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Ulke J, Chopra S, Kadiri OL, Geserick P, Stein V, Cheshmeh S, Kleinridders A, Kappert K. PTPRJ is a negative regulator of insulin signaling in neuronal cells, impacting protein biosynthesis, and neurite outgrowth. J Neuroendocrinol 2024; 36:e13446. [PMID: 39253900 PMCID: PMC11646663 DOI: 10.1111/jne.13446] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2024] [Revised: 07/29/2024] [Accepted: 08/28/2024] [Indexed: 09/11/2024]
Abstract
Central insulin resistance has been linked to the development of neurodegenerative diseases and mood disorders. Various proteins belonging to the enzyme family of protein tyrosine phosphatases (PTPs) act as inhibitors of insulin signaling. Protein tyrosine phosphatase receptor type J (PTPRJ) has been identified as a negative regulator in insulin signaling in the periphery. However, the impact of PTPRJ on insulin signaling and its functional role in neuronal cells is largely unknown. Therefore, we generated a Ptprj knockout (KO) cell model in the murine neuroblast cell line Neuro2a by CRISPR-Cas9 gene editing. Ptprj KO cells displayed enhanced insulin signaling, as shown by increased phosphorylation of the insulin receptor (INSR), IRS-1, AKT, and ERK1/2. Further, proximity ligation assays (PLA) revealed both direct interaction of PTPRJ with the INSR and recruitment of this phosphatase to the receptor upon insulin stimulation. By RNA sequencing gene expression analysis, we identified multiple gene clusters responsible for glucose uptake and metabolism, and genes involved in the synthesis of various lipids being mainly upregulated under PTPRJ deficiency. Furthermore, multiple Ca2+ transporters were differentially expressed along with decreased protein biosynthesis. This was accompanied by an increase in endoplasmic reticulum (ER) stress markers. On a functional level, PTPRJ deficiency compromised cell differentiation and neurite outgrowth, suggesting a role in nervous system development. Taken together, PTPRJ emerges as a negative regulator of central insulin signaling, impacting neuronal metabolism and neurite outgrowth.
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Affiliation(s)
- Jannis Ulke
- Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt‐Universität zu Berlin, Institute of Diagnostic Laboratory Medicine, Clinical Chemistry and PathobiochemistryBerlinGermany
- Max Rubner Center (MRC) for Cardiovascular Metabolic Renal ResearchCharité—Universitätsmedizin BerlinBerlinGermany
| | - Simran Chopra
- Department of Molecular and Experimental Nutritional Medicine, Institute of Nutritional ScienceUniversity of PotsdamNuthetalGermany
| | - Otsuware Linda‐Josephine Kadiri
- Department of Molecular and Experimental Nutritional Medicine, Institute of Nutritional ScienceUniversity of PotsdamNuthetalGermany
| | - Peter Geserick
- Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt‐Universität zu Berlin, Institute of Diagnostic Laboratory Medicine, Clinical Chemistry and PathobiochemistryBerlinGermany
- Max Rubner Center (MRC) for Cardiovascular Metabolic Renal ResearchCharité—Universitätsmedizin BerlinBerlinGermany
| | - Vanessa Stein
- Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt‐Universität zu Berlin, Institute of Diagnostic Laboratory Medicine, Clinical Chemistry and PathobiochemistryBerlinGermany
- Max Rubner Center (MRC) for Cardiovascular Metabolic Renal ResearchCharité—Universitätsmedizin BerlinBerlinGermany
| | - Sahar Cheshmeh
- Department of Molecular and Experimental Nutritional Medicine, Institute of Nutritional ScienceUniversity of PotsdamNuthetalGermany
| | - André Kleinridders
- Department of Molecular and Experimental Nutritional Medicine, Institute of Nutritional ScienceUniversity of PotsdamNuthetalGermany
| | - Kai Kappert
- Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt‐Universität zu Berlin, Institute of Diagnostic Laboratory Medicine, Clinical Chemistry and PathobiochemistryBerlinGermany
- Max Rubner Center (MRC) for Cardiovascular Metabolic Renal ResearchCharité—Universitätsmedizin BerlinBerlinGermany
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D'Antoni S, Spatuzza M, Bonaccorso CM, Catania MV. Role of fragile X messenger ribonucleoprotein 1 in the pathophysiology of brain disorders: a glia perspective. Neurosci Biobehav Rev 2024; 162:105731. [PMID: 38763180 DOI: 10.1016/j.neubiorev.2024.105731] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Revised: 05/14/2024] [Accepted: 05/15/2024] [Indexed: 05/21/2024]
Abstract
Fragile X messenger ribonucleoprotein 1 (FMRP) is a widely expressed RNA binding protein involved in several steps of mRNA metabolism. Mutations in the FMR1 gene encoding FMRP are responsible for fragile X syndrome (FXS), a leading genetic cause of intellectual disability and autism spectrum disorder, and fragile X-associated tremor-ataxia syndrome (FXTAS), a neurodegenerative disorder in aging men. Although FMRP is mainly expressed in neurons, it is also present in glial cells and its deficiency or altered expression can affect functions of glial cells with implications for the pathophysiology of brain disorders. The present review focuses on recent advances on the role of glial subtypes, astrocytes, oligodendrocytes and microglia, in the pathophysiology of FXS and FXTAS, and describes how the absence or reduced expression of FMRP in these cells can impact on glial and neuronal functions. We will also briefly address the role of FMRP in radial glial cells and its effects on neural development, and gliomas and will speculate on the role of glial FMRP in other brain disorders.
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Affiliation(s)
- S D'Antoni
- Institute for Biomedical Research and Innovation (IRIB), National Research Council (CNR), Via Paolo Gaifami 18, Catania 95126, Italy
| | - M Spatuzza
- Institute for Biomedical Research and Innovation (IRIB), National Research Council (CNR), Via Paolo Gaifami 18, Catania 95126, Italy
| | - C M Bonaccorso
- Oasi Research Institute - IRCCS, via Conte Ruggero 73, Troina 94018, Italy
| | - M V Catania
- Institute for Biomedical Research and Innovation (IRIB), National Research Council (CNR), Via Paolo Gaifami 18, Catania 95126, Italy.
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3
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Napier M, Reynolds K, Scott AL. Glial-mediated dysregulation of neurodevelopment in Fragile X Syndrome. INTERNATIONAL REVIEW OF NEUROBIOLOGY 2023; 173:187-215. [PMID: 37993178 DOI: 10.1016/bs.irn.2023.08.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2023]
Abstract
Astrocytes are highly involved in a multitude of developmental processes that are known to be dysregulated in Fragile X Syndrome. Here, we examine these processes individually and review the roles astrocytes play in contributing to the pathology of this syndrome. As a growing area of interest in the field, new and exciting insight is continually emerging. Understanding these glial-mediated roles is imperative for elucidating the underlying molecular mechanisms at play, not only in Fragile X Syndrome, but also other ASD-related disorders. Understanding these roles will be central to the future development of effective, clinically-relevant treatments of these disorders.
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Affiliation(s)
- M Napier
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada
| | - K Reynolds
- Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada; Department of Neuroscience, Tufts University School of Medicine, Boston, United States
| | - A L Scott
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada.
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4
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Ren B, Burkovetskaya M, Jung Y, Bergdolt L, Totusek S, Martinez-Cerdeno V, Stauch K, Korade Z, Dunaevsky A. Dysregulated cholesterol metabolism, aberrant excitability and altered cell cycle of astrocytes in fragile X syndrome. Glia 2023; 71:1176-1196. [PMID: 36594399 PMCID: PMC10023374 DOI: 10.1002/glia.24331] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2022] [Revised: 12/19/2022] [Accepted: 12/21/2022] [Indexed: 01/04/2023]
Abstract
Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. While neuronal contribution to FXS has been extensively studied in both animal and human-based models of FXS, the roles of astrocytes, a type of glial cells in the brain, are largely unknown. Here, we generated a human-based FXS model via differentiation of astrocytes from human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) and characterized their development, function, and proteomic profiles. We identified shortened cell cycle, enhanced Ca2+ signaling, impaired sterol biosynthesis, and pervasive alterations in the proteome of FXS astrocytes. Our work identified astrocytic impairments that could contribute to the pathogenesis of FXS and highlight astrocytes as a novel therapeutic target for FXS treatment.
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Affiliation(s)
- Baiyan Ren
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Maria Burkovetskaya
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Yoosun Jung
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Lara Bergdolt
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Steven Totusek
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Veronica Martinez-Cerdeno
- Department of Pathology and Laboratory Medicine, MIND Institute, and Institute for Pediatric Regenerative Medicine at UC Davis School of Medicine, and Shriners Hospitals for Children of Northern California, Sacramento, California, USA
| | - Kelly Stauch
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Zeljka Korade
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA
- Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, USA
- Department of Pediatrics, CHRI, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Anna Dunaevsky
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA
- Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Sharma SD, Reddy BK, Pal R, Ritakari TE, Cooper JD, Selvaraj BT, Kind PC, Chandran S, Wyllie DJA, Chattarji S. Astrocytes mediate cell non-autonomous correction of aberrant firing in human FXS neurons. Cell Rep 2023; 42:112344. [PMID: 37018073 PMCID: PMC10157295 DOI: 10.1016/j.celrep.2023.112344] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2022] [Revised: 12/12/2022] [Accepted: 03/21/2023] [Indexed: 04/06/2023] Open
Abstract
Pre-clinical studies of fragile X syndrome (FXS) have focused on neurons, with the role of glia remaining largely underexplored. We examined the astrocytic regulation of aberrant firing of FXS neurons derived from human pluripotent stem cells. Human FXS cortical neurons, co-cultured with human FXS astrocytes, fired frequent short-duration spontaneous bursts of action potentials compared with less frequent, longer-duration bursts of control neurons co-cultured with control astrocytes. Intriguingly, bursts fired by FXS neurons co-cultured with control astrocytes are indistinguishable from control neurons. Conversely, control neurons exhibit aberrant firing in the presence of FXS astrocytes. Thus, the astrocyte genotype determines the neuronal firing phenotype. Strikingly, astrocytic-conditioned medium, and not the physical presence of astrocytes, is capable of determining the firing phenotype. The mechanistic basis of this effect indicates that the astroglial-derived protein, S100β, restores normal firing by reversing the suppression of a persistent sodium current in FXS neurons.
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Affiliation(s)
- Shreya Das Sharma
- National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India; University of Trans-Disciplinary Health Science and Technology, Bangalore 560064, India; Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India; Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh EH16 4SB, UK
| | - Bharath Kumar Reddy
- National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India; Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India
| | - Rakhi Pal
- National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India; Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India
| | - Tuula E Ritakari
- Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh EH16 4SB, UK
| | - James D Cooper
- Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh EH16 4SB, UK
| | - Bhuvaneish T Selvaraj
- Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh EH16 4SB, UK
| | - Peter C Kind
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India; Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK; Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK; Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK
| | - Siddharthan Chandran
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India; Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh EH16 4SB, UK; Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK; Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK
| | - David J A Wyllie
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India; Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK; Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK; Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK.
| | - Sumantra Chattarji
- National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India; Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India; Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK; Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK.
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6
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Krzisch MA, Wu H, Yuan B, Whitfield TW, Liu XS, Fu D, Garrett-Engele CM, Khalil AS, Lungjangwa T, Shih J, Chang AN, Warren S, Cacace A, Andrykovich KR, Rietjens RGJ, Wallace O, Sur M, Jain B, Jaenisch R. Fragile X Syndrome Patient-Derived Neurons Developing in the Mouse Brain Show FMR1-Dependent Phenotypes. Biol Psychiatry 2023; 93:71-81. [PMID: 36372569 DOI: 10.1016/j.biopsych.2022.08.020] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 08/11/2022] [Accepted: 08/11/2022] [Indexed: 01/10/2023]
Abstract
BACKGROUND Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. METHODS To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. RESULTS The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. CONCLUSIONS This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.
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Affiliation(s)
- Marine A Krzisch
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.
| | - Hao Wu
- Full Circles Therapeutics, Inc., Cambridge, Massachusetts
| | - Bingbing Yuan
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Troy W Whitfield
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - X Shawn Liu
- Department of Physiology and Cellular Biophysics, Columbia University Medical Center, New York, New York
| | - Dongdong Fu
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | | | - Andrew S Khalil
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Tenzin Lungjangwa
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Jennifer Shih
- Picower Institute for Learning and Memory, Cambridge, Massachusetts
| | | | - Stephen Warren
- Departments of Human Genetics, Biochemistry, and Pediatrics, Emory University School of Medicine, Atlanta, Georgia
| | | | | | | | | | - Mriganka Sur
- Picower Institute for Learning and Memory, Cambridge, Massachusetts
| | - Bhav Jain
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Rudolf Jaenisch
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.
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7
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Talvio K, Minkeviciene R, Townsley KG, Achuta VS, Huckins LM, Corcoran P, Brennand KJ, Castrén ML. Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome. Front Cell Dev Biol 2022; 10:1034679. [PMID: 36506088 PMCID: PMC9731341 DOI: 10.3389/fcell.2022.1034679] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Accepted: 11/04/2022] [Indexed: 11/22/2022] Open
Abstract
Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.
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Affiliation(s)
- Karo Talvio
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Rimante Minkeviciene
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Kayla G. Townsley
- Pamela Sklar Division of Psychiatric Genomics, Department of Genetics and Genomics, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States,Nash Family Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States,Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai, New York, NY, United States
| | | | - Laura M. Huckins
- Pamela Sklar Division of Psychiatric Genomics, Department of Genetics and Genomics, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States,Division of Molecular Psychiatry, Department of Psychiatry, Yale University, New Haven, CT, United States
| | - Padraic Corcoran
- Array and Analysis Facility, Department of Medical Sciences, Uppsala University, Uppsala, Sweden
| | - Kristen J. Brennand
- Pamela Sklar Division of Psychiatric Genomics, Department of Genetics and Genomics, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States,Nash Family Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States,Division of Molecular Psychiatry, Department of Psychiatry, Yale University, New Haven, CT, United States,Department of Genetics, Yale University, New Haven, CT, United States
| | - Maija L. Castrén
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland,*Correspondence: Maija L. Castrén,
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Lewis S, DePass A, Hagerman RJ, Lozano R. Case Reports of Aortic Aneurism in Fragile X Syndrome. Genes (Basel) 2022; 13:1560. [PMID: 36140728 PMCID: PMC9498845 DOI: 10.3390/genes13091560] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 08/24/2022] [Accepted: 08/26/2022] [Indexed: 11/17/2022] Open
Abstract
Fragile X syndrome (FXS) is an inherited genetic condition that is the leading known cause of inherited intellectual developmental disability. Phenotypically, individuals with FXS also present with distinct physical features including, elongated face, prominent ears, pectus excavatum, macroorchidism, and joint laxity, which suggests connective tissue dysplasia. In addition to mitral valve prolapse, aortic dilatation has been identified within individuals with FXS. Abnormal elastin fiber networks have been found in the skin, valves, and aorta in individual cases. Aortic dilatation has been described in other connective tissue disorders, particularly Marfan syndrome. However, while aortic aneurysms are characteristic of Marfan syndrome, no similar cases have been reported in FXS patients to date. This case report details the presentation of two patients with FXS and aortic aneurysm. Our two cases highlight the risks of aortic pathology in FXS, and the need for monitoring in asymptomatic patients with significant aortic dilatation.
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Affiliation(s)
- Sivan Lewis
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Andrew DePass
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Randi J. Hagerman
- MIND Institute and Department of Pediatrics, University of California Davis Health, Sacramento, CA 95817, USA
| | - Reymundo Lozano
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
- Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
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9
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Kuznitsov-Yanovsky L, Shapira G, Gildin L, Shomron N, Ben-Yosef D. Transcriptomic Analysis of Human Fragile X Syndrome Neurons Reveals Neurite Outgrowth Modulation by the TGFβ/BMP Pathway. Int J Mol Sci 2022; 23:ijms23169278. [PMID: 36012539 PMCID: PMC9409179 DOI: 10.3390/ijms23169278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2022] [Revised: 08/11/2022] [Accepted: 08/14/2022] [Indexed: 11/25/2022] Open
Abstract
Fragile X Syndrome (FXS) is the main genetic reason for intellectual disability and is caused by the silencing of fragile X mental retardation protein (FMRP), an RNA-binding protein regulating the translation of many neuronal mRNAs. Neural differentiation of FX human embryonic stem cells (hESC) mimics the neurodevelopment of FXS fetuses and thus serves as a good model to explore the mechanisms underlining the development of FXS. Isogenic hESC clones with and without the FX mutation that share the same genetic background were in vitro differentiated into neurons, and their transcriptome was analyzed by RNA sequencing. FX neurons inactivating FMR1 expression presented delayed neuronal development and maturation, concomitant with dysregulation of the TGFβ/BMP signaling pathway, and genes related to the extracellular matrix. Migration assay showed decreased neurite outgrowth in FX neurons that was rescued by inhibition of the TGFβ/BMP signaling pathway. Our results provide new insights into the molecular pathway by which loss of FMRP affects neuronal network development. In FX neurons, the lack of FMRP dysregulates members of the BMP signaling pathway associated with ECM organization which, in a yet unknown mechanism, reduces the guidance of axonal growth cones, probably leading to the aberrant neuronal network function seen in FXS.
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Affiliation(s)
- Liron Kuznitsov-Yanovsky
- Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital Tel-Aviv Sourasky Medical Center, Tel Aviv 64239, Israel
- Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel
| | - Guy Shapira
- Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel
| | - Lital Gildin
- Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital Tel-Aviv Sourasky Medical Center, Tel Aviv 64239, Israel
- Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel
| | - Noam Shomron
- Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel
- Sagol School of Neuroscience, Tel-Aviv University, Tel Aviv 69978, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital Tel-Aviv Sourasky Medical Center, Tel Aviv 64239, Israel
- Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel
- Sagol School of Neuroscience, Tel-Aviv University, Tel Aviv 69978, Israel
- Correspondence:
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10
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Lee A, Xu J, Wen Z, Jin P. Across Dimensions: Developing 2D and 3D Human iPSC-Based Models of Fragile X Syndrome. Cells 2022; 11:1725. [PMID: 35681419 PMCID: PMC9179297 DOI: 10.3390/cells11111725] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Revised: 05/11/2022] [Accepted: 05/13/2022] [Indexed: 02/01/2023] Open
Abstract
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism spectrum disorder. FXS is caused by a cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in the untranslated region of the FMR1 gene leading to the functional loss of the gene's protein product FMRP. Various animal models of FXS have provided substantial knowledge about the disorder. However, critical limitations exist in replicating the pathophysiological mechanisms. Human induced pluripotent stem cells (hiPSCs) provide a unique means of studying the features and processes of both normal and abnormal human neurodevelopment in large sample quantities in a controlled setting. Human iPSC-based models of FXS have offered a better understanding of FXS pathophysiology specific to humans. This review summarizes studies that have used hiPSC-based two-dimensional cellular models of FXS to reproduce the pathology, examine altered gene expression and translation, determine the functions and targets of FMRP, characterize the neurodevelopmental phenotypes and electrophysiological features, and, finally, to reactivate FMR1. We also provide an overview of the most recent studies using three-dimensional human brain organoids of FXS and end with a discussion of current limitations and future directions for FXS research using hiPSCs.
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Affiliation(s)
- Azalea Lee
- Neuroscience Graduate Program, Emory University, Atlanta, GA 30322, USA;
- MD/PhD Program, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Jie Xu
- Genetics and Molecular Biology Graduate Program, Emory University, Atlanta, GA 30322, USA;
| | - Zhexing Wen
- Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Peng Jin
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
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11
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Impaired Functional Connectivity Underlies Fragile X Syndrome. Int J Mol Sci 2022; 23:ijms23042048. [PMID: 35216162 PMCID: PMC8878121 DOI: 10.3390/ijms23042048] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2021] [Revised: 02/06/2022] [Accepted: 02/07/2022] [Indexed: 01/15/2023] Open
Abstract
Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by a developmentally regulated silencing of the FMR1 gene, but its effect on human neuronal network development and function is not fully understood. Here, we isolated isogenic human embryonic stem cell (hESC) subclones—one with a full FX mutation and one that is free of the mutation (control) but shares the same genetic background—differentiated them into induced neurons (iNs) by forced expression of NEUROG-1, and compared the functional properties of the derived neuronal networks. High-throughput image analysis demonstrates that FX-iNs have significantly smaller cell bodies and reduced arborizations than the control. Both FX- and control-neurons can discharge repetitive action potentials, and FX neuronal networks are also able to generate spontaneous excitatory synaptic currents with slight differences from the control, demonstrating that iNs generate more mature neuronal networks than the previously used protocols. MEA analysis demonstrated that FX networks are hyperexcitable with significantly higher spontaneous burst-firing activity compared to the control. Most importantly, cross-correlation analysis enabled quantification of network connectivity to demonstrate that the FX neuronal networks are significantly less synchronous than the control, which can explain the origin of the development of intellectual dysfunction associated with FXS.
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12
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Deng PY, Avraham O, Cavalli V, Klyachko VA. Hyperexcitability of Sensory Neurons in Fragile X Mouse Model. Front Mol Neurosci 2022; 14:796053. [PMID: 35002623 PMCID: PMC8727524 DOI: 10.3389/fnmol.2021.796053] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Accepted: 11/17/2021] [Indexed: 01/18/2023] Open
Abstract
Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model.
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Affiliation(s)
- Pan-Yue Deng
- Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States
| | - Oshri Avraham
- Department of Neuroscience, Washington University School of Medicine, St. Louis, MO, United States
| | - Valeria Cavalli
- Department of Neuroscience, Washington University School of Medicine, St. Louis, MO, United States.,Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, United States.,Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, United States
| | - Vitaly A Klyachko
- Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States.,Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, United States
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13
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Liu X, Kumar V, Tsai NP, Auerbach BD. Hyperexcitability and Homeostasis in Fragile X Syndrome. Front Mol Neurosci 2022; 14:805929. [PMID: 35069112 PMCID: PMC8770333 DOI: 10.3389/fnmol.2021.805929] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Accepted: 12/14/2021] [Indexed: 01/13/2023] Open
Abstract
Fragile X Syndrome (FXS) is a leading inherited cause of autism and intellectual disability, resulting from a mutation in the FMR1 gene and subsequent loss of its protein product FMRP. Despite this simple genetic origin, FXS is a phenotypically complex disorder with a range of physical and neurocognitive disruptions. While numerous molecular and cellular pathways are affected by FMRP loss, there is growing evidence that circuit hyperexcitability may be a common convergence point that can account for many of the wide-ranging phenotypes seen in FXS. The mechanisms for hyperexcitability in FXS include alterations to excitatory synaptic function and connectivity, reduced inhibitory neuron activity, as well as changes to ion channel expression and conductance. However, understanding the impact of FMR1 mutation on circuit function is complicated by the inherent plasticity in neural circuits, which display an array of homeostatic mechanisms to maintain activity near set levels. FMRP is also an important regulator of activity-dependent plasticity in the brain, meaning that dysregulated plasticity can be both a cause and consequence of hyperexcitable networks in FXS. This makes it difficult to separate the direct effects of FMR1 mutation from the myriad and pleiotropic compensatory changes associated with it, both of which are likely to contribute to FXS pathophysiology. Here we will: (1) review evidence for hyperexcitability and homeostatic plasticity phenotypes in FXS models, focusing on similarities/differences across brain regions, cell-types, and developmental time points; (2) examine how excitability and plasticity disruptions interact with each other to ultimately contribute to circuit dysfunction in FXS; and (3) discuss how these synaptic and circuit deficits contribute to disease-relevant behavioral phenotypes like epilepsy and sensory hypersensitivity. Through this discussion of where the current field stands, we aim to introduce perspectives moving forward in FXS research.
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Affiliation(s)
- Xiaopeng Liu
- Deparment of Molecular & Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
- Beckman Institute for Advanced Science & Technology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
| | - Vipendra Kumar
- Deparment of Molecular & Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
| | - Nien-Pei Tsai
- Deparment of Molecular & Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
| | - Benjamin D. Auerbach
- Deparment of Molecular & Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
- Beckman Institute for Advanced Science & Technology, University of Illinois at Urbana-Champaign, Urbana, IL, United States
- *Correspondence: Benjamin D. Auerbach
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14
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Regulation of mRNA translation in stem cells; links to brain disorders. Cell Signal 2021; 88:110166. [PMID: 34624487 DOI: 10.1016/j.cellsig.2021.110166] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2021] [Revised: 08/09/2021] [Accepted: 09/29/2021] [Indexed: 11/22/2022]
Abstract
Translational control of gene expression is emerging as a cardinal step in the regulation of protein abundance. Especially for embryonic (ESC) and neuronal stem cells (NSC), regulation of mRNA translation is involved in the maintenance of pluripotency but also differentiation. For neuronal stem cells this regulation is linked to the various neuronal subtypes that arise in the developing brain and is linked to numerous brain disorders. Herein, we review translational control mechanisms in ESCs and NSCs during development and differentiation, and briefly discuss their link to brain disorders.
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15
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Kang Y, Zhou Y, Li Y, Han Y, Xu J, Niu W, Li Z, Liu S, Feng H, Huang W, Duan R, Xu T, Raj N, Zhang F, Dou J, Xu C, Wu H, Bassell GJ, Warren ST, Allen EG, Jin P, Wen Z. A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies. Nat Neurosci 2021; 24:1377-1391. [PMID: 34413513 PMCID: PMC8484073 DOI: 10.1038/s41593-021-00913-6] [Citation(s) in RCA: 110] [Impact Index Per Article: 27.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2020] [Accepted: 07/15/2021] [Indexed: 02/07/2023]
Abstract
Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that can regulate the translation of specific mRNAs. In this study, we developed an FXS human forebrain organoid model and observed that the loss of FMRP led to dysregulated neurogenesis, neuronal maturation and neuronal excitability. Bulk and single-cell gene expression analyses of FXS forebrain organoids revealed that the loss of FMRP altered gene expression in a cell-type-specific manner. The developmental deficits in FXS forebrain organoids could be rescued by inhibiting the phosphoinositide 3-kinase pathway but not the metabotropic glutamate pathway disrupted in the FXS mouse model. We identified a large number of human-specific mRNAs bound by FMRP. One of these human-specific FMRP targets, CHD2, contributed to the altered gene expression in FXS organoids. Collectively, our study revealed molecular, cellular and electrophysiological abnormalities associated with the loss of FMRP during human brain development.
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Affiliation(s)
- Yunhee Kang
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Ying Zhou
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Yujing Li
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Yanfei Han
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Jie Xu
- The Graduate Program in Genetics and Molecular Biology, Emory University, GA 30322, USA
| | - Weibo Niu
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Ziyi Li
- Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
| | - Shiying Liu
- Department of Population and Quantitative Health Sciences, Case Western Reserve University, OH 44106, USA
| | - Hao Feng
- Department of Population and Quantitative Health Sciences, Case Western Reserve University, OH 44106, USA
| | - Wen Huang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, 410078, China
| | - Ranhui Duan
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, 410078, China
| | - Tianmin Xu
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, China
| | - Nisha Raj
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Feiran Zhang
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Juan Dou
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Chongchong Xu
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Hao Wu
- Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Gary J Bassell
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Stephen T Warren
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Emily G Allen
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Peng Jin
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA;,To whom correspondence should be addressed: (P.J.) and (Z.W.)
| | - Zhexing Wen
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA;,To whom correspondence should be addressed: (P.J.) and (Z.W.)
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16
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Nomura T. Interneuron Dysfunction and Inhibitory Deficits in Autism and Fragile X Syndrome. Cells 2021; 10:2610. [PMID: 34685590 PMCID: PMC8534049 DOI: 10.3390/cells10102610] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 09/27/2021] [Accepted: 09/28/2021] [Indexed: 01/18/2023] Open
Abstract
The alteration of excitatory-inhibitory (E-I) balance has been implicated in various neurological and psychiatric diseases, including autism spectrum disorder (ASD). Fragile X syndrome (FXS) is a single-gene disorder that is the most common known cause of ASD. Understanding the molecular and physiological features of FXS is thought to enhance our knowledge of the pathophysiology of ASD. Accumulated evidence implicates deficits in the inhibitory circuits in FXS that tips E-I balance toward excitation. Deficits in interneurons, the main source of an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), have been reported in FXS, including a reduced number of cells, reduction in intrinsic cellular excitability, or weaker synaptic connectivity. Manipulating the interneuron activity ameliorated the symptoms in the FXS mouse model, which makes it reasonable to conceptualize FXS as an interneuronopathy. While it is still poorly understood how the developmental profiles of the inhibitory circuit go awry in FXS, recent works have uncovered several developmental alterations in the functional properties of interneurons. Correcting disrupted E-I balance by potentiating the inhibitory circuit by targeting interneurons may have a therapeutic potential in FXS. I will review the recent evidence about the inhibitory alterations and interneuron dysfunction in ASD and FXS and will discuss the future directions of this field.
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Affiliation(s)
- Toshihiro Nomura
- Department of Neuroscience, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
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17
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Telias M, Ben-Yosef D. Pharmacological Manipulation of Wnt/β-Catenin Signaling Pathway in Human Neural Precursor Cells Alters Their Differentiation Potential and Neuronal Yield. Front Mol Neurosci 2021; 14:680018. [PMID: 34421534 PMCID: PMC8371257 DOI: 10.3389/fnmol.2021.680018] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2021] [Accepted: 07/15/2021] [Indexed: 11/13/2022] Open
Abstract
The canonical Wnt/β-catenin pathway is a master-regulator of cell fate during embryonic and adult neurogenesis and is therefore a major pharmacological target in basic and clinical research. Chemical manipulation of Wnt signaling during in vitro neuronal differentiation of stem cells can alter both the quantity and the quality of the derived neurons. Accordingly, the use of Wnt activators and blockers has become an integral part of differentiation protocols applied to stem cells in recent years. Here, we investigated the effects of the glycogen synthase kinase-3β inhibitor CHIR99021, which upregulates β-catenin agonizing Wnt; and the tankyrase-1/2 inhibitor XAV939, which downregulates β-catenin antagonizing Wnt. Both drugs and their potential neurogenic and anti-neurogenic effects were studied using stable lines human neural precursor cells (hNPCs), derived from embryonic stem cells, which can be induced to generate mature neurons by chemically-defined conditions. We found that Wnt-agonism by CHIR99021 promotes induction of neural differentiation, while also reducing cell proliferation and survival. This effect was not synergistic with those of pro-neural growth factors during long-term neuronal differentiation. Conversely, antagonism of Wnt by XAV939 consistently prevented neuronal progression of hNPCs. We show here how these two drugs can be used to manipulate cell fate and how self-renewing hNPCs can be used as reliable human in vitro drug-screening platforms.
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Affiliation(s)
- Michael Telias
- Wolfe PGD-SC Lab, Racine IVF Unit, Department of Cell and Developmental Biology, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Sackler Medical School, Tel-Aviv University, Tel Aviv, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD-SC Lab, Racine IVF Unit, Department of Cell and Developmental Biology, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Sackler Medical School, Tel-Aviv University, Tel Aviv, Israel
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18
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Sahni G, Chang S, Meng JTC, Tan JZY, Fatien JJC, Bonnard C, Utami KH, Chan PW, Tan TT, Altunoglu U, Kayserili H, Pouladi M, Reversade B, Toh Y. A Micropatterned Human-Specific Neuroepithelial Tissue for Modeling Gene and Drug-Induced Neurodevelopmental Defects. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:2001100. [PMID: 33717833 PMCID: PMC7927627 DOI: 10.1002/advs.202001100] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 09/22/2020] [Indexed: 05/05/2023]
Abstract
The generation of structurally standardized human pluripotent stem cell (hPSC)-derived neural embryonic tissues has the potential to model genetic and environmental mediators of early neurodevelopmental defects. Current neural patterning systems have so far focused on directing cell fate specification spatio-temporally but not morphogenetic processes. Here, the formation of a structurally reproducible and highly-organized neuroepithelium (NE) tissue is directed from hPSCs, which recapitulates morphogenetic cellular processes relevant to early neurulation. These include having a continuous, polarized epithelium and a distinct invagination-like folding, where primitive ectodermal cells undergo E-to-N-cadherin switching and apical constriction as they acquire a NE fate. This is accomplished by spatio-temporal patterning of the mesoendoderm, which guides the development and self-organization of the adjacent primitive ectoderm into the NE. It is uncovered that TGFβ signaling emanating from endodermal cells support tissue folding of the prospective NE. Evaluation of NE tissue structural dysmorphia, which is uniquely achievable in the model, enables the detection of apical constriction and cell adhesion dysfunctions in patient-derived hPSCs as well as differentiating between different classes of neural tube defect-inducing drugs.
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Affiliation(s)
- Geetika Sahni
- Department of Biomedical EngineeringNational University of SingaporeSingapore117583Singapore
- NUS Tissue Engineering ProgramNational University of SingaporeSingapore119077Singapore
| | - Shu‐Yung Chang
- Department of Biomedical EngineeringNational University of SingaporeSingapore117583Singapore
- Institute for Health Innovation & Technology (iHealthTech)National University of SingaporeSingapore117599Singapore
| | - Jeremy Teo Choon Meng
- Divison of EngineeringNew York UniversityAbu Dhabi129188United Arab Emirates
- Department of Mechanical EngineeringNew York UniversityNew YorkNY11201USA
| | - Jerome Zu Yao Tan
- Department of Biomedical EngineeringNational University of SingaporeSingapore117583Singapore
- NUS Tissue Engineering ProgramNational University of SingaporeSingapore119077Singapore
| | - Jean Jacques Clement Fatien
- Department of Biomedical EngineeringNational University of SingaporeSingapore117583Singapore
- NUS Tissue Engineering ProgramNational University of SingaporeSingapore119077Singapore
| | - Carine Bonnard
- Institute of Medical BiologyHuman Genetics and Embryology LaboratoryA*STARSingapore138648Singapore
| | - Kagistia Hana Utami
- Translational Laboratory in Genetic Medicine (TLGM)Agency for Science, Technology, and Research (A*STAR)Singapore138648Singapore
| | - Puck Wee Chan
- Istanbul Medical FacultyMedical Genetics DepartmentIstanbul34093Turkey
| | - Thong Teck Tan
- Institute of Medical BiologyHuman Genetics and Embryology LaboratoryA*STARSingapore138648Singapore
| | - Umut Altunoglu
- Istanbul Medical FacultyMedical Genetics DepartmentIstanbul34093Turkey
| | - Hülya Kayserili
- Istanbul Medical FacultyMedical Genetics DepartmentIstanbul34093Turkey
- Koç University School of MedicineMedical Genetics DepartmentIstanbul34010Turkey
| | - Mahmoud Pouladi
- Translational Laboratory in Genetic Medicine (TLGM)Agency for Science, Technology, and Research (A*STAR)Singapore138648Singapore
- Department of MedicineNational University of SingaporeSingapore119228Singapore
| | - Bruno Reversade
- Institute of Medical BiologyHuman Genetics and Embryology LaboratoryA*STARSingapore138648Singapore
- Koç University School of MedicineMedical Genetics DepartmentIstanbul34010Turkey
- Institute of Molecular and Cellular BiologyA*STARSingapore138673Singapore
- Amsterdam Reproduction and DevelopmentAcademic Medical Centre and VU University Medical CenterAmsterdam1105the Netherlands
- National University of SingaporeDepartment of PediatricsSingapore119228Singapore
| | - Yi‐Chin Toh
- Department of Biomedical EngineeringNational University of SingaporeSingapore117583Singapore
- NUS Tissue Engineering ProgramNational University of SingaporeSingapore119077Singapore
- Institute for Health Innovation & Technology (iHealthTech)National University of SingaporeSingapore117599Singapore
- The N.1 Institute for HealthNational University of SingaporeSingapore117456Singapore
- School of MechanicalMedical and Process EngineeringQueensland University of TechnologyBrisbaneQueensland4000Australia
- Institute of Health and Biomedical InnovationQueensland University of TechnologyKelvin GroveQueensland4059Australia
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19
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Patient-Derived Induced Pluripotent Stem Cells (iPSCs) and Cerebral Organoids for Drug Screening and Development in Autism Spectrum Disorder: Opportunities and Challenges. Pharmaceutics 2021; 13:pharmaceutics13020280. [PMID: 33669772 PMCID: PMC7922555 DOI: 10.3390/pharmaceutics13020280] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 02/17/2021] [Accepted: 02/17/2021] [Indexed: 12/23/2022] Open
Abstract
Autism spectrum disorder (ASD) represents a group of neurodevelopmental diseases characterized by persistent deficits in social communication, interaction, and repetitive patterns of behaviors, interests, and activities. The etiopathogenesis is multifactorial with complex interactions between genetic and environmental factors. The clinical heterogeneity and complex etiology of this pediatric disorder have limited the development of pharmacological therapies. The major limit to ASD research remains a lack of relevant human disease models which can faithfully recapitulate key features of the human pathology and represent its genetic heterogeneity. Recent advances in induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of patients into all types of patient-specific neural cells, have provided a promising cellular tool for disease modeling and development of novel drug treatments. The iPSCs technology allowed not only a better investigation of the disease etiopathogenesis but also opened up the potential for personalized therapies and offered new opportunities for drug discovery, pharmacological screening, and toxicity assessment. Moreover, iPSCs can be differentiated and organized into three-dimensional (3D) organoids, providing a model which mimics the complexity of the brain’s architecture and more accurately recapitulates tissue- and organ-level disease pathophysiology. The aims of this review were to describe the current state of the art of the use of human patient-derived iPSCs and brain organoids in modeling ASD and developing novel therapeutic strategies and to discuss the opportunities and major challenges in this rapidly moving field.
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20
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Adhya D, Chennell G, Crowe JA, Valencia-Alarcón EP, Seyforth J, Hosny NA, Yasvoina MV, Forster R, Baron-Cohen S, Vernon AC, Srivastava DP. Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids. Mol Autism 2021; 12:4. [PMID: 33482917 PMCID: PMC7821651 DOI: 10.1186/s13229-021-00413-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2020] [Accepted: 01/08/2021] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.
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Affiliation(s)
- Dwaipayan Adhya
- Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK
- Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, UK
| | - George Chennell
- Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK
| | - James A Crowe
- Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK
- MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK
| | - Eva P Valencia-Alarcón
- Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK
- MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK
| | - James Seyforth
- M Squared Life Ltd., The Surrey Technology Centre, 40 Occam Road, Guildford, UK
| | - Neveen A Hosny
- M Squared Life Ltd., The Surrey Technology Centre, 40 Occam Road, Guildford, UK
| | - Marina V Yasvoina
- Department of Neuroimaging, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK
| | - Robert Forster
- M Squared Life Ltd., The Surrey Technology Centre, 40 Occam Road, Guildford, UK
| | - Simon Baron-Cohen
- Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, UK
| | - Anthony C Vernon
- Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK
- MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK
| | - Deepak P Srivastava
- MRC Centre for Neurodevelopmental Disorders, King's College London, London, UK.
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21
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Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay. Nat Cell Biol 2021; 23:40-48. [PMID: 33420492 PMCID: PMC8273690 DOI: 10.1038/s41556-020-00618-1] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 11/29/2020] [Indexed: 01/28/2023]
Abstract
Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.
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22
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Cheffer A, Flitsch LJ, Krutenko T, Röderer P, Sokhranyaeva L, Iefremova V, Hajo M, Peitz M, Schwarz MK, Brüstle O. Human stem cell-based models for studying autism spectrum disorder-related neuronal dysfunction. Mol Autism 2020; 11:99. [PMID: 33308283 PMCID: PMC7733257 DOI: 10.1186/s13229-020-00383-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Accepted: 09/22/2020] [Indexed: 12/13/2022] Open
Abstract
The controlled differentiation of pluripotent stem cells (PSCs) into neurons and glia offers a unique opportunity to study early stages of human central nervous system development under controlled conditions in vitro. With the advent of cell reprogramming and the possibility to generate induced pluripotent stem cells (iPSCs) from any individual in a scalable manner, these studies can be extended to a disease- and patient-specific level. Autism spectrum disorder (ASD) is considered a neurodevelopmental disorder, with substantial evidence pointing to early alterations in neurogenesis and network formation as key pathogenic drivers. For that reason, ASD represents an ideal candidate for stem cell-based disease modeling. Here, we provide a concise review on recent advances in the field of human iPSC-based modeling of syndromic and non-syndromic forms of ASD, with a particular focus on studies addressing neuronal dysfunction and altered connectivity. We further discuss recent efforts to translate stem cell-based disease modeling to 3D via brain organoid and cell transplantation approaches, which enable the investigation of disease mechanisms in a tissue-like context. Finally, we describe advanced tools facilitating the assessment of altered neuronal function, comment on the relevance of iPSC-based models for the assessment of pharmaceutical therapies and outline potential future routes in stem cell-based ASD research.
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Affiliation(s)
- Arquimedes Cheffer
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Lea Jessica Flitsch
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Tamara Krutenko
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Pascal Röderer
- Life & Brain GmbH, Platform Cellomics, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Liubov Sokhranyaeva
- Institute of Experimental Epileptology and Cognition Research, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Vira Iefremova
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Mohamad Hajo
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Michael Peitz
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
- Life & Brain GmbH, Platform Cellomics, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
- Cell Programming Core Facility, University of Bonn Medical Faculty, Bonn, Germany
| | - Martin Karl Schwarz
- Life & Brain GmbH, Platform Cellomics, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
- Institute of Experimental Epileptology and Cognition Research, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany.
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23
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Utami KH, Skotte NH, Colaço AR, Yusof NABM, Sim B, Yeo XY, Bae HG, Garcia-Miralles M, Radulescu CI, Chen Q, Chaldaiopoulou G, Liany H, Nama S, Peteri UKA, Sampath P, Castrén ML, Jung S, Mann M, Pouladi MA. Integrative Analysis Identifies Key Molecular Signatures Underlying Neurodevelopmental Deficits in Fragile X Syndrome. Biol Psychiatry 2020; 88:500-511. [PMID: 32653109 DOI: 10.1016/j.biopsych.2020.05.005] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/10/2019] [Revised: 03/26/2020] [Accepted: 05/02/2020] [Indexed: 12/23/2022]
Abstract
BACKGROUND Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Efforts to understand the molecular underpinnings of the disease have been largely performed in rodent or nonisogenic settings. A detailed examination of the impact of FMRP loss on cellular processes and neuronal properties in the context of isogenic human neurons remains lacking. METHODS Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to introduce indels in exon 3 of FMR1, we generated an isogenic human pluripotent stem cell model of FXS that shows complete loss of FMRP expression. We generated neuronal cultures and performed genome-wide transcriptome and proteome profiling followed by functional validation of key dysregulated processes. We further analyzed neurodevelopmental and neuronal properties, including neurite length and neuronal activity, using multielectrode arrays and patch clamp electrophysiology. RESULTS We showed that the transcriptome and proteome profiles of isogenic FMRP-deficient neurons demonstrate perturbations in synaptic transmission, neuron differentiation, cell proliferation and ion transmembrane transporter activity pathways, and autism spectrum disorder-associated gene sets. We uncovered key deficits in FMRP-deficient cells demonstrating abnormal neural rosette formation and neural progenitor cell proliferation. We further showed that FMRP-deficient neurons exhibit a number of additional phenotypic abnormalities, including neurite outgrowth and branching deficits and impaired electrophysiological network activity. These FMRP-deficient related impairments have also been validated in additional FXS patient-derived human-induced pluripotent stem cell neural cells. CONCLUSIONS Using isogenic human pluripotent stem cells as a model to investigate the pathophysiology of FXS in human neurons, we reveal key neural abnormalities arising from the loss of FMRP.
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Affiliation(s)
- Kagistia Hana Utami
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Niels H Skotte
- Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen N, Denmark
| | - Ana R Colaço
- Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen N, Denmark
| | | | - Bernice Sim
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Xin Yi Yeo
- Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Han-Gyu Bae
- Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Marta Garcia-Miralles
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Carola I Radulescu
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore; UK Dementia Research Institute, Department of Brain Sciences, Imperial College London, London, United Kingdom
| | - Qiyu Chen
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Georgia Chaldaiopoulou
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Herty Liany
- Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Srikanth Nama
- Institute of Medical Biology, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Ulla-Kaisa A Peteri
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Prabha Sampath
- Institute of Medical Biology, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore
| | - Maija L Castrén
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Sangyong Jung
- Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Matthias Mann
- Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen N, Denmark
| | - Mahmoud A Pouladi
- Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A∗STAR), Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
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24
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Ribosomes: An Exciting Avenue in Stem Cell Research. Stem Cells Int 2020; 2020:8863539. [PMID: 32695182 PMCID: PMC7362291 DOI: 10.1155/2020/8863539] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Revised: 06/12/2020] [Accepted: 06/16/2020] [Indexed: 02/07/2023] Open
Abstract
Stem cell research has focused on genomic studies. However, recent evidence has indicated the involvement of epigenetic regulation in determining the fate of stem cells. Ribosomes play a crucial role in epigenetic regulation, and thus, we focused on the role of ribosomes in stem cells. Majority of living organisms possess ribosomes that are involved in the translation of mRNA into proteins and promote cellular proliferation and differentiation. Ribosomes are stable molecular machines that play a role with changes in the levels of RNA during translation. Recent research suggests that specific ribosomes actively regulate gene expression in multiple cell types, such as stem cells. Stem cells have the potential for self-renewal and differentiation into multiple lineages and, thus, require high efficiency of translation. Ribosomes induce cellular transdifferentiation and reprogramming, and disrupted ribosome synthesis affects translation efficiency, thereby hindering stem cell function leading to cell death and differentiation. Stem cell function is regulated by ribosome-mediated control of stem cell-specific gene expression. In this review, we have presented a detailed discourse on the characteristics of ribosomes in stem cells. Understanding ribosome biology in stem cells will provide insights into the regulation of stem cell function and cellular reprogramming.
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25
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Das Sharma S, Pal R, Reddy BK, Selvaraj BT, Raj N, Samaga KK, Srinivasan DJ, Ornelas L, Sareen D, Livesey MR, Bassell GJ, Svendsen CN, Kind PC, Chandran S, Chattarji S, Wyllie DJA. Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns. Mol Autism 2020; 11:52. [PMID: 32560741 PMCID: PMC7304215 DOI: 10.1186/s13229-020-00351-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Accepted: 05/11/2020] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP). METHODS Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced. RESULTS Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca2+-activated K+ currents and the persistent Na+ current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na+ channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines. CONCLUSIONS Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.
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Affiliation(s)
- Shreya Das Sharma
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,The University of Trans-Displinary Health Sciences and Technology, Bangalore, 560064, India
| | - Rakhi Pal
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India
| | - Bharath Kumar Reddy
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India
| | - Bhuvaneish T Selvaraj
- Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh, EH16 4SB, UK.,UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh, EH16 4SB, UK
| | - Nisha Raj
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Krishna Kumar Samaga
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India
| | - Durga J Srinivasan
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,The University of Trans-Displinary Health Sciences and Technology, Bangalore, 560064, India
| | - Loren Ornelas
- The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,iPSC Core, The David Janet Polak Foundation Stem Cell Core Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,Cedars-Sinai Biomanufacturing Center, West Hollywood, CA, 90069, USA
| | - Dhruv Sareen
- The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,iPSC Core, The David Janet Polak Foundation Stem Cell Core Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,Cedars-Sinai Biomanufacturing Center, West Hollywood, CA, 90069, USA.,Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA
| | - Matthew R Livesey
- Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK
| | - Gary J Bassell
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Clive N Svendsen
- The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA
| | - Peter C Kind
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.,Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK
| | - Siddharthan Chandran
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh, EH16 4SB, UK.,UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh, EH16 4SB, UK.,Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK
| | - Sumantra Chattarji
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India. .,Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bangalore, 560065, India.
| | - David J A Wyllie
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India. .,Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.
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26
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Telias M. Pharmacological Treatments for Fragile X Syndrome Based on Synaptic Dysfunction. Curr Pharm Des 2020; 25:4394-4404. [PMID: 31682210 DOI: 10.2174/1381612825666191102165206] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Accepted: 10/31/2019] [Indexed: 12/29/2022]
Abstract
BACKGROUND Fragile X syndrome (FXS) is the most common form of monogenic hereditary cognitive impairment, including intellectual disability, autism, hyperactivity, and epilepsy. METHODS This article reviews the literature pertaining to the role of synaptic dysfunction in FXS. RESULTS In FXS, synaptic dysfunction alters the excitation-inhibition ratio, dysregulating molecular and cellular processes underlying cognition, learning, memory, and social behavior. Decades of research have yielded important hypotheses that could explain, at least in part, the development of these neurological disorders in FXS patients. However, the main goal of translating lab research in animal models to pharmacological treatments in the clinic has been so far largely unsuccessful, leaving FXS a still incurable disease. CONCLUSION In this concise review, we summarize and analyze the main hypotheses proposed to explain synaptic dysregulation in FXS, by reviewing the scientific evidence that led to pharmaceutical clinical trials and their outcome.
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Affiliation(s)
- Michael Telias
- Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States
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27
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Li M, Shin J, Risgaard RD, Parries MJ, Wang J, Chasman D, Liu S, Roy S, Bhattacharyya A, Zhao X. Identification of FMR1-regulated molecular networks in human neurodevelopment. Genome Res 2020; 30:361-374. [PMID: 32179589 PMCID: PMC7111522 DOI: 10.1101/gr.251405.119] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2019] [Accepted: 02/21/2020] [Indexed: 12/17/2022]
Abstract
RNA-binding proteins (RNA-BPs) play critical roles in development and disease to regulate gene expression. However, genome-wide identification of their targets in primary human cells has been challenging. Here, we applied a modified CLIP-seq strategy to identify genome-wide targets of the FMRP translational regulator 1 (FMR1), a brain-enriched RNA-BP, whose deficiency leads to Fragile X Syndrome (FXS), the most prevalent inherited intellectual disability. We identified FMR1 targets in human dorsal and ventral forebrain neural progenitors and excitatory and inhibitory neurons differentiated from human pluripotent stem cells. In parallel, we measured the transcriptomes of the same four cell types upon FMR1 gene deletion. We discovered that FMR1 preferentially binds long transcripts in human neural cells. FMR1 targets include genes unique to human neural cells and associated with clinical phenotypes of FXS and autism. Integrative network analysis using graph diffusion and multitask clustering of FMR1 CLIP-seq and transcriptional targets reveals critical pathways regulated by FMR1 in human neural development. Our results demonstrate that FMR1 regulates a common set of targets among different neural cell types but also operates in a cell type-specific manner targeting distinct sets of genes in human excitatory and inhibitory neural progenitors and neurons. By defining molecular subnetworks and validating specific high-priority genes, we identify novel components of the FMR1 regulation program. Our results provide new insights into gene regulation by a critical neuronal RNA-BP in human neurodevelopment.
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Affiliation(s)
- Meng Li
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Junha Shin
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Ryan D Risgaard
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Molly J Parries
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Jianyi Wang
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Deborah Chasman
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Shuang Liu
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Sushmita Roy
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Anita Bhattacharyya
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
| | - Xinyu Zhao
- Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.,Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
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28
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Ribosome and Translational Control in Stem Cells. Cells 2020; 9:cells9020497. [PMID: 32098201 PMCID: PMC7072746 DOI: 10.3390/cells9020497] [Citation(s) in RCA: 50] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2020] [Revised: 02/11/2020] [Accepted: 02/17/2020] [Indexed: 12/16/2022] Open
Abstract
Embryonic stem cells (ESCs) and adult stem cells (ASCs) possess the remarkable capacity to self-renew while remaining poised to differentiate into multiple progenies in the context of a rapidly developing embryo or in steady-state tissues, respectively. This ability is controlled by complex genetic programs, which are dynamically orchestrated at different steps of gene expression, including chromatin remodeling, mRNA transcription, processing, and stability. In addition to maintaining stem cell homeostasis, these molecular processes need to be rapidly rewired to coordinate complex physiological modifications required to redirect cell fate in response to environmental clues, such as differentiation signals or tissue injuries. Although chromatin remodeling and mRNA expression have been extensively studied in stem cells, accumulating evidence suggests that stem cell transcriptomes and proteomes are poorly correlated and that stem cell properties require finely tuned protein synthesis. In addition, many studies have shown that the biogenesis of the translation machinery, the ribosome, is decisive for sustaining ESC and ASC properties. Therefore, these observations emphasize the importance of translational control in stem cell homeostasis and fate decisions. In this review, we will provide the most recent literature describing how ribosome biogenesis and translational control regulate stem cell functions and are crucial for accommodating proteome remodeling in response to changes in stem cell fate.
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29
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Using Two- and Three-Dimensional Human iPSC Culture Systems to Model Psychiatric Disorders. ADVANCES IN NEUROBIOLOGY 2020; 25:237-257. [PMID: 32578150 DOI: 10.1007/978-3-030-45493-7_9] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Psychiatric disorders are among the most challenging human diseases to understand at a mechanistic level due to the heterogeneity of symptoms within established diagnostic categories, the general absence of focal pathology, and the genetic complexity inherent in these mostly polygenic disorders. Each of these features presents unique challenges to disease modeling for biological discovery, drug development, or improved diagnostics. In addition, live human neural tissue has been largely inaccessible to experimentation, leaving gaps in our knowledge derived from animal models that cannot fully recapitulate the features of the disease, indirect measures of brain function in human patients, and from analyses of postmortem tissue that can be confounded by comorbid conditions and medication history.
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30
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Freel BA, Sheets JN, Francis KR. iPSC modeling of rare pediatric disorders. J Neurosci Methods 2019; 332:108533. [PMID: 31811832 DOI: 10.1016/j.jneumeth.2019.108533] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Revised: 11/25/2019] [Accepted: 11/27/2019] [Indexed: 12/20/2022]
Abstract
Discerning the underlying pathological mechanisms and the identification of therapeutic strategies to treat individuals affected with rare neurological diseases has proven challenging due to a host of factors. For instance, rare diseases affecting the nervous system are inherently lacking in appropriate patient sample availability compared to more common diseases, while animal models often do not accurately recapitulate specific disease phenotypes. These challenges impede research that may otherwise illuminate aspects of disease initiation and progression, leading to the ultimate identification of potential therapeutics. The establishment of induced pluripotent stem cells (iPSCs) as a human cellular model with defined genetics has provided the unique opportunity to study rare diseases within a controlled environment. iPSC models enable researchers to define mutational effects on specific cell types and signaling pathways within increasingly complex systems. Among rare diseases, pediatric diseases affecting neurodevelopment and neurological function highlight the critical need for iPSC-based disease modeling due to the inherent difficulty associated with collecting human neural tissue and the complexity of the mammalian nervous system. Rare neurodevelopmental disorders are therefore ideal candidates for utilization of iPSC-based in vitro studies. In this review, we address both the state of the iPSC field in the context of their utility and limitations for neurodevelopmental studies, as well as speculating about the future applications and unmet uses for iPSCs in rare diseases.
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Affiliation(s)
- Bethany A Freel
- Cellular Therapies and Stem Cell Biology Group, Sanford Research, Sioux Falls, SD, USA
| | - Jordan N Sheets
- Cellular Therapies and Stem Cell Biology Group, Sanford Research, Sioux Falls, SD, USA
| | - Kevin R Francis
- Cellular Therapies and Stem Cell Biology Group, Sanford Research, Sioux Falls, SD, USA; Department of Pediatrics, University of South Dakota Sanford School of Medicine, Sioux Falls, SD, USA.
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31
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Preisler L, Ben-Yosef D, Mayshar Y. Adenomatous Polyposis Coli as a Major Regulator of Human Embryonic Stem Cells Self-Renewal. Stem Cells 2019; 37:1505-1515. [PMID: 31461190 DOI: 10.1002/stem.3084] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Revised: 07/22/2019] [Accepted: 08/08/2019] [Indexed: 12/13/2022]
Abstract
Human embryonic stem cells (hESCs) provide an essential tool to investigate early human development, study disease pathogenesis, and examine therapeutic interventions. Adenomatous polyposis coli (APC) is a negative regulator of Wnt/β-catenin signaling, implicated in the majority of sporadic colorectal cancers and in the autosomal dominant inherited syndrome familial adenomatous polyposis (FAP). Studies into the role of Wnt/β-catenin signaling in hESCs arrived at conflicting results, due at least in part to variations in culture conditions and the use of external inhibitors and agonists. Here, we directly targeted APC in hESCs carrying a germline APC mutation, derived from affected blastocysts following preimplantation genetic diagnosis (PGD) for FAP, in order to answer open questions regarding the role of APC in regulating pluripotency and differentiation potential of hESCs. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), we generated second hit APC mutations in FAP-hESCs. Despite high CRISPR/Cas9 targeting efficiency and the successful isolation of many clones, none of the isolated clones carried a loss of function mutation in the wild-type (WT) APC allele. Using a fluorescent β-catenin reporter and analysis of mutated-allele frequencies in the APC locus, we show that APC double mutant hESCs robustly activate Wnt/β-catenin signaling that results in rapid differentiation to endodermal and mesodermal lineages. Here, we provide direct evidence for a strict requirement for constant β-catenin degradation through the APC destruction complex in order to maintain pluripotency, highlighting a fundamental role for APC in self-renewal of hESCs. Stem Cells 2019;37:1505-1515.
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Affiliation(s)
- Livia Preisler
- Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.,Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.,Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
| | - Yoav Mayshar
- Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel
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32
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Yang G, Shcheglovitov A. Probing disrupted neurodevelopment in autism using human stem cell-derived neurons and organoids: An outlook into future diagnostics and drug development. Dev Dyn 2019; 249:6-33. [PMID: 31398277 DOI: 10.1002/dvdy.100] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2019] [Revised: 07/23/2019] [Accepted: 07/31/2019] [Indexed: 12/11/2022] Open
Abstract
Autism spectrum disorders (ASDs) represent a spectrum of neurodevelopmental disorders characterized by impaired social interaction, repetitive or restrictive behaviors, and problems with speech. According to a recent report by the Centers for Disease Control and Prevention, one in 68 children in the US is diagnosed with ASDs. Although ASD-related diagnostics and the knowledge of ASD-associated genetic abnormalities have improved in recent years, our understanding of the cellular and molecular pathways disrupted in ASD remains very limited. As a result, no specific therapies or medications are available for individuals with ASDs. In this review, we describe the neurodevelopmental processes that are likely affected in the brains of individuals with ASDs and discuss how patient-specific stem cell-derived neurons and organoids can be used for investigating these processes at the cellular and molecular levels. Finally, we propose a discovery pipeline to be used in the future for identifying the cellular and molecular deficits and developing novel personalized therapies for individuals with idiopathic ASDs.
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Affiliation(s)
- Guang Yang
- Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah.,Neuroscience Graduate Program, University of Utah, Salt Lake City, Utah
| | - Alex Shcheglovitov
- Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah.,Neuroscience Graduate Program, University of Utah, Salt Lake City, Utah
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33
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Pal R, Bhattacharya A. Modelling Protein Synthesis as A Biomarker in Fragile X Syndrome Patient-Derived Cells. Brain Sci 2019; 9:E59. [PMID: 30862080 PMCID: PMC6468675 DOI: 10.3390/brainsci9030059] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 02/27/2019] [Accepted: 03/06/2019] [Indexed: 12/26/2022] Open
Abstract
The most conserved molecular phenotype of Fragile X Syndrome (FXS) is aberrant protein synthesis. This has been validated in a variety of experimental model systems from zebrafish to rats, patient-derived lymphoblasts and fibroblasts. With the advent of personalized medicine paradigms, patient-derived cells and their derivatives are gaining more translational importance, not only to model disease in a dish, but also for biomarker discovery. Here we review past and current practices of measuring protein synthesis in FXS, studies in patient derived cells and the inherent challenges in measuring protein synthesis in them to offer usable avenues of modeling this important metabolic metric for further biomarker development.
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Affiliation(s)
- Rakhi Pal
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, GKVK Post, Bellary Road, Bengaluru 560065, India.
| | - Aditi Bhattacharya
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, GKVK Post, Bellary Road, Bengaluru 560065, India.
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34
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Telias M. Molecular Mechanisms of Synaptic Dysregulation in Fragile X Syndrome and Autism Spectrum Disorders. Front Mol Neurosci 2019; 12:51. [PMID: 30899214 PMCID: PMC6417395 DOI: 10.3389/fnmol.2019.00051] [Citation(s) in RCA: 57] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Accepted: 02/12/2019] [Indexed: 12/21/2022] Open
Abstract
Fragile X syndrome (FXS) is the most common form of monogenic hereditary cognitive impairment. FXS patient exhibit a high comorbidity rate with autism spectrum disorders (ASDs). This makes FXS a model disease for understanding how synaptic dysregulation alters neuronal excitability, learning and memory, social behavior, and more. Since 1991, with the discovery of fragile X mental retardation 1 (FMR1) as the sole gene that is mutated in FXS, thousands of studies into the function of the gene and its encoded protein FMR1 protein (FMRP), have been conducted, yielding important information regarding the pathophysiology of the disease, as well as insight into basic synaptic mechanisms that control neuronal networking and circuitry. Among the most important, are molecular mechanisms directly involved in plasticity, including glutamate and γ-aminobutyric acid (GABA) receptors, which can control synaptic transmission and signal transduction, including short- and long-term plasticity. More recently, several novel mechanisms involving growth factors, enzymatic cascades and transcription factors (TFs), have been proposed to have the potential of explaining some of the synaptic dysregulation in FXS. In this review article, I summarize the main mechanisms proposed to underlie synaptic disruption in FXS and ASDs. I focus on studies conducted on the Fmr1 knock-out (KO) mouse model and on FXS-human pluripotent stem cells (hPSCs), emphasizing the differences and even contradictions between mouse and human, whenever possible. As FXS and ASDs are both neurodevelopmental disorders that follow a specific time-course of disease progression, I highlight those studies focusing on the differential developmental regulation of synaptic abnormalities in these diseases.
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Affiliation(s)
- Michael Telias
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
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35
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Kumari D, Gazy I, Usdin K. Pharmacological Reactivation of the Silenced FMR1 Gene as a Targeted Therapeutic Approach for Fragile X Syndrome. Brain Sci 2019; 9:brainsci9020039. [PMID: 30759772 PMCID: PMC6406686 DOI: 10.3390/brainsci9020039] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2019] [Revised: 02/07/2019] [Accepted: 02/08/2019] [Indexed: 12/22/2022] Open
Abstract
More than ~200 CGG repeats in the 5′ untranslated region of the FMR1 gene results in transcriptional silencing and the absence of the FMR1 encoded protein, FMRP. FMRP is an RNA-binding protein that regulates the transport and translation of a variety of brain mRNAs in an activity-dependent manner. The loss of FMRP causes dysregulation of many neuronal pathways and results in an intellectual disability disorder, fragile X syndrome (FXS). Currently, there is no effective treatment for FXS. In this review, we discuss reactivation of the FMR1 gene as a potential approach for FXS treatment with an emphasis on the use of small molecules to inhibit the pathways important for gene silencing.
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Affiliation(s)
- Daman Kumari
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Inbal Gazy
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Karen Usdin
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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36
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Kuznitsov-Yanovsky L, Mayshar Y, Ben-Yosef D. Modeling FXS: Human Pluripotent Stem Cells and In Vitro Neural Differentiation. Methods Mol Biol 2019; 1942:89-100. [PMID: 30900178 DOI: 10.1007/978-1-4939-9080-1_8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
In fragile X syndrome (FXS) embryos FMRP is widely expressed during early stages of embryogenesis however it is inactivated by the end of the first trimester. In the same manner, human embryonic stem cell (hESC) lines from FXS blastocysts, bearing the full CGG expansion mutation, express FMRP in their pluripotent stage and in neurons derived following in vitro differentiation, FMR1 is completely silenced. Therefore, in vitro neural differentiation of FX-hESC lines serves as a uniquely valuable model system to study the developmental mechanisms underlying FXS, together with the proper differentiation protocol to mimic the neurodevelopmental process occurs in vivo.
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Affiliation(s)
- Liron Kuznitsov-Yanovsky
- Wolfe PGD Stem Cell Lab, Racine IVF Unit at Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.,Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - Yoav Mayshar
- Wolfe PGD Stem Cell Lab, Racine IVF Unit at Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD Stem Cell Lab, Racine IVF Unit at Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel.
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37
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Zhao X, Bhattacharyya A. Human Models Are Needed for Studying Human Neurodevelopmental Disorders. Am J Hum Genet 2018; 103:829-857. [PMID: 30526865 DOI: 10.1016/j.ajhg.2018.10.009] [Citation(s) in RCA: 109] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Accepted: 10/09/2018] [Indexed: 12/19/2022] Open
Abstract
The analysis of animal models of neurological disease has been instrumental in furthering our understanding of neurodevelopment and brain diseases. However, animal models are limited in revealing some of the most fundamental aspects of development, genetics, pathology, and disease mechanisms that are unique to humans. These shortcomings are exaggerated in disorders that affect the brain, where the most significant differences between humans and animal models exist, and could underscore failures in targeted therapeutic interventions in affected individuals. Human pluripotent stem cells have emerged as a much-needed model system for investigating human-specific biology and disease mechanisms. However, questions remain regarding whether these cell-culture-based models are sufficient or even necessary. In this review, we summarize human-specific features of neurodevelopment and the most common neurodevelopmental disorders, present discrepancies between animal models and human diseases, demonstrate how human stem cell models can provide meaningful information, and discuss the challenges that exist in our pursuit to understand distinctively human aspects of neurodevelopment and brain disease. This information argues for a more thoughtful approach to disease modeling through consideration of the valuable features and limitations of each model system, be they human or animal, to mimic disease characteristics.
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Affiliation(s)
- Xinyu Zhao
- Waisman Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison WI 53705, USA.
| | - Anita Bhattacharyya
- Waisman Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison WI 53705, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison WI 53705, USA.
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38
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D'Souza MN, Gowda NKC, Tiwari V, Babu RO, Anand P, Dastidar SG, Singh R, James OG, Selvaraj B, Pal R, Ramesh A, Chattarji S, Chandran S, Gulyani A, Palakodeti D, Muddashetty RS. FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation. iScience 2018; 9:399-411. [PMID: 30469012 PMCID: PMC6249352 DOI: 10.1016/j.isci.2018.11.007] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Revised: 09/28/2018] [Accepted: 11/01/2018] [Indexed: 12/19/2022] Open
Abstract
FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2’O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2’O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.
FMRP binds to C/D Box snoRNAs in the nucleus Differential 2’O-methylation on rRNA contributes to ribosome heterogeneity in a cell 2’O-Methylation pattern on ribosomal RNA is altered in the absence of FMRP FMRP recognizes 2’O-methylation on rRNA, which may determine interaction with ribosomes
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Affiliation(s)
- Michelle Ninochka D'Souza
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India; The University of Trans-Disciplinary Health Sciences & Technology (TDU), Bengaluru, Karnataka 560064, India
| | | | - Vishal Tiwari
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India; National Centre for Biological Sciences, Bengaluru, Karnataka 560065, India
| | | | - Praveen Anand
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India
| | - Sudhriti Ghosh Dastidar
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India; Manipal Academy of Higher Education, Madhav Nagar, Manipal, Karnataka 576104, India
| | - Randhir Singh
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India
| | - Owen G James
- Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at Edinburgh, University of Edinburgh, Edinburgh EH16 4SB, UK
| | - Bhuvaneish Selvaraj
- Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at Edinburgh, University of Edinburgh, Edinburgh EH16 4SB, UK
| | - Rakhi Pal
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India
| | - Arati Ramesh
- National Centre for Biological Sciences, Bengaluru, Karnataka 560065, India
| | - Sumantra Chattarji
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India; National Centre for Biological Sciences, Bengaluru, Karnataka 560065, India; Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK
| | - Siddharthan Chandran
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India; Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK; UK Dementia Research Institute at Edinburgh, University of Edinburgh, Edinburgh EH16 4SB, UK
| | - Akash Gulyani
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India
| | - Dasaradhi Palakodeti
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India
| | - Ravi S Muddashetty
- Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru 560065, India.
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39
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Haenfler JM, Skariah G, Rodriguez CM, Monteiro da Rocha A, Parent JM, Smith GD, Todd PK. Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells. Front Mol Neurosci 2018; 11:282. [PMID: 30158855 PMCID: PMC6104480 DOI: 10.3389/fnmol.2018.00282] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2018] [Accepted: 07/25/2018] [Indexed: 12/15/2022] Open
Abstract
Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.
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Affiliation(s)
- Jill M Haenfler
- Department of Neurology, University of Michigan, Ann Arbor, MI, United States.,Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, United States
| | - Geena Skariah
- Department of Neurology, University of Michigan, Ann Arbor, MI, United States
| | - Caitlin M Rodriguez
- Department of Neurology, University of Michigan, Ann Arbor, MI, United States
| | - Andre Monteiro da Rocha
- Department of Internal Medicine, Center for Arrhythmia Research, University of Michigan, Ann Arbor, MI, United States
| | - Jack M Parent
- Department of Neurology, University of Michigan, Ann Arbor, MI, United States.,Veterans Administration Ann Arbor Healthcare System, Ann Arbor, MI, United States
| | - Gary D Smith
- Departments of Obstetrics/Gynecology, Physiology, and Urology, University of Michigan, Ann Arbor, MI, United States
| | - Peter K Todd
- Department of Neurology, University of Michigan, Ann Arbor, MI, United States.,Veterans Administration Ann Arbor Healthcare System, Ann Arbor, MI, United States
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40
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Zhang Z, Marro SG, Zhang Y, Arendt KL, Patzke C, Zhou B, Fair T, Yang N, Südhof TC, Wernig M, Chen L. The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling. Sci Transl Med 2018; 10:eaar4338. [PMID: 30068571 PMCID: PMC6317709 DOI: 10.1126/scitranslmed.aar4338] [Citation(s) in RCA: 66] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Accepted: 07/12/2018] [Indexed: 11/02/2022]
Abstract
Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.
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Affiliation(s)
- Zhenjie Zhang
- Departments of Neurosurgery, and Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305-5453, USA
| | - Samuele G Marro
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
- Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Yingsha Zhang
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Kristin L Arendt
- Departments of Neurosurgery, and Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305-5453, USA
| | - Christopher Patzke
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Bo Zhou
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Tyler Fair
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
- Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Nan Yang
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
- Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Thomas C Südhof
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA.
- Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Marius Wernig
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5453, USA.
- Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5453, USA
| | - Lu Chen
- Departments of Neurosurgery, and Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305-5453, USA.
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41
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Zhou R, Jiang G, Tian X, Wang X. Progress in the molecular mechanisms of genetic epilepsies using patient-induced pluripotent stem cells. Epilepsia Open 2018; 3:331-339. [PMID: 30187003 PMCID: PMC6119748 DOI: 10.1002/epi4.12238] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/25/2018] [Indexed: 12/29/2022] Open
Abstract
Research findings on the molecular mechanisms of epilepsy almost always originate from animal experiments, and the development of induced pluripotent stem cell (iPSC) technology allows the use of human cells with genetic defects for studying the molecular mechanisms of genetic epilepsy (GE) for the first time. With iPSC technology, terminally differentiated cells collected from GE patients with specific genetic etiologies can be differentiated into many relevant cell subtypes that carry all of the GE patient's genetic information. iPSCs have opened up a new research field involving the pathogenesis of GE. Using this approach, studies have found that gene mutations induce GE by altering the balance between neuronal excitation and inhibition, which is associated. among other factors, with neuronal developmental disturbances, ion channel abnormalities, and synaptic dysfunction. Simultaneously, astrocyte activation, mitochondrial dysfunction, and abnormal signaling pathway activity are also important factors in the molecular mechanisms of GE.
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Affiliation(s)
- Ruijiao Zhou
- Department of Neurology the First Affiliated Hospital of Chongqing Medical University Chongqing Key Laboratory of Neurology Chongqing China
| | - Guohui Jiang
- Department of Neurology Institute of Neurological Diseases Affiliated Hospital of North Sichuan Medical College Nanchong China
| | - Xin Tian
- Department of Neurology the First Affiliated Hospital of Chongqing Medical University Chongqing Key Laboratory of Neurology Chongqing China
| | - Xuefeng Wang
- Department of Neurology the First Affiliated Hospital of Chongqing Medical University Chongqing Key Laboratory of Neurology Chongqing China
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42
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Bustos F, Segarra-Fas A, Chaugule VK, Brandenburg L, Branigan E, Toth R, Macartney T, Knebel A, Hay RT, Walden H, Findlay GM. RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation. Cell Rep 2018; 23:1599-1611. [PMID: 29742418 PMCID: PMC5976579 DOI: 10.1016/j.celrep.2018.04.022] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2017] [Revised: 03/13/2018] [Accepted: 04/03/2018] [Indexed: 11/29/2022] Open
Abstract
X-linked intellectual disability (XLID) is a heterogeneous syndrome affecting mainly males. Human genetics has identified >100 XLID genes, although the molecular and developmental mechanisms underpinning this disorder remain unclear. Here, we employ an embryonic stem cell model to explore developmental functions of a recently identified XLID gene, the RNF12/RLIM E3 ubiquitin ligase. We show that RNF12 catalytic activity is required for proper stem cell maintenance and neural differentiation, and this is disrupted by patient-associated XLID mutation. We further demonstrate that RNF12 XLID mutations specifically impair ubiquitylation of developmentally relevant substrates. XLID mutants disrupt distinct RNF12 functional modules by either inactivating the catalytic RING domain or interfering with a distal regulatory region required for efficient ubiquitin transfer. Our data thereby uncover a key function for RNF12 E3 ubiquitin ligase activity in stem cell and neural development and identify mechanisms by which this is disrupted in intellectual disability.
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Affiliation(s)
- Francisco Bustos
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Anna Segarra-Fas
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Viduth K Chaugule
- Institute of Molecular Cell and Systems Biology, The University of Glasgow, Glasgow G12 8QQ, UK
| | - Lennart Brandenburg
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Emma Branigan
- Centre for Gene Regulation and Expression, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Rachel Toth
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Thomas Macartney
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Axel Knebel
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Ronald T Hay
- Centre for Gene Regulation and Expression, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Helen Walden
- Institute of Molecular Cell and Systems Biology, The University of Glasgow, Glasgow G12 8QQ, UK
| | - Greg M Findlay
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK.
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43
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Banerjee A, Ifrim MF, Valdez AN, Raj N, Bassell GJ. Aberrant RNA translation in fragile X syndrome: From FMRP mechanisms to emerging therapeutic strategies. Brain Res 2018; 1693:24-36. [PMID: 29653083 PMCID: PMC7377270 DOI: 10.1016/j.brainres.2018.04.008] [Citation(s) in RCA: 76] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Revised: 03/30/2018] [Accepted: 04/06/2018] [Indexed: 02/07/2023]
Abstract
Research in the past decades has unfolded the multifaceted role of Fragile X mental retardation protein (FMRP) and how its absence contributes to the pathophysiology of Fragile X syndrome (FXS). Excess signaling through group 1 metabotropic glutamate receptors is commonly observed in mouse models of FXS, which in part is attributed to dysregulated translation and downstream signaling. Considering the wide spectrum of cellular and physiologic functions that loss of FMRP can affect in general, it may be advantageous to pursue disease mechanism based treatments that directly target translational components or signaling factors that regulate protein synthesis. Various FMRP targets upstream and downstream of the translational machinery are therefore being investigated to further our understanding of the molecular mechanism of RNA and protein synthesis dysregulation in FXS as well as test their potential role as therapeutic interventions to alleviate FXS associated symptoms. In this review, we will broadly discuss recent advancements made towards understanding the role of FMRP in translation regulation, new pre-clinical animal models with FMRP targets located at different levels of the translational and signal transduction pathways for therapeutic intervention as well as future use of stem cells to model FXS associated phenotypes.
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Affiliation(s)
- Anwesha Banerjee
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
| | - Marius F Ifrim
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Arielle N Valdez
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Nisha Raj
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Gary J Bassell
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA; Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA.
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44
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Dahlhaus R. Of Men and Mice: Modeling the Fragile X Syndrome. Front Mol Neurosci 2018; 11:41. [PMID: 29599705 PMCID: PMC5862809 DOI: 10.3389/fnmol.2018.00041] [Citation(s) in RCA: 87] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2017] [Accepted: 01/31/2018] [Indexed: 12/26/2022] Open
Abstract
The Fragile X Syndrome (FXS) is one of the most common forms of inherited intellectual disability in all human societies. Caused by the transcriptional silencing of a single gene, the fragile x mental retardation gene FMR1, FXS is characterized by a variety of symptoms, which range from mental disabilities to autism and epilepsy. More than 20 years ago, a first animal model was described, the Fmr1 knock-out mouse. Several other models have been developed since then, including conditional knock-out mice, knock-out rats, a zebrafish and a drosophila model. Using these model systems, various targets for potential pharmaceutical treatments have been identified and many treatments have been shown to be efficient in preclinical studies. However, all attempts to turn these findings into a therapy for patients have failed thus far. In this review, I will discuss underlying difficulties and address potential alternatives for our future research.
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Affiliation(s)
- Regina Dahlhaus
- Institute for Biochemistry, Emil-Fischer Centre, University of Erlangen-Nürnberg, Erlangen, Germany
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45
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Abstract
Neural stem cells (NSCs) give rise to the entire nervous system. Animal models suggest that defects in NSC proliferation and differentiation contribute to several brain disorders (e.g., microcephaly, macrocephaly, autism, schizophrenia, and Huntington's disease). However, animal models of such diseases do not fully recapitulate all disease-related phenotypes because of substantial differences in brain development between rodents and humans. Therefore, additional human-based evidence is required to understand the mechanisms that are involved in the development of neurological diseases that result from human NSC (hNSC) dysfunction. Human-induced pluripotent stem cells provide a new model to investigate the contribution of hNSCs to various neurological pathologies. In this chapter, we review the role of hNSCs in both neurodevelopment- and neurodegeneration-related human brain pathologies, with an emphasis on recent evidence that has been obtained using embryonic stem cell- or induced pluripotent stem cell-derived hNSCs and progenitors.
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Affiliation(s)
- Ewa Liszewska
- International Institute of Molecular and Cell Biology, Warsaw, Poland.
| | - Jacek Jaworski
- International Institute of Molecular and Cell Biology, Warsaw, Poland.
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46
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Belagodu AP, Fleming S, Galvez R. Neocortical developmental analysis of vasculature and their growth factors offer new insight into fragile X syndrome abnormalities. Dev Neurobiol 2017; 77:1321-1333. [DOI: 10.1002/dneu.22514] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Revised: 05/22/2017] [Accepted: 07/13/2017] [Indexed: 01/19/2023]
Affiliation(s)
- Amogh P. Belagodu
- Neuroscience Program, University of Illinois at Urbana‐ChampaignUrbana IL61801
- Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana‐ChampaignUrbana IL61801
| | - Stephen Fleming
- Psychology DepartmentUniversity of Illinois at Urbana‐ChampaignUrbana IL61801
| | - Roberto Galvez
- Neuroscience Program, University of Illinois at Urbana‐ChampaignUrbana IL61801
- Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana‐ChampaignUrbana IL61801
- Psychology DepartmentUniversity of Illinois at Urbana‐ChampaignUrbana IL61801
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47
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Sourial M, Doering LC. Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus. Brain Res 2017; 1666:58-69. [PMID: 28442243 DOI: 10.1016/j.brainres.2017.04.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Revised: 03/31/2017] [Accepted: 04/18/2017] [Indexed: 10/19/2022]
Abstract
The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST+CD15+CD133+) and an increased proportion of neuroblasts (PSA-NCAM+CD15+) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G2/M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X.
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Affiliation(s)
- Mary Sourial
- McMaster Integrative Neuroscience Discovery and Study, McMaster University, Hamilton, Ontario, Canada; Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
| | - Laurie C Doering
- McMaster Integrative Neuroscience Discovery and Study, McMaster University, Hamilton, Ontario, Canada; Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
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48
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Boland MJ, Nazor KL, Tran HT, Szücs A, Lynch CL, Paredes R, Tassone F, Sanna PP, Hagerman RJ, Loring JF. Molecular analyses of neurogenic defects in a human pluripotent stem cell model of fragile X syndrome. Brain 2017; 140:582-598. [PMID: 28137726 DOI: 10.1093/brain/aww357] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Accepted: 12/03/2016] [Indexed: 11/13/2022] Open
Abstract
New research suggests that common pathways are altered in many neurodevelopmental disorders including autism spectrum disorder; however, little is known about early molecular events that contribute to the pathology of these diseases. The study of monogenic, neurodevelopmental disorders with a high incidence of autistic behaviours, such as fragile X syndrome, has the potential to identify genes and pathways that are dysregulated in autism spectrum disorder as well as fragile X syndrome. In vitro generation of human disease-relevant cell types provides the ability to investigate aspects of disease that are impossible to study in patients or animal models. Differentiation of human pluripotent stem cells recapitulates development of the neocortex, an area affected in both fragile X syndrome and autism spectrum disorder. We have generated induced human pluripotent stem cells from several individuals clinically diagnosed with fragile X syndrome and autism spectrum disorder. When differentiated to dorsal forebrain cell fates, our fragile X syndrome human pluripotent stem cell lines exhibited reproducible aberrant neurogenic phenotypes. Using global gene expression and DNA methylation profiling, we have analysed the early stages of neurogenesis in fragile X syndrome human pluripotent stem cells. We discovered aberrant DNA methylation patterns at specific genomic regions in fragile X syndrome cells, and identified dysregulated gene- and network-level correlates of fragile X syndrome that are associated with developmental signalling, cell migration, and neuronal maturation. Integration of our gene expression and epigenetic analysis identified altered epigenetic-mediated transcriptional regulation of a distinct set of genes in fragile X syndrome. These fragile X syndrome-aberrant networks are significantly enriched for genes associated with autism spectrum disorder, giving support to the idea that underlying similarities exist among these neurodevelopmental diseases.
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Affiliation(s)
- Michael J Boland
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA
| | - Kristopher L Nazor
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA
| | - Ha T Tran
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA
| | - Attila Szücs
- BioCircuits Institute, University of California San Diego, La Jolla, CA, USA.,MTA-ELTE NAP-B Neuronal Cell Biology Group, Eötvös Lóránd University, Budapest, Hungary
| | - Candace L Lynch
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA
| | - Ryder Paredes
- CIRM Bridges to Stem Cells Program, California State University Channel Islands, Camarillo, CA, USA
| | - Flora Tassone
- Department of Biochemistry and Molecular Medicine, University of California Davis, Sacramento, CA, USA.,MIND Institute, University of California Davis, Sacramento, CA, USA
| | - Pietro Paolo Sanna
- Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, CA, USA
| | - Randi J Hagerman
- MIND Institute, University of California Davis, Sacramento, CA, USA.,Department of Pediatrics, University of California Davis Medical Center, Sacramento, CA, USA
| | - Jeanne F Loring
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA
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49
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Zorio DAR, Jackson CM, Liu Y, Rubel EW, Wang Y. Cellular distribution of the fragile X mental retardation protein in the mouse brain. J Comp Neurol 2017; 525:818-849. [PMID: 27539535 PMCID: PMC5558202 DOI: 10.1002/cne.24100] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2016] [Revised: 08/10/2016] [Accepted: 08/11/2016] [Indexed: 11/07/2022]
Abstract
The fragile X mental retardation protein (FMRP) plays an important role in normal brain development. Absence of FMRP results in abnormal neuronal morphologies in a selected manner throughout the brain, leading to intellectual deficits and sensory dysfunction in the fragile X syndrome (FXS). Despite FMRP importance for proper brain function, its overall expression pattern in the mammalian brain at the resolution of individual neuronal cell groups is not known. In this study we used FMR1 knockout and isogenic wildtype mice to systematically map the distribution of FMRP expression in the entire mouse brain. Using immunocytochemistry and cellular quantification analyses, we identified a large number of prominent cell groups expressing high levels of FMRP at the subcortical levels, in particular sensory and motor neurons in the brainstem and thalamus. In contrast, many cell groups in the midbrain and hypothalamus exhibit low FMRP levels. More important, we describe differential patterns of FMRP distribution in both cortical and subcortical brain regions. Almost all major brain areas contain high and low levels of FMRP cell groups adjacent to each other or between layers of the same cortical areas. These differential patterns indicate that FMRP expression appears to be specific to individual neuronal cell groups instead of being associated with all neurons in distinct brain regions, as previously considered. Taken together, these findings support the notion of FMRP differential neuronal regulation and strongly implicate the contribution of fundamental sensory and motor processing at subcortical levels to FXS pathology. J. Comp. Neurol. 525:818-849, 2017. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Diego A. R. Zorio
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
| | - Christine M. Jackson
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
| | - Yong Liu
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
| | - Edwin W Rubel
- Virginia Merrill Bloedel Hearing Research Center, Department of Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Box 357923, Seattle, WA 98195, USA
| | - Yuan Wang
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
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50
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Vershkov D, Benvenisty N. Human pluripotent stem cells in modeling human disorders: the case of fragile X syndrome. Regen Med 2017; 12:53-68. [DOI: 10.2217/rme-2016-0100] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Human pluripotent stem cells (PSCs) generated from affected blastocysts or from patient-derived somatic cells are an emerging platform for disease modeling and drug discovery. Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, was one of the first disorders modeled in both embryonic stem cells and induced PCSs and can serve as an exemplary case for the utilization of human PSCs in the study of human diseases. Over the past decade, FXS-PSCs have been used to address the fundamental questions regarding the pathophysiology of FXS. In this review we summarize the methodologies for generation of FXS-PSCs, discuss their advantages and disadvantages compared with existing modeling systems and describe their utilization in the study of FXS pathogenesis and in the development of targeted treatment.
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Affiliation(s)
- Dan Vershkov
- The Azrieli Center for Stem Cells & Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells & Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel
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