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

项目摘要 TBCK基因的常染色体隐性突变导致不同严重程度的智力残疾。我有 进一步表征了具有纯合无效突变的波多黎各儿童的神经系统表型 （p.R126X），我们将其命名为Boricua突变。这一现象背后的生物学机制 基因型-表型相关性仍不清楚。在一个极端，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，其他患者 双等位基因TBCK突变具有自闭症和/或智力残疾的临床诊断，没有证据表明 神经变性TBCK蛋白的功能尚不清楚，但以前的研究表明， TBCK导致mTORC 1信号转导的下调。mTORC 1通路调节自噬，包括 线粒体的定向降解（线粒体自噬）。我最近报告说，自噬通量增加， TBCKE患者成纤维细胞中受损的糖蛋白降解，通过激活mTORC 1来挽救 与L-亮氨酸的信号传导。我们的成纤维细胞研究表明，TBCKE患者有线粒体功能障碍， 线粒体DNA（mtDNA）缺失。此外，线粒体DNA缺失的程度预测了 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 导致线粒体DNA耗竭和线粒体功能障碍，由于过度的自噬清除 线粒体为了在更多的疾病相关模型中检验这一假设，我建议生成TBCK空模型。 诱导多能干细胞（iPSC）衍生的神经元（iNeu）和tbck-/-斑马鱼。本提案的目的是 解决TBCK功能丧失是否影响人类神经元中的线粒体功能，以及 确定mtDNA缺失是否调节TBCK疾病中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 （目的1）缺乏TBCK蛋白的人神经元是否存在过度的线粒体自噬？（目标2） TBCK的功能是什么？我们能定义它在神经元中的蛋白质-蛋白质相互作用吗？(Aim 3）TBCK- 零斑马鱼模型TBCK疾病的可变严重程度？线粒体DNA缺失是否可以调节 体内表型？本提案中概述的实验将确定线粒体在TBCKE中的作用。 以及mtDNA缺失是否足以驱动神经退行性表型，或者仅仅是一种 附带现象这项工作也将提供新技术的培训，用于测定神经元线粒体 在原位疾病模型中发挥作用。我还将学习开发和表征斑马鱼模型， 神经发育疾病从这个K 02奖的支持将有助于提高我的独立性 研究计划作为一个医生科学家在一个极好的机构环境对我的长期目标， 研究线粒体功能障碍在小儿神经退行性疾病中的作用。

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.