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

发育和分化中的线粒体DNA异质性：一种体外方法

• 批准号: 8497267
• 负责人: Shilpa Iyer
• 金额: $ 11.52万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8497267
• 关键词: Address; Affect; Age; Aging; Alzheimer' s Disease; Applications Grants; Autoimmune Diseases; Bioenergetics; Biogenesis; Bioinformatics; Biological Models; Cardiovascular Diseases; Cell Death; Cell Line; Cell model; Cells; Child; Childhood; Clinical; Clonal Expansion; Complex; Defect; Development; Diabetes Mellitus; Diagnostic; Disease; Elderly; Energy Metabolism; Event; Exhibits; Foundations; Future; Genetic; Genome; Genotype; Health; Healthcare; Human; In Situ; In Vitro; Individual; Kearns-Sayre syndrome; Leber' s Hereditary Optic Neuropathy; Leigh Disease; Life; Metabolic Control; Metabolic Diseases; Methodology; Methods; Mission; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Monitor; Mutation; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Outcome; Oxidative Stress; Parkinson Disease; Play; Pluripotent Stem Cells; Point Mutation; Population; Production; Property; Research; Role; Sampling; Severities; Severity of illness; Source; Stem cells; Syndrome; Testing; Time; Tissues; Transfection; Undifferentiated; United States National Institutes of Health; Work; age related; base; cell type; human stem cells; improved; innovation; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial genome; nerve stem cell; neuron development; neuron loss; next generation sequencing; novel; relating to nervous system; research and development; response; self-renewal; tumorigenic

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.

描述(申请人提供)：人类线粒体DNA(MtDNA)疾病影响多个组织，临床复杂，往往是致命的。这些疾病代表了一大组具有不同临床和病理表现的疾病，其特征是特定神经元功能不正常，有时甚至是不可逆转的损害。其中许多疾病的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，但源于mtDNA突变或能量水平下降。临床严重程度可受病变细胞中致病线粒体DNA基因组与正常线粒体DNA基因组的百分比的影响(异质性)。异质性的起源和时间尚不清楚，但可能包括随着时间的推移，由于致病线粒体DNA的未知机制而导致的非常高比例的细胞内克隆性扩张(同质)。此外，无法直接在原位操纵mtDNA一直是理解致病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)来源的人类神经前体细胞(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基因方法将外源mtDNA导入hNPs方面的专业知识，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。最重要的假设是，hNPs中的mtDNA突变会克隆性地扩大，当超过临界阈值时，将导致HNP的异常自我更新，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的目标来检验整个假设，并调查致病mtDNA(LS-Leigh‘s综合征；LHON-Leber’s遗传性视神经病变；KSSKearns Sayers综合征)负担与各种表现线粒体突变或生物能量学改变的已知年龄相关疾病的影响。目的1验证一个假设，即引入致病的mtDNA(来自LHON、LS和KSS)将影响hNPs在超过特定阈值后的自我更新特性。目的2验证致病线粒体DNA水平升高会影响LHON、LS、KSS-hNPs向神经元分化的假说。目的3验证致病线粒体DNA水平升高将改变LHON、LS、KSS-HNP来源神经元线粒体功能的假说。通过干细胞模型系统、下一代测序和线粒体功能表征的互补方法，我们希望捕捉和分析致病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了线粒体转基因方法，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。另一个影响是使用严格的下一代测序方法来量化神经元分化过程中的异质性。更广泛地说，虽然这里首先针对神经线粒体疾病，但其他研究领域，包括代谢性疾病、糖尿病、衰老、自身免疫和心血管疾病研究，可能会在未来受益。