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Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders / L. S. NGUYEN in Molecular Autism, 9 (2018)
[article]
Titre : Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders Type de document : Texte imprimé et/ou numérique Auteurs : L. S. NGUYEN, Auteur ; J. FREGEAC, Auteur ; C. BOLE-FEYSOT, Auteur ; N. CAGNARD, Auteur ; A. IYER, Auteur ; J. ANINK, Auteur ; E. ARONICA, Auteur ; O. ALIBEU, Auteur ; P. NITSCHKE, Auteur ; L. COLLEAUX, Auteur Article en page(s) : 38p. Langues : Anglais (eng) Mots-clés : Autism Spectrum Disorder/genetics/metabolism Cell Line Cell Lineage Child Female Humans Male MicroRNAs/genetics/metabolism Neural Stem Cells/cytology/metabolism Neurogenesis Temporal Lobe/cytology/metabolism Up-Regulation Autism spectrum disorders Human neural stem cell Transcriptome microRNA Index. décimale : PER Périodiques Résumé : Background: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have emerged as important modulators of brain development and neuronal function and are implicated in several neurological diseases. Previous studies found miR-146a upregulation is the most common miRNA deregulation event in neurodevelopmental disorders such as autism spectrum disorder (ASD), epilepsy, and intellectual disability (ID). Yet, how miR-146a upregulation affects the developing fetal brain remains unclear. Methods: We analyzed the expression of miR-146a in the temporal lobe of ASD children using Taqman assay. To assess the role of miR-146a in early brain development, we generated and characterized stably induced H9 human neural stem cell (H9 hNSC) overexpressing miR-146a using various cell and molecular biology techniques. Results: We first showed that miR-146a upregulation occurs early during childhood in the ASD brain. In H9 hNSC, miR-146a overexpression enhances neurite outgrowth and branching and favors differentiation into neuronal like cells. Expression analyses revealed that 10% of the transcriptome was deregulated and organized into two modules critical for cell cycle control and neuronal differentiation. Twenty known or predicted targets of miR-146a were significantly deregulated in the modules, acting as potential drivers. The two modules also display distinct transcription profiles during human brain development, affecting regions relevant for ASD including the neocortex, amygdala, and hippocampus. Cell type analyses indicate markers for pyramidal, and interneurons are highly enriched in the deregulated gene list. Up to 40% of known markers of newly defined neuronal lineages were deregulated, suggesting that miR-146a could participate also in the acquisition of neuronal identities. Conclusion: Our results demonstrate the dynamic roles of miR-146a in early neuronal development and provide new insight into the molecular events that link miR-146a overexpression to impaired neurodevelopment. This, in turn, may yield new therapeutic targets and strategies. En ligne : https://dx.doi.org/10.1186/s13229-018-0219-3 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=371
in Molecular Autism > 9 (2018) . - 38p.[article] Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders [Texte imprimé et/ou numérique] / L. S. NGUYEN, Auteur ; J. FREGEAC, Auteur ; C. BOLE-FEYSOT, Auteur ; N. CAGNARD, Auteur ; A. IYER, Auteur ; J. ANINK, Auteur ; E. ARONICA, Auteur ; O. ALIBEU, Auteur ; P. NITSCHKE, Auteur ; L. COLLEAUX, Auteur . - 38p.
Langues : Anglais (eng)
in Molecular Autism > 9 (2018) . - 38p.
Mots-clés : Autism Spectrum Disorder/genetics/metabolism Cell Line Cell Lineage Child Female Humans Male MicroRNAs/genetics/metabolism Neural Stem Cells/cytology/metabolism Neurogenesis Temporal Lobe/cytology/metabolism Up-Regulation Autism spectrum disorders Human neural stem cell Transcriptome microRNA Index. décimale : PER Périodiques Résumé : Background: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have emerged as important modulators of brain development and neuronal function and are implicated in several neurological diseases. Previous studies found miR-146a upregulation is the most common miRNA deregulation event in neurodevelopmental disorders such as autism spectrum disorder (ASD), epilepsy, and intellectual disability (ID). Yet, how miR-146a upregulation affects the developing fetal brain remains unclear. Methods: We analyzed the expression of miR-146a in the temporal lobe of ASD children using Taqman assay. To assess the role of miR-146a in early brain development, we generated and characterized stably induced H9 human neural stem cell (H9 hNSC) overexpressing miR-146a using various cell and molecular biology techniques. Results: We first showed that miR-146a upregulation occurs early during childhood in the ASD brain. In H9 hNSC, miR-146a overexpression enhances neurite outgrowth and branching and favors differentiation into neuronal like cells. Expression analyses revealed that 10% of the transcriptome was deregulated and organized into two modules critical for cell cycle control and neuronal differentiation. Twenty known or predicted targets of miR-146a were significantly deregulated in the modules, acting as potential drivers. The two modules also display distinct transcription profiles during human brain development, affecting regions relevant for ASD including the neocortex, amygdala, and hippocampus. Cell type analyses indicate markers for pyramidal, and interneurons are highly enriched in the deregulated gene list. Up to 40% of known markers of newly defined neuronal lineages were deregulated, suggesting that miR-146a could participate also in the acquisition of neuronal identities. Conclusion: Our results demonstrate the dynamic roles of miR-146a in early neuronal development and provide new insight into the molecular events that link miR-146a overexpression to impaired neurodevelopment. This, in turn, may yield new therapeutic targets and strategies. En ligne : https://dx.doi.org/10.1186/s13229-018-0219-3 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=371 CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons / Y. ZHOU in Molecular Autism, 7 (2016)
[article]
Titre : CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons Type de document : Texte imprimé et/ou numérique Auteurs : Y. ZHOU, Auteur ; D. KUMARI, Auteur ; N. SCIASCIA, Auteur ; K. USDIN, Auteur Article en page(s) : 42p. Langues : Anglais (eng) Mots-clés : 5' Untranslated Regions Alleles Cell Differentiation Cell Line DNA Methylation Embryonic Stem Cells/metabolism/pathology Fragile X Mental Retardation Protein/genetics/metabolism Fragile X Syndrome/genetics/metabolism/pathology Gene Silencing Humans Induced Pluripotent Stem Cells/metabolism/pathology Male Neurons/metabolism/pathology Primary Cell Culture Time Factors Trinucleotide Repeat Expansion Fragile X syndrome Repeat contractions Repeat expansion mutation Repeat-mediated gene silencing Stem cells Index. décimale : PER Périodiques Résumé : BACKGROUND: Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. METHODS: We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. RESULTS: We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. CONCLUSIONS: Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats. En ligne : http://dx.doi.org/10.1186/s13229-016-0105-9 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=329
in Molecular Autism > 7 (2016) . - 42p.[article] CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons [Texte imprimé et/ou numérique] / Y. ZHOU, Auteur ; D. KUMARI, Auteur ; N. SCIASCIA, Auteur ; K. USDIN, Auteur . - 42p.
Langues : Anglais (eng)
in Molecular Autism > 7 (2016) . - 42p.
Mots-clés : 5' Untranslated Regions Alleles Cell Differentiation Cell Line DNA Methylation Embryonic Stem Cells/metabolism/pathology Fragile X Mental Retardation Protein/genetics/metabolism Fragile X Syndrome/genetics/metabolism/pathology Gene Silencing Humans Induced Pluripotent Stem Cells/metabolism/pathology Male Neurons/metabolism/pathology Primary Cell Culture Time Factors Trinucleotide Repeat Expansion Fragile X syndrome Repeat contractions Repeat expansion mutation Repeat-mediated gene silencing Stem cells Index. décimale : PER Périodiques Résumé : BACKGROUND: Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. METHODS: We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. RESULTS: We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. CONCLUSIONS: Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats. En ligne : http://dx.doi.org/10.1186/s13229-016-0105-9 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=329 Increased expression of the PI3K catalytic subunit p110delta underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family / A. C. POOPAL in Molecular Autism, 7 (2016)
[article]
Titre : Increased expression of the PI3K catalytic subunit p110delta underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family Type de document : Texte imprimé et/ou numérique Auteurs : A. C. POOPAL, Auteur ; L. M. SCHROEDER, Auteur ; P. S. HORN, Auteur ; Gary J. BASSELL, Auteur ; C. GROSS, Auteur Article en page(s) : 3p. Langues : Anglais (eng) Mots-clés : Adenine/analogs & derivatives/pharmacology Autistic Disorder/enzymology/genetics/pathology Biomarkers Cell Line Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors/biosynthesis/genetics/physiology Diseases in Twins Enzyme-Linked Immunosorbent Assay Family Health Female Humans Lymphocytes/enzymology Male Molecular Targeted Therapy Nerve Tissue Proteins/genetics/metabolism Phosphorylation Protein Processing, Post-Translational Quinazolines/pharmacology Ribosomal Protein S6 Kinases/metabolism Signal Transduction/genetics TOR Serine-Threonine Kinases/physiology Autism Biomarker Ic87114 PI3K/mTOR signaling S6 phosphorylation p110delta Index. décimale : PER Périodiques Résumé : BACKGROUND: Dysfunctions in the PI3K/mTOR pathway have gained a lot of attention in autism research. This was initially based on the discovery of several monogenic autism spectrum disorders with mutations or defects in PI3K/mTOR signaling components. Recent genetic studies corroborate that defective PI3K/mTOR signaling might be a shared pathomechanism in autism disorders of so far unknown etiology, but functional molecular analyses in human cells are rare. The goals of this study were to perform a functional screen of cell lines from patients with idiopathic autism for defects in PI3K/mTOR signaling, to test if further functional analyses are suitable to detect underlying molecular mechanisms, and to evaluate this approach as a biomarker tool to identify therapeutic targets. METHODS: We performed phospho-S6- and S6-specific ELISA experiments on 21 lymphoblastoid cell lines from the AGRE collection and on 37 lymphoblastoid cell lines from the Simons Simplex Collection and their healthy siblings. Cell lines from one individual with increased S6 phosphorylation and his multiplex family were analyzed in further detail to identify upstream defects in PI3K signaling associated with autism diagnosis. RESULTS: We detected significantly increased S6 phosphorylation in 3 of the 21 lymphoblastoid cell lines from AGRE compared to a healthy control and in 1 of the 37 lymphoblastoid cell lines from the Simons Simplex Collection compared to the healthy sibling. Further analysis of cells from one individual with elevated S6 phosphorylation showed increased expression of the PI3K catalytic subunit p110delta, which was also observed in lymphoblastoid cells from other autistic siblings but not unaffected members in his multiplex family. The p110delta-selective inhibitor IC87114 reduced elevated S6 phosphorylation and protein synthesis in this cell line. CONCLUSIONS: Our results suggest that functional analysis of PI3K/mTOR signaling is a biomarker tool to identify disease-associated molecular defects that could serve as therapeutic targets in autism. Using this approach, we discovered impaired signaling and protein synthesis through the PI3K catalytic subunit p110delta as an underlying molecular defect and potential treatment target in select autism spectrum disorders. Increased p110delta activity was recently associated with schizophrenia, and our results suggest that p110delta may also be implicated in autism. En ligne : http://dx.doi.org/10.1186/s13229-015-0066-4 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=329
in Molecular Autism > 7 (2016) . - 3p.[article] Increased expression of the PI3K catalytic subunit p110delta underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family [Texte imprimé et/ou numérique] / A. C. POOPAL, Auteur ; L. M. SCHROEDER, Auteur ; P. S. HORN, Auteur ; Gary J. BASSELL, Auteur ; C. GROSS, Auteur . - 3p.
Langues : Anglais (eng)
in Molecular Autism > 7 (2016) . - 3p.
Mots-clés : Adenine/analogs & derivatives/pharmacology Autistic Disorder/enzymology/genetics/pathology Biomarkers Cell Line Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors/biosynthesis/genetics/physiology Diseases in Twins Enzyme-Linked Immunosorbent Assay Family Health Female Humans Lymphocytes/enzymology Male Molecular Targeted Therapy Nerve Tissue Proteins/genetics/metabolism Phosphorylation Protein Processing, Post-Translational Quinazolines/pharmacology Ribosomal Protein S6 Kinases/metabolism Signal Transduction/genetics TOR Serine-Threonine Kinases/physiology Autism Biomarker Ic87114 PI3K/mTOR signaling S6 phosphorylation p110delta Index. décimale : PER Périodiques Résumé : BACKGROUND: Dysfunctions in the PI3K/mTOR pathway have gained a lot of attention in autism research. This was initially based on the discovery of several monogenic autism spectrum disorders with mutations or defects in PI3K/mTOR signaling components. Recent genetic studies corroborate that defective PI3K/mTOR signaling might be a shared pathomechanism in autism disorders of so far unknown etiology, but functional molecular analyses in human cells are rare. The goals of this study were to perform a functional screen of cell lines from patients with idiopathic autism for defects in PI3K/mTOR signaling, to test if further functional analyses are suitable to detect underlying molecular mechanisms, and to evaluate this approach as a biomarker tool to identify therapeutic targets. METHODS: We performed phospho-S6- and S6-specific ELISA experiments on 21 lymphoblastoid cell lines from the AGRE collection and on 37 lymphoblastoid cell lines from the Simons Simplex Collection and their healthy siblings. Cell lines from one individual with increased S6 phosphorylation and his multiplex family were analyzed in further detail to identify upstream defects in PI3K signaling associated with autism diagnosis. RESULTS: We detected significantly increased S6 phosphorylation in 3 of the 21 lymphoblastoid cell lines from AGRE compared to a healthy control and in 1 of the 37 lymphoblastoid cell lines from the Simons Simplex Collection compared to the healthy sibling. Further analysis of cells from one individual with elevated S6 phosphorylation showed increased expression of the PI3K catalytic subunit p110delta, which was also observed in lymphoblastoid cells from other autistic siblings but not unaffected members in his multiplex family. The p110delta-selective inhibitor IC87114 reduced elevated S6 phosphorylation and protein synthesis in this cell line. CONCLUSIONS: Our results suggest that functional analysis of PI3K/mTOR signaling is a biomarker tool to identify disease-associated molecular defects that could serve as therapeutic targets in autism. Using this approach, we discovered impaired signaling and protein synthesis through the PI3K catalytic subunit p110delta as an underlying molecular defect and potential treatment target in select autism spectrum disorders. Increased p110delta activity was recently associated with schizophrenia, and our results suggest that p110delta may also be implicated in autism. En ligne : http://dx.doi.org/10.1186/s13229-015-0066-4 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=329