TBCK Encephaloneuronopathy: establishing the role of mitochondrial dysfunction in promoting neurodegeneration

TBCK 脑神经病：确定线粒体功能障碍在促进神经退行性变中的作用

基本信息

批准号：
10439444
负责人：
XILMA R ORTIZ-GONZALEZ
金额：
$ 19.19万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10439444
关键词：
Address Affect Age Atrophic Autism Diagnosis Automobile Driving Autophagocytosis Biogenesis Biological Biological Assay Biology Brain Branched-Chain Amino Acids CRISPR/Cas technology Child Childhood Clinical Co-Immunoprecipitations Complex DNA copy number Data Disease Disease model Down-Regulation Encephalopathies Environment Epilepsy FRAP1 gene Face Fibroblasts Fishes Frameshift Mutation Frequencies Functional disorder Future Genes Genetic Diseases Genotype Glycoprotein Degradation Pathway Goals Homidium Bromide Human Impairment In Situ Independent Scientist Award Induced pluripotent stem cell derived neurons Intellectual functioning disability Label Lead Learning Leucine Measures Mitochondria Mitochondrial DNA Modeling Motor Neurons Mutation NADH Names Nerve Degeneration Nervous System Physiology Neurodegenerative Disorders Neurologic Neurologic Symptoms Neurons Neurosciences Pathway interactions Patients Phenotype Phosphotransferases Physicians Physiological Play Positioning Attribute Protein Kinase Proteins Proteomics Puerto Rican Quality Control Reporting Research Respiration Respiratory Chain Role Scientist Seizures Severities Signal Transduction Spinal Muscular Atrophy Symptoms Syndrome Techniques Testing Therapeutic Training Treatment Efficacy Work Zebrafish autistic clinical diagnosis experimental study fluorescence lifetime imaging gene replacement in vivo insight loss of function mitochondrial dysfunction nervous system disorder neurodegenerative phenotype neurogenetics neuromuscular new therapeutic target novel novel therapeutics null mutation overexpression programs protein expression protein protein interaction tool vector

项目摘要

PROJECT SUMMARY Autosomal recessive mutations in the TBCK gene cause intellectual disability of variable severity. I have further characterized the neurologic phenotype of Puerto Rican children with a homozygous null mutation (p.R126X) in TBCK, which we designated the Boricua mutation. The biological mechanism underlying the genotype-phenotype correlations remain unclear. On one extreme, patients with the severe Boricua mutation develop progressive brain, cerebellar and motor neuron atrophy, coarse facial features and epilepsy. We named this severe syndrome TBCK-encephaloneuronopathy (TBCKE). On the other hand, other patients with biallelic TBCK mutations have clinical diagnosis of autism and/or intellectual disability, without evidence of neurodegeneration. The function of TBCK protein is unknown, but previous studies have shown absence of TBCK leads to downregulation of mTORC1 signaling. The mTORC1 pathway regulates autophagy, including the targeted degradation of mitochondria (mitophagy). I recently reported increased autophagic flux and impaired glycoprotein degradation in TBCKE patients’ fibroblasts, which was rescued by activating mTORC1 signaling with L-leucine. Our fibroblasts studies suggests that TBCKE patients have mitochondrial dysfunction and mitochondrial DNA (mtDNA) depletion. Furthermore, the degree of mtDNA depletion predicts the neurologic severity of TBCK disease. Therefore, I hypothesize that loss of function of TBCK in human neurons leads to mtDNA depletion and mitochondrial dysfunction due to excessive autophagic clearance of mitochondria. To test this hypothesis in more disease relevant models, I propose to generate TBCK-null induced pluripotent stem cell (iPSC) derived neurons (iNeu) and tbck-/- zebrafish. The goal of this proposal is to address whether loss of function of TBCK affects mitochondrial function in human neurons, and to determine whether mtDNA depletion modulates the severity of neurodegeneration in TBCK disease. Using novel disease models and unique tools to assay mitochondrial function, I propose to address the following questions: (Aim 1) Do human neurons lacking TBCK protein have excessive mitophagy? (Aim2) What is the function of TBCK? Can we define its protein-protein interactions in neurons? (Aim 3) Can TBCK- null zebrafish model the variable severity of TBCK disease? Can mtDNA depletion modulate the severity of the phenotype in vivo? The experiments outlined in this proposal will determine the role of mitochondria in TBCKE and whether mtDNA depletion is sufficient to drive the neurodegenerative phenotype or merely an epiphenomenon. This work will also provide training in novel techniques for assaying neuronal mitochondrial function in disease models in situ. I will also learn to develop and characterize zebrafish models of neurodevelopmental disease. Support from this K02 award will be instrumental in growing my independent research program as a physician scientist in a superb institutional environment towards my long-term goal of studying the role of mitochondrial dysfunction in pediatric neurodegenerative disorders.

项目概要 TBCK 基因的常染色体隐性突变导致我患有不同程度的智力障碍。进一步表征了具有纯合无效突变的波多黎各儿童的神经表型（p.R126X）在 TBCK 中，我们将其命名为 Boricua 突变的生物学机制。基因型与表型的相关性仍不清楚。在一种极端情况下，患有严重 Boricua 突变的患者。出现进行性大脑、小脑和运动神经元萎缩、面部特征粗糙和癫痫。将这种严重综合征命名为 TBCK 脑神经病 (TBCKE)。双等位基因 TBCK 突变具有自闭症和/或智力障碍的临床诊断，但没有证据表明 TBCK 蛋白的功能尚不清楚，但之前的研究表明其不存在。 TBCK 导致 mTORC1 信号传导下调 mTORC1 通路调节自噬，包括。我最近报告了线粒体的定向降解（线粒体自噬）和自噬通量的增加。 TBCKE 患者成纤维细胞中糖蛋白降解受损，可通过激活 mTORC1 来挽救我们的成纤维细胞研究表明 TBCKE 患者存在线粒体功能障碍。此外，mtDNA 耗尽的程度可预测。 TBCK 疾病的神经系统严重性因此，我对人类神经元中 TBCK 功能的丧失感到困扰。由于过度自噬清除，导致 mtDNA 耗竭和线粒体功能障碍为了在更多疾病相关模型中测试这一假设，我建议生成 TBCK-null。诱导多能干细胞 (iPSC) 衍生神经元 (iNeu) 和 tbck-/- 斑马鱼该提案的目标是解决 TBCK 功能丧失是否影响人类神经元线粒体功能的问题，并确定 mtDNA 耗竭是否会调节 TBCK 疾病神经退行性变的严重程度。我建议使用新的疾病模型和独特的线粒体检测功能工具来解决以下问题：（目标 1）缺乏 TBCK 蛋白的人类神经元是否存在过度线粒体自噬？ TBCK 的功能是什么？我们能否定义其在神经元中的蛋白质-蛋白质相互作用？（目标 3）TBCK- 可以吗？ null 斑马鱼模型 TBCK 疾病的不同严重程度线粒体 DNA 缺失能否调节该疾病的严重程度？本提案中概述的实验将确定线粒体在 TBCKE 中的作用以及 mtDNA 耗尽是否足以驱动神经退行性表型或仅仅是一种这项工作还将提供检测神经元线粒体新技术的培训。我还将学习开发和表征斑马鱼模型。神经发育疾病。K02 奖的支持将有助于增强我的独立性。作为一名医师科学家，在一流的机构环境中进行研究计划，以实现我的长期目标研究线粒体功能障碍在儿科神经退行性疾病中的作用。