Cortical neurons obtained from patient-derived iPSCs with GNAO1 p.G203R variant show altered differentiation and functional properties
Maria Cristina Benedetti,
Tiziano D'andrea,
Alessio Colantoni,
Denis Silachev,
Valeria de Turris,
Zaira Boussadia,
Valentina A. Babenko,
Egor A. Volovikov,
Lilia Belikova,
Alexandra N. Bogomazova,
Rita Pepponi,
Dosh Whye,
Elizabeth D. Buttermore,
Gian Gaetano Tartaglia,
Maria A. Lagarkova,
Vladimir L. Katanaev,
Ilya Musayev,
Simone Martinelli,
Sergio Fucile,
Alessandro Rosa
Affiliations
Maria Cristina Benedetti
Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome, Rome, Italy; Center for Life Nano- & Neuro-Science, Fondazione Istituto Italiano di Tecnologia (IIT), Rome, Italy
Tiziano D'andrea
Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy
Alessio Colantoni
Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome, Rome, Italy; Center for Life Nano- & Neuro-Science, Fondazione Istituto Italiano di Tecnologia (IIT), Rome, Italy
Denis Silachev
School of Medicine and Life Sciences, Far Eastern Federal University, 690090, Vladivostok, Russia; A.N. Belozersky Research Institute of Physico-Chemical Biology, Moscow State University, 119992, Moscow, Russia; Department of Cell Physiology and Metabolism, Faculty of Medicine, Translational Research Center in Oncohaematology, University of Geneva, 1211, Geneva, Switzerland
Valeria de Turris
Center for Life Nano- & Neuro-Science, Fondazione Istituto Italiano di Tecnologia (IIT), Rome, Italy
Zaira Boussadia
National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy
Valentina A. Babenko
A.N. Belozersky Research Institute of Physico-Chemical Biology, Moscow State University, 119992, Moscow, Russia; Department of Cell Physiology and Metabolism, Faculty of Medicine, Translational Research Center in Oncohaematology, University of Geneva, 1211, Geneva, Switzerland
Egor A. Volovikov
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia
Lilia Belikova
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia
Alexandra N. Bogomazova
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia
Rita Pepponi
National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy
Dosh Whye
Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center and F.M. Kirby Neurobiology Department, Boston Children's Hospital, Boston, MA, USA
Elizabeth D. Buttermore
Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center and F.M. Kirby Neurobiology Department, Boston Children's Hospital, Boston, MA, USA
Gian Gaetano Tartaglia
Center for Human Technologies (CHT), Istituto Italiano di Tecnologia (IIT), Genova, Italy
Maria A. Lagarkova
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435, Moscow, Russia
Vladimir L. Katanaev
School of Medicine and Life Sciences, Far Eastern Federal University, 690090, Vladivostok, Russia; Department of Cell Physiology and Metabolism, Faculty of Medicine, Translational Research Center in Oncohaematology, University of Geneva, 1211, Geneva, Switzerland
Ilya Musayev
Child's Cure Genetic Research, Fremont, CA, USA
Simone Martinelli
Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
Sergio Fucile
Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy; IRCCS Neuromed, Pozzilli, Italy
Alessandro Rosa
Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome, Rome, Italy; Center for Life Nano- & Neuro-Science, Fondazione Istituto Italiano di Tecnologia (IIT), Rome, Italy; Corresponding author. Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome, Rome, Italy.
Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Gαo, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients’ cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca2+]i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.
