mtDNA heteroplasmy in development and differentiation: an in vitro approach

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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.