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.
Mitochondria play a critical role in the life of the cell as they control metabolic rates, energy production and cell death. 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 within mitochondria 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 |
