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RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome / N. L. PACHECO in Molecular Autism, 8 (2017)
[article]
Titre : RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome Type de document : Texte imprimé et/ou numérique Auteurs : N. L. PACHECO, Auteur ; M. R. HEAVEN, Auteur ; L. M. HOLT, Auteur ; D. K. CROSSMAN, Auteur ; K. J. BOGGIO, Auteur ; S. A. SHAFFER, Auteur ; D. L. FLINT, Auteur ; M. L. OLSEN, Auteur Article en page(s) : 56p. Langues : Anglais (eng) Mots-clés : Multi-cellular deficits Proteome Rett syndrome Transcriptome Index. décimale : PER Périodiques Résumé : BACKGROUND: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. METHODS: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity(R) Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. RESULTS: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. CONCLUSIONS: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT. En ligne : http://dx.doi.org/10.1186/s13229-017-0174-4 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=330
in Molecular Autism > 8 (2017) . - 56p.[article] RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome [Texte imprimé et/ou numérique] / N. L. PACHECO, Auteur ; M. R. HEAVEN, Auteur ; L. M. HOLT, Auteur ; D. K. CROSSMAN, Auteur ; K. J. BOGGIO, Auteur ; S. A. SHAFFER, Auteur ; D. L. FLINT, Auteur ; M. L. OLSEN, Auteur . - 56p.
Langues : Anglais (eng)
in Molecular Autism > 8 (2017) . - 56p.
Mots-clés : Multi-cellular deficits Proteome Rett syndrome Transcriptome Index. décimale : PER Périodiques Résumé : BACKGROUND: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. METHODS: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity(R) Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. RESULTS: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. CONCLUSIONS: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT. En ligne : http://dx.doi.org/10.1186/s13229-017-0174-4 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=330 Synaptic vesicle dynamic changes in a model of fragile X / Jantine A.C. BROEK in Molecular Autism, 7 (2016)
[article]
Titre : Synaptic vesicle dynamic changes in a model of fragile X Type de document : Texte imprimé et/ou numérique Auteurs : Jantine A.C. BROEK, Auteur ; Z. LIN, Auteur ; H. M. DE GRUITER, Auteur ; H. VAN 'T SPIJKER, Auteur ; E. D. HAASDIJK, Auteur ; David COX, Auteur ; S. OZCAN, Auteur ; G. W. A. VAN CAPPELLEN, Auteur ; A. B. HOUTSMULLER, Auteur ; R. WILLEMSEN, Auteur ; C. I. DE ZEEUW, Auteur ; S. BAHN, Auteur Article en page(s) : 17p. Langues : Anglais (eng) Mots-clés : Animals Animals, Congenic Cells, Cultured Cerebellum/pathology/physiopathology Fluorescent Dyes Fragile X Mental Retardation Protein/genetics/physiology Fragile X Syndrome/genetics/metabolism/physiopathology Hippocampus/pathology/physiopathology Intravital Microscopy Male Mass Spectrometry/methods Mice Mice, Inbred C57BL Mice, Knockout Mice, Neurologic Mutants Microscopy, Electron Models, Animal Nerve Tissue Proteins/analysis Presynaptic Terminals/secretion Proteome Purkinje Cells/physiology/ultrastructure Pyridinium Compounds Quaternary Ammonium Compounds Signal Transduction Synaptic Transmission Synaptic Vesicles/metabolism Synaptosomes/metabolism Electron microscopy Fragile X syndrome (FXS) Mass spectrometry (MS) Quantitative live-cell imaging Index. décimale : PER Périodiques Résumé : BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. METHODS: Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. RESULTS: Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. CONCLUSIONS: Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS. En ligne : http://dx.doi.org/10.1186/s13229-016-0080-1 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=328
in Molecular Autism > 7 (2016) . - 17p.[article] Synaptic vesicle dynamic changes in a model of fragile X [Texte imprimé et/ou numérique] / Jantine A.C. BROEK, Auteur ; Z. LIN, Auteur ; H. M. DE GRUITER, Auteur ; H. VAN 'T SPIJKER, Auteur ; E. D. HAASDIJK, Auteur ; David COX, Auteur ; S. OZCAN, Auteur ; G. W. A. VAN CAPPELLEN, Auteur ; A. B. HOUTSMULLER, Auteur ; R. WILLEMSEN, Auteur ; C. I. DE ZEEUW, Auteur ; S. BAHN, Auteur . - 17p.
Langues : Anglais (eng)
in Molecular Autism > 7 (2016) . - 17p.
Mots-clés : Animals Animals, Congenic Cells, Cultured Cerebellum/pathology/physiopathology Fluorescent Dyes Fragile X Mental Retardation Protein/genetics/physiology Fragile X Syndrome/genetics/metabolism/physiopathology Hippocampus/pathology/physiopathology Intravital Microscopy Male Mass Spectrometry/methods Mice Mice, Inbred C57BL Mice, Knockout Mice, Neurologic Mutants Microscopy, Electron Models, Animal Nerve Tissue Proteins/analysis Presynaptic Terminals/secretion Proteome Purkinje Cells/physiology/ultrastructure Pyridinium Compounds Quaternary Ammonium Compounds Signal Transduction Synaptic Transmission Synaptic Vesicles/metabolism Synaptosomes/metabolism Electron microscopy Fragile X syndrome (FXS) Mass spectrometry (MS) Quantitative live-cell imaging Index. décimale : PER Périodiques Résumé : BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. METHODS: Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. RESULTS: Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. CONCLUSIONS: Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS. En ligne : http://dx.doi.org/10.1186/s13229-016-0080-1 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=328