclusions: We demonstrate that DM1 impacts astrocyte cell biology, possibly compromising the support and regulation of synaptic function through defective neuroglia interplay

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Abstract Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types, we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading and migration. RNA-dependent toxicity and phenotypes was also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses revealed extensive expression and accumulation of toxic RNA in astrocytes, which resulted in RNA spliceopathy that was remarkably more severe than in neurons. Astrocyte missplicing affected primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis, and it was confirmed in human brain tissue. We demonstrate for the first time that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function