A Pharmaco-Epigenomic Intervention for the Mitochondrial Disorder MELAS

 描述（由申请人提供）：最常见的线粒体呼吸系统疾病是MELAS（线粒体脑病乳酸酸中毒伴卒中样癫痫），这是一种儿童早期发病的不可治愈的进行性神经退行性疾病。这种罕见病引起异质性临床症状，如脑病、癫痫发作、中风样发作、认知障碍、慢性乳酸酸中毒和肌病。大多数MELAS患者在唾液酸编码的tRNALeu/UUR基因中存在母系遗传突变（A3243G），该突变影响负责ATP合成的氧化磷酸化（OXPHOS）系统。在MELAS细胞中，多拷贝线粒体基因组群体是异质的，突变型mtDNA和野生型（WT）mtDNA的比例可变，这种状态称为异质性。只有当突变负荷超过异质性的特定阈值时，具有MELAS突变的个体才会出现症状。目前，没有治疗选择可用于预防疾病的进展，导致严重残疾，预后不良和过早死亡。我们提出的新型药理学方法使用丁酸盐通过促进线粒体生物发生和最大化残留ATP输出来促进核和线粒体代谢重编程。使用来自两名MELAS患者皮肤活检的原代成纤维细胞，我们进行了基于Affymetrix的全基因组微阵列分析，揭示了暴露于丁酸盐后线粒体生物发生和生物能量学途径的富集。从活细胞共聚焦显微镜，我们发现丁酸盐恢复线粒体质量和池的生物能胜任的线粒体在MELAS成纤维细胞。在健康的神经元细胞中，我们发现丁酸盐通过表达必需的核编码调节剂诱导线粒体生物合成，并增加生物能线粒体和ATP水平。因此，我们的集体初步数据验证了我们的药物表观基因组方法，并为拟议的研究建立了原理证明。我们假设丁酸盐可以增加20例MELAS患者皮肤成纤维细胞的功能性线粒体质量。来自这些患者的成纤维细胞，每个都具有不同的核背景和异质性负荷，将确保研究结果超出病例研究，成为统计学上合理和广泛适用的报告。我们将测试丁酸盐是否：（目的1）诱导有利的线粒体生物发生，从而将异质性向健康的线粒体转移;和（目的2）通过优化OXPHOS活性和向脂肪酸β氧化的代谢转移来最大化ATP输出。我们期望确定最有希望的丁酸盐候选物，以减轻MELAS疾病的症状。这项拟议中的研究将为未来与儿童国家医学中心和北 美国线粒体疾病联盟，与NINDS转化研究办公室针对R21探索性项目发布的PAR-13 - 023一致。

 DESCRIPTION (provided by applicant): The most common mitochondrial respiratory disorder is MELAS (Mitochondrial Encephalopathy Lactic Acidosis with Stroke-like Episodes), an incurable progressive neurodegenerative disease with early childhood onset. This orphan disease causes heterogeneous clinical symptoms, such as encephalopathy, seizures, stroke-like episodes, cognitive impairment, chronic lactic acidosis, and myopathy. Most MELAS patients harbor a maternally inherited mutation (A3243G) in the mitochondrial-encoded tRNALeu/UUR gene, which affects the oxidative phosphorylation (OXPHOS) system responsible for ATP synthesis. In MELAS cells, the multi-copy mitochondrial genome population is heterogeneous, with variable ratios of mutant mtDNAs and wild type (WT) mtDNAs, a state known as heteroplasmy. Individuals with the MELAS mutation become symptomatic only when the mutant load exceeds a certain threshold of heteroplasmy. Currently, no therapeutic options are available to prevent the progression of the disease, resulting in significant disability, a poo prognosis, and premature death. Our proposed novel pharmacological approach uses butyrates to promote nuclear and mitochondrial metabolic reprogramming by boosting mitochondrial biogenesis and maximizing residual ATP output. Using primary fibroblasts derived from a skin biopsy of two MELAS patients, we performed an Affymetrix-based genome-wide microarray analysis that revealed enrichment of pathways for mitochondrial biogenesis and bioenergetics upon exposure to butyrates. From live cell confocal microscopy, we found that butyrates restored the mitochondrial mass and the pool of bioenergetically competent mitochondria in MELAS fibroblasts. In healthy neuronal cells, we found that butyrates induced mitochondrial biogenesis via expression of essential nuclear-encoded regulators and augmented the pool of bioenergetic mitochondria and ATP levels. Thus, our collective preliminary data validate our pharmaco-epigenomic approach and establish proof-of-principle for the proposed studies. We hypothesize that butyrates can augment the functional mitochondrial mass in skin fibroblasts from 20 MELAS patients. Fibroblasts from this number of patients, each with different nuclear backgrounds and heteroplasmic loads, will ensure the findings extend beyond a case study into a statistically sound and broadly applicable report. We will test whether butyrates: (Aim 1) induce favorable mitochondrial biogenesis, thereby shifting heteroplasmy toward healthy mitochondria; and (Aim 2) maximize ATP output via optimization of OXPHOS activity and a metabolic shift toward fatty acid beta oxidation. We anticipate identifying the most promising butyrate candidate for alleviating the symptoms of the MELAS disease. The proposed study will set the stage for future clinical studies with the Children's National Medical Center and the North American Mitochondrial Disease Consortium, in concordance with the PAR-13-023 issued by the NINDS Office of Translational Research for R21 exploratory projects.