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 expressionscharacterized by improper functions of and sometimes irreversible damage to specialized neurons. They arefundamentally 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, althoughnot 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 percentageof intracellular clonal expansion (homoplasmy) by unknown mechanisms of pathogenic mtDNA's over time. Inaddition, inability to manipulate mtDNA directly in situ has been an impediment to understanding the effects ofpathogenic 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 anovel mitochondrial transfection methodology for delivering exogenous mtDNA into hNPs, provides a strongfoundation for analyzing the effects of heteroplasmy on neuronal development and neurodegeneration. Theoverarching hypothesis is that mtDNA mutations in hNPs will clonally expand and upon exceeding a criticalthreshold, will cause abnormal hNP self-renewal, affect differentiation potential and contribute to mitochondrialdysfunction in differentiated neurons. We propose three specific aims to test the overall hypothesis and investigate the effects of pathogenicmtDNA (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) willaffect self-renewal properties in hNPs after they cross a specific threshold.
Aim 2 will test the hypothesis thatincreased 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 ofLHON, LS, KSS-hNP derived neurons. Through complementary approaches involving novel in vitro stem cellmodel systems, next generation sequencing methodologies and mitochondrial functional characterizations, weexpect to develop innovative approaches to capture and analyze the threshold effects of pathogenic mtDNA onneuronal differentiation and bioenergetics. The scientific impact of this study is the availability of a novel mitochondrial transfection methodologyfor production of disease-specific stem cell progenitors with mtDNA mutations that will for the first time, enableus to monitor and quantitate mitochondrial DNA dynamics during neuronal differentiation. An additional impactis 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 basicresearch fields, including metabolic disease, diabetes, aging, autoimmune and cardiovascular disease re-search, are likely to benefit from the proposed experimental approaches.
Mitochondria play a critical role in the life of the cell as they control metabolic rates; energy production and celldeath. Many devastating diseases arise from mutations or deletions in small; circular mitochondrial DNA(mtDNA) that reside within our mitochondria. Each cell contains hundreds to thousands of copies of mtDNA.This project will explore how pathogenic mtDNA mutations can assume dominance over normal mtDNA withinmitochondria and influence disease severity.
| Alsayegh, Khaled N; Sheridan, Steven D; Iyer, Shilpa et al. (2018) Knockdown of CDK2AP1 in human embryonic stem cells reduces the threshold of differentiation. PLoS One 13:e0196817 |
| Rao, Raj R; Iyer, Shilpa (2015) Stem cells, neural progenitors, and engineered stem cells. Methods Mol Biol 1254:255-67 |
| Alsayegh, Khaled N; Gadepalli, Venkat S; Iyer, Shilpa et al. (2015) Knockdown of CDK2AP1 in primary human fibroblasts induces p53 dependent senescence. PLoS One 10:e0120782 |
| Iyer, Shilpa (2013) Novel therapeutic approaches for Leber's hereditary optic neuropathy. Discov Med 15:141-9 |
