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Author (down) Zhou, Y.; Kumari, D.; Sciascia, N.; Usdin, K.
Title CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons Type Journal Article
Year 2016 Publication Molecular Autism Abbreviated Journal Mol Autism
Volume 7 Issue Pages 42
Keywords Fragile X syndrome; Repeat contractions; Repeat expansion mutation; Repeat-mediated gene silencing; Stem cells
Abstract 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.
Address Section on Gene Structure and Disease, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD USA
Corporate Author Thesis
Publisher Place of Publication Editor
Language English Summary Language Original Title
Series Editor Series Title Abbreviated Series Title
Series Volume Series Issue Edition
ISSN 2040-2392 ISBN Medium
Area Expedition Conference
Notes PMID:27713816; PMCID:PMC5053128 Approved no
Call Number refbase @ user @ Serial 16656
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Author (down) Zhang, Z.-N.; Freitas, B.C.; Qian, H.; Lux, J.; Acab, A.; Trujillo, C.A.; Herai, R.H.; Nguyen Huu, V.A.; Wen, J.H.; Joshi-Barr, S.; Karpiak, J.V.; Engler, A.J.; Fu, X.-D.; Muotri, A.R.; Almutairi, A.
Title Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction Type Journal Article
Year 2016 Publication Proceedings of the National Academy of Sciences of the United States of America Abbreviated Journal Proc Natl Acad Sci U S A
Volume 113 Issue 12 Pages 3185-3190
Keywords *Cell Movement; Humans; *Hydrogels; Induced Pluripotent Stem Cells/*cytology; Methyl-CpG-Binding Protein 2/*physiology; Neurons/*metabolism; 3D RTT modeling; 3D hydrogels; neuronal migration and maturation
Abstract Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.
Address Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093; aalmutairi@ucsd.edu muotri@ucsd.edu
Corporate Author Thesis
Publisher Place of Publication Editor
Language English Summary Language Original Title
Series Editor Series Title Abbreviated Series Title
Series Volume Series Issue Edition
ISSN 0027-8424 ISBN Medium
Area Expedition Conference
Notes PMID:26944080; PMCID:PMC4812712 Approved no
Call Number refbase @ user @ Serial 16699
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Author (down) Zhang, W.-B.; Ross, P.J.; Tu, Y.S.; Wang, Y.; Beggs, S.; Sengar, A.S.; Ellis, J.; Salter, M.W.
Title Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons Type Journal Article
Year 2016 Publication Scientific Reports Abbreviated Journal Sci Rep
Volume 6 Issue Pages 23837
Keywords
Abstract NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39-57), but not a Src-interfering peptide, Src(40-58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.
Address Department of Physiology, University of Toronto, Toronto, ON, Canada
Corporate Author Thesis
Publisher Place of Publication Editor
Language English Summary Language Original Title
Series Editor Series Title Abbreviated Series Title
Series Volume Series Issue Edition
ISSN 2045-2322 ISBN Medium
Area Expedition Conference
Notes PMID:27040756; PMCID:PMC4819183 Approved no
Call Number refbase @ user @ Serial 16692
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Author (down) Young-Pearse, T.L.; Morrow, E.M.
Title Modeling developmental neuropsychiatric disorders with iPSC technology: challenges and opportunities Type Journal Article
Year 2016 Publication Current Opinion in Neurobiology Abbreviated Journal Curr Opin Neurobiol
Volume 36 Issue Pages 66-73
Keywords Animals; Autism Spectrum Disorder/*metabolism/physiopathology; Autistic Disorder/*metabolism/physiopathology; Cellular Reprogramming Techniques; Dendrites/metabolism/physiology; Humans; In Vitro Techniques; Induced Pluripotent Stem Cells/*metabolism/physiology; Long QT Syndrome/*metabolism/physiopathology; Models, Neurological; Neuroglia/metabolism/physiology; Neurons/metabolism/physiology; Phenotype; Reproducibility of Results; Schizophrenia/*metabolism/physiopathology; Synapses/metabolism/physiology; Syndactyly/*metabolism/physiopathology
Abstract The development of cellular reprogramming methods to generate human induced pluripotent stem cells (iPSC) has led to the establishment of lines from hundreds of patients with a variety of neurologic and psychiatric diseases. One of the fundamental powers of iPSC technology lies in the competency of these cells to be directed to become any cell type in the body, thus allowing researchers to examine disease mechanisms and identify and test novel therapeutics in relevant cell types. The field has now exited the phase of 'proof-of-principle' studies showing the potential of the model systems, and it has now entered an exciting new era where iPSC studies are contributing to the field's understanding of mechanisms of disease. Here, we describe the challenges of iPSC modeling of neuropsychiatric disorders, and highlight studies where some of these challenges have been addressed to provide novel insights into disease mechanisms.
Address Department of Molecular Biology, Cell Biology and Biochemistry (MCB), and Institute for Brain Science, Brown University, 70 Ship Street, Providence, RI 02912, USA; Developmental Disorders Genetics Research Program, Emma Pendleton Bradley Hospital and Department of Psychiatry and Human Behavior, Brown University Medical School, Providence, RI 02912, USA. Electronic address: Eric_Morrow@brown.edu
Corporate Author Thesis
Publisher Place of Publication Editor
Language English Summary Language Original Title
Series Editor Series Title Abbreviated Series Title
Series Volume Series Issue Edition
ISSN 0959-4388 ISBN Medium
Area Expedition Conference
Notes PMID:26517284; PMCID:PMC4738093 Approved no
Call Number refbase @ user @ Serial 16727
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Author (down) Yi, F.; Danko, T.; Botelho, S.C.; Patzke, C.; Pak, C.H.; Wernig, M.; Sudhof, T.C.
Title Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons Type Journal Article
Year 2016 Publication Science (New York, N.Y.) Abbreviated Journal Science
Volume 352 Issue 6286 Pages aaf2669
Keywords Action Potentials; Animals; Autism Spectrum Disorder/*genetics; Cells, Cultured; Channelopathies/*genetics; Chromosome Deletion; Chromosome Disorders/genetics; Chromosomes, Human, Pair 22/genetics; Embryonic Stem Cells/metabolism; Gene Deletion; Genetic Engineering; *Genetic Predisposition to Disease; Haploinsufficiency/*genetics; Humans; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism; Mice; Mice, Knockout; Mutagenesis; Nerve Tissue Proteins/*genetics/metabolism; Neurons/*metabolism; Synapses/physiology; Synaptic Transmission
Abstract Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired hyperpolarization-activated cation (Ih) channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) that form Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest that SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.
Address Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA. Howard Hughes Medical Institute, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA. tcs1@stanford.edu
Corporate Author Thesis
Publisher Place of Publication Editor
Language English Summary Language Original Title
Series Editor Series Title Abbreviated Series Title
Series Volume Series Issue Edition
ISSN 0036-8075 ISBN Medium
Area Expedition Conference
Notes PMID:26966193; PMCID:PMC4901875 Approved no
Call Number refbase @ user @ Serial 16697
Permanent link to this record