mtDNA heteroplasmy in development and differentiation: an in vitro approach

项目总结 人类线粒体DNA(MtDNA)疾病累及多个组织，临床复杂，常致人死亡。 这些疾病代表了一大组具有不同临床和病理表现的疾病。 以特殊神经元的不正常功能为特征，有时对其造成不可逆转的损害。他们是 从根本上无法治疗，最常见的是损害代谢活跃的组织，如肌肉、视网膜和大脑。 许多这些疾病中神经细胞死亡和相关缺陷的原因和机制，尽管 尚未完全了解，源于线粒体DNA突变或能量水平下降。临床严重程度可能会影响- 根据患病细胞中存在的致病线粒体DNA基因组与正常线粒体DNA基因组的百分比进行鉴定 (异质性)。异质性的起源和时间尚不清楚，但可能包括非常高的比例。 随着时间的推移，由未知的致病线粒体DNA机制引起的细胞内克隆性扩张(同质)。在……里面 此外，无法直接在原位操纵mtDNA一直是理解 致病线粒体DNA对自我更新和分化造成负担。 我们在(A)人类多样性的自我更新和分化方面的最新研究进展和专业知识- 潜在干细胞(HPSC)来源的人神经前体细胞(HNPs)和(B)a的开发和利用 新的线粒体转染法将外源mtDNA导入hNPs，提供了一种强有力的 为分析异质性对神经元发育和神经变性的影响奠定了基础。这个 最重要的假设是hNPs中的mtDNA突变将克隆性扩大，当超过临界 阈值，将导致HNP异常自我更新，影响分化潜能，并有助于线粒体 分化神经元功能障碍。 我们提出了三个具体目标来检验整个假设并调查致病因素的影响。 线粒体DNA(LS-Leigh综合征；LHON-Leber遗传性视神经病变；KSS-Kearns Sayers综合征) 与各种已知的与年龄相关的疾病相匹配的负担，这些疾病表现出线粒体突变或生物变化- 能量学。Aim 1将检验这样的假设：引入致病mtDNA(来自LHON、LS和KSS)将 在hNPs超过特定阈值后影响其自我续订属性。目标2将检验这一假设 致病线粒体DNA水平升高会影响LHON、LS、KSS-hNPs向神经元分化的潜能。 目的3将测试假设，即致病线粒体DNA水平增加将改变线粒体功能 LHON、LS、KSS-HNP来源的神经元。通过涉及新的体外干细胞的互补方法 模型系统、下一代测序方法和线粒体功能表征，我们 希望开发创新的方法来捕获和分析致病线粒体DNA对 神经元分化和生物能量学。 这项研究的科学影响在于一种新的线粒体转染法的可用性。 为了生产具有mtDNA突变的疾病特异性干细胞前体细胞，将首次启用 美国监测和量化线粒体DNA在神经元分化过程中的动力学。一个额外的影响 是基于使用严格的下一代测序方法来定量测定神经营养不良期间的异质性。 区域分化。更广泛地说，虽然神经线粒体疾病首先是这里的目标，但其他基础 研究领域包括代谢性疾病、糖尿病、衰老、自身免疫和心血管疾病 搜索，可能会从拟议的实验方法中受益。

PROJECT SUMMARY Human mitochondrial DNA (mtDNA) disorders affect multiple tissues, are clinically complex and often fatal. These disorders represent a large group of diseases with heterogeneous clinical and pathological expressions characterized by improper functions of and sometimes irreversible damage to specialized neurons. They are fundamentally untreatable and most often impair metabolically active tissues such as muscle, retina and brain. The causes and mechanisms of neuronal cell death and related defects in many of these disorders, although not fully understood, derive from mutations in mtDNA or decline in energy levels. Clinical severity can be influ- enced by the percentage of pathogenic versus normal mtDNA genomes present in affected cells (heteroplasmy). The origins and timing of heteroplasmy are not clear, but may include a very high percentage of intracellular clonal expansion (homoplasmy) by unknown mechanisms of pathogenic mtDNA's over time. In addition, inability to manipulate mtDNA directly in situ has been an impediment to understanding the effects of pathogenic mtDNA burdens on self-renewal and differentiation. Our recent research developments and expertise in (a) self-renewal and differentiation of human pluri- potent stem cell (hPSC)-derived human neural progenitors (hNPs) and (b) development and utilization of a novel mitochondrial transfection methodology for delivering exogenous mtDNA into hNPs, provides a strong foundation for analyzing the effects of heteroplasmy on neuronal development and neurodegeneration. The overarching hypothesis is that mtDNA mutations in hNPs will clonally expand and upon exceeding a critical threshold, will cause abnormal hNP self-renewal, affect differentiation potential and contribute to mitochondrial dysfunction in differentiated neurons. We propose three specific aims to test the overall hypothesis and investigate the effects of pathogenic mtDNA (LS- Leigh's syndrome; LHON- Leber's hereditary optic neuropathy; KSS- Kearns sayers syndrome) burdens which match various known age-related diseases that exhibit mitochondrial mutations or altered bio- energetics. Aim 1 will test the hypothesis that introduced pathogenic mtDNA (from LHON, LS and KSS) will affect self-renewal properties in hNPs after they cross a specific threshold. Aim 2 will test the hypothesis that increased pathogenic mtDNA levels will affect differentiation potential of LHON, LS, KSS-hNPs into neurons. Aim 3 will test the hypothesis that increased pathogenic mtDNA levels will alter the mitochondrial function of LHON, LS, KSS-hNP derived neurons. Through complementary approaches involving novel in vitro stem cell model systems, next generation sequencing methodologies and mitochondrial functional characterizations, we expect to develop innovative approaches to capture and analyze the threshold effects of pathogenic mtDNA on neuronal differentiation and bioenergetics. The scientific impact of this study is the availability of a novel mitochondrial transfection methodology for production of disease-specific stem cell progenitors with mtDNA mutations that will for the first time, enable us to monitor and quantitate mitochondrial DNA dynamics during neuronal differentiation. An additional impact is based on use of stringent next generation sequencing approaches to quantitate heteroplasmy during neu- ronal differentiation. More broadly, while neuro-mitochondrial disorders are targeted here first, other basic research fields, including metabolic disease, diabetes, aging, autoimmune and cardiovascular disease re- search, are likely to benefit from the proposed experimental approaches.