Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neural cells

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects

Etude in vitro de l'impact de la drogue ABX sur les macrophages primaires humains issus de monocytes du sang périphérique dans un contexte inflammatoire : Implication du micro-ARN 124

Macrophages are heterogeneous cell mediators, which play a crucial role in inflammation. A chemical molecule ABX has been developed by the private company ABIVAX and is currently undergoing phase II clinical trials (ulcerative colitis, rheumatoid arthritis). In collaboration with the CNRS, it has been observed that drug ABX is a potent anti-inflammatory drug able to specifically induce the overexpression of the miR-124, described for its anti-inflammatory properties. The aim of my PhD project was to characterize in vitro the phenotypic modulations and molecular mechanisms involved by drug ABX, on Human Monocyte-Derived Macrophages (Hu MDM) of healthy donors in an inflammatory context, and pretreated with drug ABX versus control. The data obtained on six Hu MDM show on the one hand, a significant decrease in the membrane receptors of the M1 phenotype (CD86, CD274) and on the other hand, a significant decrease in the factors involved in the TLR4 signaling pathway (p65/RelA, IkBζ/NFKBIZ, TRAF6). In addition, the secretory profile shows a significant decrease of a set of pro-inflammatory proteins such as cytokines (IL-6, TNFα, IL-1β), proteins involved in cell adhesion and leukocyte recruitment (ICAM-1, CXCL1), as well as the chemokine responsible for the infiltration of macrophages (MCP-1/ CCL2). Remarkably, the data obtained with 14 Hu MDM, show a significant overexpression of micro-RNA 124 of 3.4 times that of the DMSO control. All of these data suggest that drug ABX limits excessive inflammation and could play a role in the control of cellular homeostasis. The overexpression of miR-124 is consistent with in vitro data obtained with T CD4 lymphocyte and human PBMC data, as well as clinical data from ulcerative colitis patients. For the first time, the laboratory identifies the immunomodulatory potential of drug ABX in inflammation. By inducing specific overexpression of miR-124, drug ABX attenuates the pro-inflammatory profile of macrophages by a negative feedback loop of the TLR4/NF-kB signaling. MiR-124 potentiates the homeostatic effect of drug ABX. This new chemical molecule ABX could represent a new therapeutic perspective, in order to rebalance some mediators involved in innate and adaptive immunity.Les macrophages sont des médiateurs cellulaires hétérogènes, dont le rôle est crucial dans l’inflammation. Une molécule chimique ABX a été développée par la société privée ABIVAX et fait actuellement l’objet d’essais cliniques de phase II (colite ulcéreuse, polyarthrite rhumatoïde). En collaboration avec le CNRS, il a été observé que cette drogue ABX est un puissant anti-inflammatoire capable d’induire spécifiquement la surexpression du miR-124, décrit pour ses propriétés anti-inflammatoires. L’objectif de mon projet de thèse a été de caractériser in vitro, les modulations phénotypiques et les mécanismes moléculaires mis en jeu par la drogue ABX, sur des macrophages humains dérivés de monocytes (Hu MDM) de donneurs sains dans un contexte inflammatoire, et prétraités par la drogue ABX versus contrôle. Les données obtenues sur 6 Hu MDM montrent d’une part, une diminution significative des récepteurs membranaires du phénotype M1 (CD86, CD274), et d’autre part, une diminution significative des facteurs impliqués dans la voie de signalisation du TLR4 (p65/RelA, IkBζ/NFKBIZ, TRAF6). De plus l’analyse du profil sécrétoire montre une diminution significative d’un ensemble de protéines pro-inflammatoires, à savoir : des cytokines (IL-6, TNFα, IL-1β), des protéines impliquées dans l’adhésion cellulaires et le recrutement des leucocytes (ICAM-1, CXCL1), ainsi que la chimiokine responsable de l’infiltration des macrophages (MCP-1/ CCL2). Fait remarquable, les données obtenues avec 14 HuMDM, montrent une surexpression significative du micro-ARN 124 de 3,4 fois par rapport au contrôle. Somme toute, l’ensemble de ces données suggère que la drogue ABX, limite l’excès d’inflammation, et jouerait un rôle dans le contrôle de l’homéostasie cellulaire. La surexpression du miR-124 est en concordance avec les données obtenues in vitro sur les lymphocytes T CD4 et les PBMC humains, mais également avec les données cliniques obtenues avec des patients atteints de colite ulcéreuse. Finalement, le laboratoire identifie, pour la première fois le potentiel immunorégulateur de la drogue ABX dans l’inflammation. En induisant la surexpression spécifique du miR-124, la drogue ABX, atténue le profil pro-inflammatoire des macrophages par un rétrocontrôle négatif de la voie TLR4/NF-kB. Le miR-124 potentialise l’effet homéostatique de la drogue ABX. Cette nouvelle molécule chimique ABX pourrait représenter une nouvelle perspective thérapeutique, visant à rééquilibrer certains médiateurs impliqués dans l’immunité innée et adaptative

In vitro study of the impact of drug ABX on primary human macrophages derived from peripheral blood monocytes in an inflammatory context : Involvement of microRNA 124

Les macrophages sont des médiateurs cellulaires hétérogènes, dont le rôle est crucial dans l’inflammation. Une molécule chimique ABX a été développée par la société privée ABIVAX et fait actuellement l’objet d’essais cliniques de phase II (colite ulcéreuse, polyarthrite rhumatoïde). En collaboration avec le CNRS, il a été observé que cette drogue ABX est un puissant anti-inflammatoire capable d’induire spécifiquement la surexpression du miR-124, décrit pour ses propriétés anti-inflammatoires. L’objectif de mon projet de thèse a été de caractériser in vitro, les modulations phénotypiques et les mécanismes moléculaires mis en jeu par la drogue ABX, sur des macrophages humains dérivés de monocytes (Hu MDM) de donneurs sains dans un contexte inflammatoire, et prétraités par la drogue ABX versus contrôle. Les données obtenues sur 6 Hu MDM montrent d’une part, une diminution significative des récepteurs membranaires du phénotype M1 (CD86, CD274), et d’autre part, une diminution significative des facteurs impliqués dans la voie de signalisation du TLR4 (p65/RelA, IkBζ/NFKBIZ, TRAF6). De plus l’analyse du profil sécrétoire montre une diminution significative d’un ensemble de protéines pro-inflammatoires, à savoir : des cytokines (IL-6, TNFα, IL-1β), des protéines impliquées dans l’adhésion cellulaires et le recrutement des leucocytes (ICAM-1, CXCL1), ainsi que la chimiokine responsable de l’infiltration des macrophages (MCP-1/ CCL2). Fait remarquable, les données obtenues avec 14 HuMDM, montrent une surexpression significative du micro-ARN 124 de 3,4 fois par rapport au contrôle. Somme toute, l’ensemble de ces données suggère que la drogue ABX, limite l’excès d’inflammation, et jouerait un rôle dans le contrôle de l’homéostasie cellulaire. La surexpression du miR-124 est en concordance avec les données obtenues in vitro sur les lymphocytes T CD4 et les PBMC humains, mais également avec les données cliniques obtenues avec des patients atteints de colite ulcéreuse. Finalement, le laboratoire identifie, pour la première fois le potentiel immunorégulateur de la drogue ABX dans l’inflammation. En induisant la surexpression spécifique du miR-124, la drogue ABX, atténue le profil pro-inflammatoire des macrophages par un rétrocontrôle négatif de la voie TLR4/NF-kB. Le miR-124 potentialise l’effet homéostatique de la drogue ABX. Cette nouvelle molécule chimique ABX pourrait représenter une nouvelle perspective thérapeutique, visant à rééquilibrer certains médiateurs impliqués dans l’immunité innée et adaptative.Macrophages are heterogeneous cell mediators, which play a crucial role in inflammation. A chemical molecule ABX has been developed by the private company ABIVAX and is currently undergoing phase II clinical trials (ulcerative colitis, rheumatoid arthritis). In collaboration with the CNRS, it has been observed that drug ABX is a potent anti-inflammatory drug able to specifically induce the overexpression of the miR-124, described for its anti-inflammatory properties. The aim of my PhD project was to characterize in vitro the phenotypic modulations and molecular mechanisms involved by drug ABX, on Human Monocyte-Derived Macrophages (Hu MDM) of healthy donors in an inflammatory context, and pretreated with drug ABX versus control. The data obtained on six Hu MDM show on the one hand, a significant decrease in the membrane receptors of the M1 phenotype (CD86, CD274) and on the other hand, a significant decrease in the factors involved in the TLR4 signaling pathway (p65/RelA, IkBζ/NFKBIZ, TRAF6). In addition, the secretory profile shows a significant decrease of a set of pro-inflammatory proteins such as cytokines (IL-6, TNFα, IL-1β), proteins involved in cell adhesion and leukocyte recruitment (ICAM-1, CXCL1), as well as the chemokine responsible for the infiltration of macrophages (MCP-1/ CCL2). Remarkably, the data obtained with 14 Hu MDM, show a significant overexpression of micro-RNA 124 of 3.4 times that of the DMSO control. All of these data suggest that drug ABX limits excessive inflammation and could play a role in the control of cellular homeostasis. The overexpression of miR-124 is consistent with in vitro data obtained with T CD4 lymphocyte and human PBMC data, as well as clinical data from ulcerative colitis patients. For the first time, the laboratory identifies the immunomodulatory potential of drug ABX in inflammation. By inducing specific overexpression of miR-124, drug ABX attenuates the pro-inflammatory profile of macrophages by a negative feedback loop of the TLR4/NF-kB signaling. MiR-124 potentiates the homeostatic effect of drug ABX. This new chemical molecule ABX could represent a new therapeutic perspective, in order to rebalance some mediators involved in innate and adaptive immunity

Preparing sequencing grade RNAs from a small number of FACS-sorted larvae macrophages isolated from enzyme free dissociated zebrafish larvae

International audienceMacrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages.•An optimized protocol in 5 steps to extract high quality RNAs from zebrafish macrophages.•A cell dissociation method using an enzyme-free and osmotically controlled buffer to prevent the alteration of macrophage activation status and limit cell mortality.•Production of high integrity RNAs from a small number of isolated macrophages

A novel function for Cyclin A2: control of cell invasion via RhoA signaling.

International audienceCyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium

International audienceCDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also phosphorylates the C-terminal domain of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell proliferation and differentiation is unknown. Here, we find in mice that CDK8 is dispensable for regulation of gene expression, normal intestinal homeostasis, and efficient tumourigenesis, and is largely redundant with CDK19 in the control of gene expression. Their combined deletion in intestinal organoids reduces long-term proliferative capacity but is not lethal and allows differentiation. However, double-mutant organoids show mucus accumulation and increased secretion by goblet cells, as well as downregulation of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and functionality of the CFTR pathway. Pharmacological inhibition of CDK8/19 kinase activity in organoids and in mice recapitulates several of these phenotypes. Thus, the Mediator kinases are not essential for cell proliferation and differentiation in an adult tissue, but they cooperate to regulate specific transcriptional programmes

Frozen versus fresh embryos for ES cell microinjections : a comparative study with BALB/cN and C57BL/6N strains from seven transgenic facilities

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CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium

Abstract CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also promotes transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell differentiation is unknown. Here, we find that CDK8 is dispensable for RNA polII CTD phosphorylation, regulation of gene expression, normal intestinal homeostasis and efficient tumourigenesis in mice. Furthermore, CDK8 is largely redundant with CDK19 in the control of gene expression. Yet, while their combined deletion in intestinal organoids reduces long-term proliferative capacity, it is not lethal and allows differentiation. Nevertheless, in double mutant organoids, the cystic fibrosis transmembrane conductance regulator (CFTR) pathway is transcriptionally and functionally downregulated, leading to mucus accumulation and increased secretion by goblet cells. This phenotype can be recapitulated by pharmacological inhibition of CDK8/19 kinase activity. Thus, the Mediator kinases are not essential for cell proliferation and differentiation, but they cooperate to regulate tissue-specific transcriptional programmes

Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neurons

Abstract Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects