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

基本信息

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
项目类别：
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&apos 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&apos 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

项目摘要

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（由申请人提供）：人类线粒体 DNA (mtDNA) 疾病影响多个组织，临床情况复杂且常常致命。这些疾病代表了一大类具有异质临床和病理表现的疾病，其特征是特殊神经元的功能不正常，有时甚至是不可逆的损伤。尽管尚不完全清楚，但许多此类疾病中神经元细胞死亡和相关缺陷的原因和机制源自 mtDNA 突变或能量水平下降。临床严重程度可能受到受影响细胞中致病性与正常 mtDNA 基因组百分比的影响（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，致病性mtDNA的未知机制导致细胞内克隆扩张（同质性）的比例非常高。此外，无法直接原位操纵线粒体DNA一直阻碍着理解致病性线粒体DNA负荷对自我更新和分化的影响。我们在 (a) 人类多能干细胞 (hPSC) 衍生的人类神经祖细胞 (hNP) 的自我更新和分化以及 (b) 开发和利用新型线粒体转染方法将外源 mtDNA 传递到 hNP 方面的专业知识，为我们提供了强有力的支持。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。总体假设是，hNP 中的 mtDNA 突变将克隆扩展，超过临界阈值后，将导致 hNP 自我更新异常，影响分化潜力，并导致分化神经元中的线粒体功能障碍。我们提出了三个具体目标来检验总体假设并研究致病性线粒体 DNA（LS-Leigh 综合征；LHON-Leber 遗传性视神经病；KSSKearns Sayers 综合征）负担的影响，这些负担与各种已知的年龄相关疾病（表现出线粒体突变或生物能学改变）相匹配。目标 1 将检验以下假设：引入致病性 mtDNA（来自 LHON、LS 和 KSS）将在 hNP 跨越特定阈值后影响其自我更新特性。目标 2 将检验以下假设：致病性 mtDNA 水平增加将影响 LHON、LS、KSS-hNP 分化为神经元的潜力。目标 3 将检验以下假设：致病性 mtDNA 水平增加将改变 LHON、LS、KSS-hNP 衍生神经元的线粒体功能。通过涉及干细胞模型系统、下一代测序和线粒体功能表征的互补方法，我们期望捕获并分析致病性线粒体DNA对神经元分化和生物能学的阈值影响。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定量神经元分化过程中的线粒体 DNA 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这里的首要目标，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研究，未来可能会受益。