Novel mechanism of neural and muscular degeneration

Ant 1是腺嘌呤核苷酸移位酶的肌肉/心脏/中枢神经系统（CNS）同种型， 主要参与ATP/ADP跨线粒体内膜（IMM）的交换。越来越 Ant 1中的许多错义突变被发现导致影响骨骼肌的显性疾病， 中枢神经系统这些疾病通常表现为部分线粒体DNA缺失和轻度 生物能量缺陷神经肌肉损伤在疾病中的机制知之甚少。 有趣的是，我们最近在酵母和培养的人类细胞中的研究表明，突变的Ant 1是错误折叠的。 这通过一种新的机制导致细胞死亡，我们称之为线粒体前体过度积累 压力（mPOS）。mPOS的特点是毒性积累和聚集的非输入性 线粒体前体蛋白在胞质溶胶中。这些发现导致了一个核心假设，即突变的Ant 1 主要影响线粒体蛋白质输入。这导致胞质溶胶中的mPOS，其在细胞内起重要作用。 在诱导神经和肌肉退化中的作用。线粒体DNA部分缺失的发生与 核苷酸转运活性，可能是线粒体蛋白质输入减少的继发性损害。 在本申请中，我们建议在小鼠模型中直接检验这一假设。我们成功地生成了 表达Ant 1的错误折叠变体的敲入（KI）小鼠系。初步研究表明，这些老鼠 出现与神经和肌肉退化一致的表型。在具体目标1中，我们将使用这些 独特的实验模型来测试错误折叠的Ant 1诱导神经和肌肉的假设， 线粒体DNA的变性和不稳定性独立于核苷酸转运。在具体目标2中，我们将使用各种 我们在酵母、培养的人类细胞和Ant 1-KI小鼠中开发的实验工具， 错误折叠的Ant 1（或酵母中的Aac 2）导致细胞结构和功能损伤的假说。 线粒体蛋白质输入机器并诱导胞质溶胶中的mPOS。在具体目标3中，我们将确定 保护细胞免受Ant 1诱导的蛋白质输入应激和mPOS的机制。成功 项目将建立与mPOS相关的蛋白质输入应激的小鼠模型。特别是，验证 mPOS模型将有助于协调许多神经和神经系统中的线粒体和蛋白质稳定途径， 肌肉退化性疾病最后，这些结果可能对理解 和Ant 1诱导的疾病的治疗，以及直接或间接影响 线粒体蛋白输入。

Ant1 is the muscle/heart/central nervous system (CNS) isoform of adenine nucleotide translocase that is primarily involved in ATP/ADP exchange across the inner mitochondrial membrane (IMM). An increasing number of missense mutations in Ant1 are found to cause dominant diseases that affect skeletal muscle and the central nervous system. These diseases are commonly manifested by fractional mtDNA deletions and mild bioenergetic defects. The mechanism of neuromuscular damage in the diseases is poorly understood. Interestingly, our recent studies in yeast and cultured human cells suggested that the mutant Ant1 is misfolded. This leads to cell death by a novel mechanism that we named mitochondrial Precursor Overaccumulation Stress (mPOS). mPOS is characterized by the toxic accumulation and aggregation of un-imported mitochondrial preproteins in the cytosol. These findings led to the central hypothesis that the mutant Ant1 primarily affects mitochondrial protein import. This results in mPOS in the cytosol, which plays an important role in inducing neural and muscular degeneration. Fractional mtDNA deletions occur independent of nucleotide transport activity, likely as a secondary damage collateral to reduced mitochondrial protein import. In this application, we propose to directly test this hypothesis in mouse models. We successfully generated knock-in (KI) mouse lines expressing misfolded variants of Ant1. Preliminary studies indicated that these mice develop phenotypes consistent with neural and muscular degeneration. In Specific Aim 1, we will use these unique experimental models to test the hypothesis that misfolded Ant1 induces neural and muscular degeneration and mtDNA instability independent of nucleotide transport. In Specific Aim 2, we will use various experimental tools that we developed in yeast, cultured human cells and the Ant1-KI mice to test the hypothesis that the misfolded Ant1 (or Aac2 in yeast) causes structural and functional damage to the mitochondrial protein import machinery and induces mPOS in the cytosol. In Specific Aim 3, we will determine the mechanisms that protect cells against Ant1-induced protein import stress and mPOS. Success of the project will establish a mouse model of protein import stress associated with mPOS. Particularly, validation of the mPOS model would help reconciling the mitochondrial and proteostatic pathways in many neural and muscular degenerative diseases. Finally, the results could have important implications for the understanding and therapy of Ant1-induced diseases, as well as many other clinical conditions that directly or indirectly affect mitochondrial protein import.