title: Rho Kinase Inhibition Is Essential During In Vitro Neurogenesis and Promotes Phenotypic Rescue of Human Induced Pluripotent Stem Cell-Derived Neurons With Oligophrenin-1 Loss of Function.
abstract: : Rho-GTPases have relevant functions in various aspects of neuronal development, such as differentiation, migration, and synaptogenesis. Loss of function of the oligophrenin-1 gene (OPHN1) causes X-linked intellectual disability with cerebellar hypoplasia and leads to hyperactivation of the rho kinase (ROCK) pathway. ROCK mainly acts through phosphorylation of the myosin phosphatase targeting subunit 1, triggering actin-myosin contractility. We show that during in vitro neurogenesis, ROCK activity decreases from day 10 until terminal differentiation, whereas in OPHN1-deficient human induced pluripotent stem cells (h-iPSCs), the levels of ROCK are elevated throughout differentiation. ROCK inhibition favors neuronal-like appearance of h-iPSCs, in parallel with transcriptional upregulation of nuclear receptor NR4A1, which is known to induce neurite outgrowth. This study analyzed the morphological, biochemical, and functional features of OPHN1-deficient h-iPSCs and their rescue by treatment with the ROCK inhibitor fasudil, shedding light on the relevance of the ROCK pathway during neuronal differentiation and providing a neuronal model for human OPHN1 syndrome and its treatment.
  
  
  CONCLUSIONS
  The analysis of the levels of rho kinase (ROCK) activity at different stages of in vitro neurogenesis of human induced pluripotent stem cells reveals that ROCK activity decreases progressively in parallel with the appearance of neuronal-like morphology and upregulation of nuclear receptor NR4A1. These results shed light on the role of the ROCK pathway during early stages of human neurogenesis and provide a neuronal stem cell-based model for the treatment of OPHN1 syndrome and other neurological disorders due to ROCK dysfunction.
authors.affiliation: Unit of Neuromuscular and Neurodegenerative Disorders, Department of Neurosciences, Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Compagnucci
authors.affiliation: Unit of Neuromuscular and Neurodegenerative Disorders, Department of Neurosciences, Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy.
authors.forename: Sabina
authors.initials: S
authors.lastname: Barresi
authors.affiliation: Research Laboratories, Confocal Microscopy Core Facility, and Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Petrini
authors.affiliation: Department of Genetic and Development, Institut Cochin, Université Paris Descartes, Paris, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Billuart
authors.affiliation: Unit of Child Neuropsychiatry, Department of Neurosciences, Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Piccini
authors.affiliation: Institute of Human and Medical Genetics, Catholic University, Rome, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Chiurazzi
authors.affiliation: Unit of Child Neuropsychiatry, Department of Neurosciences, Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Alfieri
authors.affiliation: Unit of Neuromuscular and Neurodegenerative Disorders, Department of Neurosciences, Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Bertini
authors.affiliation: Unit of Neuromuscular and Neurodegenerative Disorders, Department of Neurosciences, Bambino Gesù Children's Hospital, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy ginevra.zanni@opbg.net.
authors.forename: Ginevra
authors.initials: G
authors.lastname: Zanni
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D048430:Cell Shape / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D008954:Models, Biological; D009154:Mutation / Q000502:physiology; D009422:Nervous System Diseases / Q000235:genetics / Q000473:pathology; D055495:Neurogenesis / Q000187:drug effects* / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D010641:Phenotype; D013577:Syndrome; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology
chemical_list: D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; C111952:OPHN1 protein, human; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases; C049347:fasudil
keywords: In vitro neurogenesis; Oligophrenin-1; ROCK inhibitors (fasudil, Y-27632); Rho-kinase signaling