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（由申请人提供）：人线粒体DNA（mtDNA）疾病影响多个组织，在临床上是复杂的，通常是致命的。这些疾病代表了一大批具有异质临床和病理表达的疾病，其特征是对专门神经元的功能不当，有时是不可逆的损害。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，尽管尚未完全理解，但它来自mtDNA中的突变或能量水平下降。临床严重程度可能受影响细胞（异质）中存在的致病性和正常mtDNA基因组的百分比。异质的起源和时机尚不清楚，但可能包括通过病原mtDNA随着时间的未知机制的极高比例的细胞内克隆膨胀（同型）。另外，无法直接在原位操纵mtDNA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（HNP）和（b）开发和利用新型线粒体转染方法的发展和利用方面的（HPSC）自我更新和分化方面的专业知识，为将外源性mtDNA输送到HNP中，为分析Neeteropery的效果分析了HETRODERY的基础。总体假设是，HNP中的mtDNA突变将在克隆上扩展并超过临界阈值后，会导致异常的HNP自我更新，影响分化潜力并导致分化神经元中线粒体功能障碍。我们提出了三个具体旨在检验整体假设并研究致病性mtDNA（LS-LEIGH综合征； Lhon-Leber的遗传性视神经病； KSSKEARNS SAIERS综合症）的影响，这些疾病与各种已知的年龄相关疾病相匹配，这些疾病表现出了线粒体突变或更改的biioenergetics。 AIM 1将检验以下假设，即引入致病性mtDNA（来自LHON，LS和KSS）将影响HNP的自我更新特性后，它们越过了特定的阈值。 AIM 2将检验以下假设：升高的致病mtDNA水平将影响LHON，LS，KSS-HNP的分化潜力到神经元中。 AIM 3将检验以下假设：升高的致病mtDNA水平将改变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的互补方法，下一代测序和线粒体功能特征，我们希望捕获和分析致病性mtDNA对神经元分化和生物能学的阈值阈值的影响。这项研究的科学影响是使用线粒体转染方法，该方法将首次将我们监测和定量神经元分化过程中的mtDNA动力学。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体。更广泛地说，尽管在这里神经界疾病首先是针对的，但其他研究领域，包括代谢疾病，糖尿病，衰老，自身免疫性和心血管疾病研究，可能会在未来受益。