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.

项目摘要 TBCK基因中的常染色体隐性突变会导致智障严重程度。我有进一步表征了具有纯合子突变的波多黎各儿童的神经系统表型（P.R126X）在TBCK中，我们设计了Boricua突变。生物学机制基因型 - 表型相关性仍然不清楚。在一个极端，患有严重的Boricua突变的患者发展进行性大脑，小脑和运动神经元萎缩，粗面特征和癫痫。我们命名为严重的综合征TBCK-脑膜炎（TBCKE）。另一方面，其他患者双重TBCK突变具有自闭症和/或智力障碍的临床诊断，而没有证据表明神经变性。 TBCK蛋白的功能尚不清楚，但先前的研究表明 TBCK导致MTORC1信号的下调。 MTORC1途径调节自噬，包括线粒体（线粒体）的靶向降解。我最近报道自噬通量增加和 TBCKE患者成纤维细胞中糖蛋白降解受损，通过激活MTORC1做出反应用L-达氨酸发出信号。我们的成纤维细胞研究表明，TBCKE患者有线粒体功能障碍和线粒体DNA（mtDNA）部署。此外，mtDNA部署预测程度 TBCK疾病的神经系统严重程度。因此，我假设人类神经元中TBCK功能的丧失由于超级自噬清除，导致mtDNA部署和线粒体功能障碍线粒体。为了在更相关的疾病模型中检验这一假设，我建议生成tbck-null 诱导多能干细胞（IPSC）衍生的神经元（INEU）和TBCK - / - 斑马鱼。该提议的目的是解决TBCK功能的丧失是否影响人类神经元中的线粒体功能以及确定mtDNA耗竭是否调节TBCK疾病中神经退行性的严重程度。使用新型疾病模型和独特的工具来测定线粒体功能，我建议解决该问题以下问题：（目标1）缺乏TBCK蛋白的人神经元是否有多余的线粒体？（AIM2） TBCK的功能是什么？我们可以定义其在神经元中的蛋白质蛋白质相互作用吗？（AIM 3）可以tbck- 无斑马鱼模型TBCK疾病的严重程度可变？ MTDNA部署可以调节体内表型？该提案中概述的实验将确定线粒体在TBCKE中的作用以及mtDNA部署是否足以驱动神经退行性表型或仅仅是 Epiphenomenon。这项工作还将提供针对分析神经元线粒体的新技术培训在原位疾病模型中的功能。我还将学会发展并表征斑马鱼模型神经发育疾病。该K02奖的支持将有助于发展我的独立在极好的机构环境中，研究计划作为物理科学家，以实现我的长期目标研究线粒体功能障碍在小儿神经退行性疾病中的作用。