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The Phenotypic Landscape of a Tbc1d24 Mutant Mouse Title Includes Convulsive Seizures Resembling Human Early Infantile Epileptic Encephalopathy( Dissertation 全文 )

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The Phenotypic Landscape of a Tbc1d24 Mutant Mouse Title Includes Convulsive Seizures Resembling Human Early Infantile Epileptic Encephalopathy( Dissertation 全文 ) The Phenotypic Landscape of a Tbc1d24 Mutant Mouse Title Includes Convulsive Seizures Resembling Human Early Infantile Epileptic Encephalopathy( Dissertation_全文 ) Author(s) Tona, Risa Citation 京都大学 Issue Date 2019-03-25 URL https://doi.org/10.14989/doctor.k21664 許諾条件により本文は2020-01-02に公開; This is a pre- copyedited, author-produced version of an article accepted for Right publication in Human Molecular Genetics peer review.The version of record is available oline. Type Thesis or Dissertation Textversion ETD Kyoto University Human Molecular Genetics General Article The Phenotypic Landscape of a Tbc1d24 Mutant Mouse Includes Convulsive Seizures Resembling Human Early Infantile Epileptic Encephalopathy Risa Tona1, 2, Wenqian Chen1, Yoko Nakano3, Laura D. Reyes4, Ronald S. Petralia5, Ya-Xian Wang5, Matthew F. Starost6, Talah T. Wafa7, Robert J. Morell8, Kevin D. Cravedi9, Johann du Hoffmann9, Takushi Miyoshi1, Jeeva P. Munasinghe10, Tracy S. Fitzgerald7, Yogita Chudasama9, 11, Koichi Omori2, Carlo Pierpaoli4, Botond Banfi3, Lijin Dong12, Inna A. Belyantseva1, Thomas B. Friedman1,* 1Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, Maryland 20892, USA 2Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto City, Kyoto 606-8507, Japan 3Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA 4Quantitative Medical Imaging Section, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA 1 5Advanced Imaging Core, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA 6Division of Veterinary Resources, National Institutes of Health, Bethesda, Maryland 20892, USA 7Mouse Auditory Testing Core Facility, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892, USA 8Genomics and Computational Biology Core, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892, USA 9Rodent Behavioral Core, National Institute of Mental Health, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, Maryland 20892, USA 10In vivo NMR Center and Mouse Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA 11Section on Behavioral Neuroscience, National Institute of Mental Health, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, Maryland 20892, USA 12Genetic Engineering Core, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA *To whom correspondence should be addressed at: The National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, Room 1F141, National Institutes of Health, Bethesda, Maryland 20892, USA. Tel: 301 496 7882; Email: [email protected] (T.B.F.) 2 Abstract Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early- infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively-spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon-utilization switch. During embryonic and early postnatal development S324Tfs*3 homozygotes produce predominantly the shorter wild- type TBC1D24 protein isoform that omits the six residues encoded by the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice-factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold- electronmicroscopy using a TBC1D24-specific antibody revealed TBC1D24 associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model 3 of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs (AEDs) ameliorating TBCID24 seizure-disorders. Keywords: Tbc1d24 mutant mouse Micro-exon Seizures SRRM3 Hippocampus Introduction The human genome encodes 42 proteins that contain TBC domains (Tre-2- Bub2-Cdc16) (1), an ancient, conserved sequence consisting of approximately 200 amino acids (2). Pathogenic variants of several genes encoding TBC domains contribute to a range of disorders in humans including epilepsy, intellectual disability, pontocerebellar hypoplasia, dysmorphic features and deafness (Table 1) (3-7). Studies of these disorders have contributed to an appreciation of the broad range of in vivo functions of TBC-containing proteins, although their pathophysiology is not fully understood. Surprisingly, different pathogenic mutations of human TBC1D24 (TBC1 domain family member 24, OMIM 613577) (Figure 1A) are associated with several distinct clinical phenotypes (Table 1), including nonsyndromic deafness segregating as a recessive (referred to as DFNB86 deafness) or dominant trait (DFNA65) (5, 8, 9), early-infantile epileptic encephalopathy 16 (EIEE16) with or without deafness (10), progressive myoclonic epilepsy (PME) (11), familial infantile 4 myoclonic epilepsy (FIME) (10) and DOORS syndrome. DOORS is a multisystem disorder characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures (3). Eight heterozygous de novo microdeletions that include TBC1D24, ATP6V0C and PDPK1, three closely linked genes on chromosome 16p12.3, in eight patients were associated with epilepsy and variable magnitudes of delayed development, intellectual disability and microcephaly (12). The human TBC1D24 gene encodes several alternatively-spliced transcripts (5). The longest transcript of TBC1D24 encodes an N-terminal TBC domain followed by 82 residues that link to a carboxyl-terminus TLDc domain (TBC, LysM, domain catalytic) (Figure 1A). This combination of TBC and TLDc domains is unique in the mammalian proteome. While some TLDc domain- containing proteins, such as OXR1 (oxidative resistance 1), have a neuro- protective role against oxidative stress (13), the function of the TLDc domain of TBC1D24 is unknown. Some TBC domain-containing proteins have well-established roles as GAPs (GTPase-activating protein) which inactivate RABs, an observation revealed first in studies of yeast (14). RABs regulate protein and lipid transport between cellular compartments as well as a variety of other functions such as the biogenesis of organelles (15). However, TBC1D24 is unlikely to be a RAB- GAP, as it lacks critical RQ finger residues that are important for GTP hydrolysis (6, 16, 17). Thus, the biochemical role and cellular functions of TBC1D24 are only partially characterized. Following the first report of a mutation of TBC1D24 associated with epilepsy (4, 10), 55 additional pathological variants of TBC1D24 have been 5 reported. The phenotype associated with each variant and their locations relative to the two protein domains of TBC1D24 are shown in Figure 1A. Presently, there is no settled association of a specific phenotype with either the location of the variant within the gene or with the variant type (missense, splice site or truncating mutation) (18). Mutations of TBC1D24 associated with each of the three different clinically distinctive phenotypes do not cluster in one or the other of the two domains of TBC1D24, but rather are distributed across the primary amino acid sequence (Figure 1A). To explain the spectrum of TBC1D24- associated disorders, it has been suggested that the pathogenic variants of TBC1D24 may damage different binding motifs for its protein partners resulting in different phenotypes (6), may affect one or more alternative transcripts encoding different TBC1D24 protein isoforms (5), or both. In addition, modifiers in the genetic background of an affected individual may influence the penetrance, severity or scope of a TBC1D24-associated disorder (19, 20). Insight into the functions of TBC1D24 can be gained by a combination of observations including its expression profiles in specific cell types, localization within an intracellular compartment, and the composition and complexity of its protein interaction network (6, 21). ARF6 (ADP-ribosylation factor) is a small GTPase crucial for membrane trafficking (10, 22) that binds TBC1D24, which may modulate ARF6 activation that regulates vesicle trafficking (10, 22). In Xenopus laevis, TBC1D24 interacts indirectly with ephrinB2 through Dishevelled and is important for cranial neural crest migration (23). Additionally, mutations of Sky, the Drosophila melanogaster ortholog of mammalian TBC1D24, result
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