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

项目总结 TBCK基因的常染色体隐性突变会导致不同严重程度的智能障碍。我有过 进一步表征纯合子零突变的波多黎各儿童的神经表型 (p.R126X)，我们命名为Boricua突变。其背后的生物学机制 基因-表型相关性尚不清楚。在一个极端情况下，患有严重Boricua突变的患者 出现进行性脑、小脑和运动神经元萎缩、面部特征粗糙和癫痫。我们 将这种严重综合征命名为TBCK-脑神经元病(TBCKE)。另一方面，其他患有 双等位基因突变有自闭症和/或智能障碍的临床诊断，但没有证据表明 神经退行性变。TBCK蛋白的功能尚不清楚，但先前的研究表明，缺乏 TBCK导致mTORC1信号的下调。MTORC1途径调节自噬，包括 线粒体的靶向降解(有丝分裂)。我最近报告了自噬通量的增加 TBCKE患者成纤维细胞糖蛋白降解受损，可通过激活mTORC1来挽救 用L-亮氨酸发出信号。我们的成纤维细胞研究表明，TBCKE患者存在线粒体功能障碍 线粒体DNA(MtDNA)耗竭。此外，线粒体DNA耗尽的程度预示着 TBCK病的神经学严重程度。因此，我假设人类神经元中TBCK功能的丧失 由于过度的自噬清除导致线粒体DNA枯竭和线粒体功能障碍 线粒体。为了在更多与疾病相关的模型中测试这一假设，我建议生成TBCK-NULL 诱导多能干细胞(IPSC)来源的神经元(INeu)和tbck-/-斑马鱼。这项提议的目标是 探讨TBCK功能丧失是否会影响人类神经元的线粒体功能，并 确定线粒体DNA缺失是否调节了TBCK病的神经退行性变的严重程度。 使用新的疾病模型和独特的工具来分析线粒体功能，我建议解决 以下问题：(目的1)缺乏TBCK蛋白的人神经元是否有过度的有丝分裂？(AIM2) TBCK的作用是什么？我们能定义它在神经元中的蛋白质-蛋白质相互作用吗？(目标3)TBCK能否- 斑马鱼模型无效，TBCK病的严重程度可变？线粒体DNA耗竭能否调节高血压的严重程度 活体表型？这项提案中概述的实验将确定线粒体在TBCKE中的作用 线粒体DNA耗竭是否足以驱动神经退行性表型，还是仅仅是一种 超常现象。这项工作还将提供分析神经元线粒体的新技术方面的培训 在原位疾病模型中的作用。我还将学习开发斑马鱼模型并对其进行表征 神经发育疾病。来自K02奖项的支持将有助于我的独立性发展 作为一名内科科学家，在一流的机构环境中进行研究，朝着我的长期目标 研究线粒体功能障碍在儿童神经退行性疾病中的作用。

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.