Barth syndrome: Evaluating cardiolipin mediated tissue specific oxidative phosphorylation dysfunction

项目摘要 Barth综合征（BTHS）是一种X连锁的先天性线粒体磷脂代谢缺陷，由以下原因引起： tafazzin基因（TAZ）。TAZ编码一种转酰酶，参与最终的重构步骤， 心磷脂（CL），一种定位于线粒体内膜的磷脂，在嵴中起关键作用 线粒体呼吸链的形成、组织和凋亡级联。TAZ和CL 在所有组织中普遍表达，但CL含量具有组织特异性;脑CL的特征在于 由包括多不饱和链在内的多种酰基链组成，而在心脏和骨骼中， 肌肉CL的主要特征是四氢化萘油酰基形式。作为典型的大多数初级线粒体 BTHS是一种多系统疾病，以心肌病、骨骼肌病和 中性粒细胞减少症等特征。然而，与许多线粒体疾病形成鲜明对比的是，BTHS 最小的神经负担。因此，了解BTHS的组织特异性机制， 有可能提供更好的了解特定CL含量对线粒体功能的作用，以及 为BTHS提供了新的治疗靶点。在我对一种新的TAZ缺陷细胞模型的初步研究中，我 发现呼吸链的复合物I在蛋白质和代谢水平上失调。在此基础上， 我假设复合物I是氧化磷酸化（OXPHOS）功能障碍的主要部位， BTHS中受影响的组织。BTHS中复合物I的失调是一个特别有趣的发现，因为 其他复杂I型疾病，包括Leigh病，对神经系统有显着影响。因此，我进一步 假设神经组织的组织特异性CL含量使其免于这种功能障碍。到 针对这一假设，我提出了两个具体目标。目的1：确定TAZ缺乏的影响， 因此CL含量异常，对复合物I功能有影响。我将首先研究减少的影响， 复合物I蛋白的表达对酶稳定性和功能的影响。然后我将决定大会是否 因子NDUFAF 1在复合物I蛋白表达减少中起着重要作用，如果减少的 NDUFAF 1的表达是由于在转录水平的失调。目标2：了解 CL含量对BTHS病理生理学的组织特异性。我将首先区分TAZ缺陷的iPSCs， iPSC TAZ 50，以表征和比较复合物I失调 通过评估NDUFAF 1蛋白表达、NADH/NAD+比率和复合物I酶稳定性/功能。然后， 我将确定与WT神经元相比，TAZ缺陷神经元是否显示改变的CL，以及是否缺乏TAZ缺陷神经元。 TAZ缺陷神经元中改变的CL含量反映了TAZ在神经元CL重塑中的较小作用。这 项目提案结合了基础科学和转化方法，以解开细胞病理生理学， 确定罕见疾病的潜在治疗靶点。我在完成这项研究时将获得的技能将 为我的职业生涯做好准备，为罕见的遗传疾病做好治疗发现。

Project Summary Barth Syndrome (BTHS), is an X-linked inborn error of mitochondrial phospholipid metabolism, caused by variants in the gene tafazzin (TAZ). TAZ encodes for a transacylase involved in the final remodeling step of cardiolipin (CL), a phospholipid localized to the inner mitochondrial membrane with key roles in cristae formation, organization of the mitochondrial respiratory chain, and in the apoptotic cascade. Both TAZ and CL are ubiquitously expressed in all tissues, however the CL content is tissue specific; brain CL is characterized by a diversified array of acyl chains including polyunsaturated chains, whereas in the cardiac and skeletal muscle CL is predominantly characterized by the tetralinoleoyl form. As is typical of most primary mitochondrial diseases, BTHS is a multisystem disorder, characterized by cardiomyopathy, skeletal myopathy, and neutropenia among other features. However, in sharp contrast to many mitochondrial diseases, BTHS has minimal neurological burden. Thus, understanding the mechanisms of tissue specificity in BTHS has the potential to provide great insight into the role of specific CL content on mitochondrial function, as well as to offer novel treatment targets for BTHS. In my preliminary work with a novel TAZ deficient cellular model, I found that complex I of the respiratory chain is dysregulated at the protein and metabolic level. Based on this, I hypothesize that complex I is a major site of oxidative phosphorylation (OXPHOS) dysfunction in tissues affected in BTHS. Dysregulation of complex I in BTHS is a particularly interesting finding, because other complex I disorders, including Leigh Disease, have a significant neurological impact. Therefore, I further hypothesize that the tissue specific CL content of neurologic tissue spares it from this dysfunction. To address this hypothesis, I propose two specific aims. Aim 1: To determine the impact of TAZ deficiency, and therefore abnormal CL content, on complex I function. I will first investigate the effect of the reduced expression of Complex I proteins on enzyme stability and function. Then I will determine whether the assembly factor NDUFAF1 plays a prominent role in the reduced expression of complex I proteins, and if the reduced NDUFAF1 expression is due to dysregulation at the transcriptional level. Aim 2: To understand the role of tissue specificity of CL content on the pathophysiology of BTHS. I will first differentiate TAZ deficient iPSCs, iPSC TAZ∆50, into cardiomyocytes and neurons in order to characterize and compare complex I dysregulation by assessing NDUFAF1 protein expression, NADH/NAD+ ratio, and complex I enzyme stability/function. Then, I will determine if TAZ deficient neurons display altered CL as compared to WT neurons, and if the lack of altered CL content in TAZ deficient neurons reflects a smaller role for TAZ in neuronal CL remodeling. This project proposal combines basic science and translational approaches to unravel cellular pathophysiology and identify potential therapeutic targets for a rare disease. The skills I will acquire as I complete this research will prepare me for a career in therapeutic discovery for rare genetic conditions.