Respiratory chain dysfunction is a class of devastating and invariably fatal neuromuscular disorders caused by defects in mitochondrial DNA (mtDNA). Previously under-recognized, these disorders have a high prevalence (~1:5000) and there are currently no generalized therapies available. Previously, it was shown that in cellular models of mtDNA disease, intracellular quality control pathways could be harnessed to restore mitochondrial function. Specifically, in heteroplasmic cells (i.e. cells containing a mixed population of normal and mutated mtDNA, which is typical of the clinical situation), the macro-autophagy pathway that degrades defective mitochondria could be induced by a glucose-free ketone body diet (ketogenic therapy) to intracellularly increase the proportion of functional mitochondria and decrease the percentage of mutated mtDNA. Ketogenic therapy is not feasible in patients, but the FDA-approved drug sirolimus/rapamycin is known to induce macro-autophagy in cells. Rapamycin (rapa) treatment of mtDNA disease cells rapidly and dramatically increased the intracellularly proportion of functional mitochondria, though surprisingly, there was no associated shift in percentage of mutated mtDNA, as expected if rapa induced macro-autophagy. Changes were observed in mitochondrial morphology, however, and thus rapa may work by promoting mitochondrial fusion and inter-organellar complementation. Based on these preliminary data, rapa was administered to a mouse model of mtDNA disease, specifically a model of mtDNA depletion syndrome (MDS). These MDS mice have mutations in a nuclear gene encoding a mitochondrial-specific thymidine kinase enzyme (TK2) that governs mtDNA integrity, and exhibit severe progressive motor and neurologic symptoms, with mortality by approx. 14 days old. Early intervention with rapa significantly delayed mortality in this mouse model, almost doubling lifespan. This research training program proposes to follow-up on these preliminary results in two ways.
Specific Aim 1 will investigate the cellular mechanism of rapa-mediated functional rescue in heteroplasmic cell models of mtDNA disease. This will be accomplished by genetically and pharmacologically inhibiting the autophagic and mitochondrial fusion pathways, independently, and then assessing mitochondrial translation and respiratory function in cell models of mtDNA disease through immunocytochemistry and innovative real-time bioenergetics analysis. The goal of Specific Aim 2 is to determine the maximum effect of rapa in rescuing mitochondrial disease, as well as the rescue mechanism. Therapy will first be optimized, and then enzymatic and immunochemical analysis of treated and control mice will be performed to assess rapa-mediated amelioration of Tk2 deficiency, mitochondrial function, and effects on downstream cellular pathways. Use of rapamycin may represent the first generalized treatment for mtDNA-based mitochondrial disorders, supporting the mission of reducing the burden neurologic disease.

Public Health Relevance

Mitochondrial DNA (mtDNA) disease is a class of devastating and invariably fatal neuromuscular disorders caused by defects in mtDNA. Previously under-recognized, these disorders actually have a high prevalence, and there are currently no generalized therapies available. The FDA/approved drug rapamycin/sirolimus shows promise in both cellular and animal models of mtDNA disease, and with further research this may represent the first generalized therapy to treat the whole class of mtDNA diseases to significantly improve the lives of all of these patients.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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NST-2 Subcommittee (NST)
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Gwinn, Katrina
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Columbia University (N.Y.)
Schools of Medicine
New York
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Siegmund, Stephanie E; Grassucci, Robert; Carter, Stephen D et al. (2018) Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography. iScience 6:83-91
Liao, Pin-Chao; Boldogh, Istvan R; Siegmund, Stephanie E et al. (2018) Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification. PLoS One 13:e0196632
Siegmund, Stephanie E; Yang, Hua; Sharma, Rohit et al. (2017) Low-dose rapamycin extends lifespan in a mouse model of mtDNA depletion syndrome. Hum Mol Genet 26:4588-4605