mtDNA heteroplasmy in development and differentiation: an in-vitro approach

描述（由申请人提供）：人类线粒体DNA（mtDNA）疾病影响多个组织，临床上复杂且通常致命。这些疾病代表了一大批具有异质临床和病理表现的疾病，其特征是特化神经元的功能不正常，有时是不可逆的损伤。许多这些疾病中神经元细胞死亡和相关缺陷的原因和机制，虽然尚未完全了解，但源于mtDNA的突变或能量水平的下降。临床严重程度可受到受影响细胞中存在的致病性与正常mtDNA基因组的百分比（异质性）的影响。异质性的起源和时间尚不清楚，但可能包括一个非常高百分比的细胞内克隆扩增（同质），由未知的致病mtDNA的机制随时间推移。此外，无法直接在原位操纵mtDNA一直是理解致病性mtDNA负担对自我更新和分化的影响的障碍。我们在(a)人类多能干细胞（hPSC）衍生的人类神经祖细胞（hNPs）的自我更新和分化以及(b)开发和利用一种新的线粒体转染方法，将外源mtDNA传递到hNPs中，为分析异质性对神经元发育和神经变性的影响提供了坚实的基础。总体假设是，hNP中的mtDNA突变会无性扩展，并在超过临界阈值时，会导致异常的hNP自我更新，影响分化潜能，并导致分化神经元的线粒体功能障碍。我们提出了三个具体目标来检验总体假设并研究致病性mtDNA （LS- Leigh's综合征；LHON- Leber's遗传性视神经病变；KSSKearns Sayers综合征）负担的影响，这些负担与各种已知的表现出线粒体突变或生物能量改变的年龄相关疾病相匹配。目的1将检验引入的致病mtDNA（来自LHON、LS和KSS）在hNPs超过特定阈值后会影响其自我更新特性的假设。目的2将检验致病mtDNA水平升高是否会影响LHON、LS、KSS-hNPs向神经元分化的潜力。目的3将检验致病mtDNA水平升高将改变LHON、LS、KSS-hNP衍生神经元线粒体功能的假设。通过包括干细胞模型系统、下一代测序和线粒体功能表征在内的互补方法，我们希望捕获和分析致病mtDNA对神经元分化和生物能量学的阈值效应。这项研究的科学影响是使用线粒体转染方法，这将首次使我们能够监测和量化神经元分化过程中的mtDNA动态。另一个影响是基于使用严格的下一代测序方法来定量神经元分化过程中的异质性。更广泛地说，虽然神经线粒体疾病是首先针对的，但其他研究领域，包括代谢疾病、糖尿病、衰老、自身免疫和心血管疾病的研究，可能会在未来受益。

DESCRIPTION (provided by applicant): 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. 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 influenced 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 expertise in (a) self-renewal and differentiation of human pluripotent 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; KSSKearns Sayers syndrome) burdens which match various known age-related diseases that exhibit mitochondrial mutations or altered bioenergetics. 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 stem cell model systems, next generation sequencing and mitochondrial functional characterizations, we expect to capture and analyze the threshold effects of pathogenic mtDNA on neuronal differentiation and bioenergetics. The scientific impact of this study is use of a mitochondrial transfection methodology that will for the first time, enable us to monitor and quantitate mtDNA dynamics during neuronal differentiation. An additional impact is based on use of stringent next generation sequencing approaches to quantitate heteroplasmy during neuronal differentiation. More broadly, while neuro-mitochondrial disorders are targeted here first, other research fields, including metabolic disease, diabetes, aging, autoimmune and cardiovascular disease research, are likely to benefit in the future.