Chemical Approaches to Rescue Human Mitochondrial Disease Mutations

摘要 线粒体编码的DNA或核编码的线粒体基因中的突变可导致线粒体DNA的缺失。 线粒体蛋白的功能表型。线粒体疾病（MD）是一组异质性的疾病， 具有导致多种器官表型的几种不同突变的病症。一般来说，线粒体 疾病的特征在于具有高能量需求的器官，例如心脏， 骨骼肌和大脑。此外，患有MD的患者通常患有牙科疾病， 独特的管理。许多麻醉药物抑制线粒体功能，这对神经外科手术提出了挑战。 MD患者的治疗。MD患者可发展为进行性呼吸衰竭和乳酸 酸中毒，麻醉后会加重。除了牙科治疗问题， 在MD患者中观察到骨健康状况差，例如骨质减少和骨质疏松。 这项拟议的研究旨在发现新的遗传靶点，这些靶点可以在药物上靶向， 使携带线粒体突变的细胞恢复功能和存活。一种高通量的小分子 在线粒体脑肌病，乳酸酸中毒， 和中风样发作（MELAS）综合征进行，以发现导致增加的化合物， 细胞在营养缺乏的条件下存活。强力霉素被确定为最热门的药物之一。 有趣的是，多西环素也在Rieske的高通量小分子筛选中被鉴定（复合物 III）使用与MELAS相同的细胞存活测定的突变成纤维细胞和ND 1（复合物I）突变胞质杂交体 屏幕在三个独立的小分子存活筛选中，只有两个家族的化合物被发现。 鉴定为阳性命中：靶向线粒体核糖体的抗生素和mTOR抑制剂。mTOR 先前已经在线粒体疾病中研究了线粒体，因此，该项目将重点关注 多西环素治疗线粒体疾病疗效基于细胞间的常见屏幕点击 线，我假设多西环素能够促进生存线粒体疾病独立的遗传 突变此外，我相信这是通过减少线粒体蛋白质合成，调节 应激反应因子，以及抑制细胞死亡途径。 拟议的实验将首先旨在确定强力霉素促进细胞存活的机制 在线粒体突变的细胞中。NDUfs4基因敲除小鼠，一种成熟的线粒体突变小鼠 具有强烈神经元退化和短寿命的患者将用强力霉素治疗，以评估 体内多西环素。这项工作将为一种新的治疗策略奠定基础，使MD受益 患者

Abstract Mutations in the mitochondrial encoded DNA or in the nuclear encoded mitochondrial genes can lead to loss of function phenotypes in mitochondrial proteins. Mitochondrial diseases (MD) are a heterogeneous group of disorders with several different mutations leading to a variety of organ phenotypes. In general, mitochondrial diseases are characterized by diminished function of organs with high energetic demands, such as the heart, skeletal muscle, and brain. Additionally, patients with MDs often present with dental disease that requires unique management. Many anesthetic drugs suppress mitochondrial function, which presents a challenge in the treatment of MD patients. Patients with MD can develop progressive respiratory failure and lactic acidosis, which are exacerbated with anesthesia. In addition to dental treatment concerns, manifestations of poor bone heath; such as osteopenia and osteoporosis, have been observed in MD patients. The proposed study aims to discover novel genetic targets that can be pharmaceutically targeted in order to restore function and survival to cells carrying a mitochondrial mutation. A high-throughput small molecule screen on a trans-mitochondrial hybrid (cybrid) model of mitochondrial encephalomyopathy, lactic-acidosis, and stroke-like episodes (MELAS) syndrome was performed to discover compounds that lead to an increase in cell survival in conditions of nutrient deprivation. Doxycycline was identified amongst the top hits. Interestingly, doxycycline was also identified in a high-throughput small molecule screen on Rieske (complex III) mutant fibroblasts and ND1 (complex I) mutant cybrids using the same cell survival assay as the MELAS screen. Across the three independent small molecule survival screens, only two families of compounds were identified as positive hits: antibiotics targeting the mitochondrial ribosome and mTOR inhibitors. mTOR inhibiton has been previously studied in mitochondrial disease, therefore, this project will focus on the efficacy of doxycycline as a therapy in mitochondrial disease. Based off the common screen hits across cell lines, I hypothesize that doxycycline is able promote survival in mitochondrial disease independent of genetic mutation. Further, I believe this is occurring through decreases in mitochondrial protein synthesis, modulation of stress response factors, and inhibition of the cell death pathway. The proposed experiments will first aim to identify the mechanism by which doxycycline promotes cell survival in cells with mitochondrial mutations. Ndufs4 knockout mice, a well-established mitochondrial mutant mouse with strong neuronal deterioration and short lifespan, will be treated with doxycycline to evaluate the efficacy of doxycycline in vivo. The proposed work will lay the groundwork for a novel therapeutic strategy to benefit MD patients.