Novel mechanism of neural and muscular degeneration

ANT1是腺嘌呤核苷酸易位酶的肌肉/心脏/中枢神经系统(CNS)亚型，即 主要参与线粒体内膜(IMM)上的ATP/ADP交换。不断增加的 ANT1错义突变的数量被发现会导致影响骨骼肌和 中枢神经系统。这些疾病通常表现为部分mtdna缺失和轻微的 生物能量缺陷。这些疾病中神经肌肉损伤的机制还知之甚少。 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ANT1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 应激(MPOS)。MPOS的特点是未进口的有毒物质积累和聚集 细胞质中的线粒体前蛋白。这些发现导致了一个中心假设，即突变的ANT1 主要影响线粒体蛋白进口。这导致mPOS在胞浆中发挥着重要的作用 在诱导神经和肌肉退化方面的作用。部分mtDNA缺失独立于 核苷酸转运活性，可能是线粒体蛋白进口减少的次要损害附带因素。 在这一应用中，我们建议直接在小鼠模型中检验这一假设。我们成功地生成了 表达ANT1错误折叠变体的敲入(KI)小鼠品系。初步研究表明，这些小鼠 形成与神经和肌肉退行性变一致的表型。在具体目标1中，我们将使用这些 独特的实验模型来验证错误折叠的ANT1诱导神经和肌肉的假说 不依赖于核苷酸转运的变性和线粒体DNA不稳定性。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ANT1-Ki小鼠中开发的实验工具来测试 假设错误折叠的ANT1(或酵母中的Aac2)会导致结构和功能上的损害 线粒体蛋白的输入机制，并诱导胞浆中的mPOS。在具体目标3中，我们将确定 保护细胞免受ANT1诱导的蛋白输入应激和mPOS的机制。成功之路 该项目将建立与mPOS相关的蛋白质进口应激小鼠模型。尤其是，验证 MPOS模型将有助于协调许多神经和蛋白质的线粒体和蛋白恒定通路 肌肉退行性疾病。最后，研究结果可能会对理解 和治疗由ANT1引起的疾病，以及许多其他直接或间接影响 线粒体蛋白进口。

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.