This project defines in vivo mechanisms of acquired mitochondrial (mt) DNA depletion caused by nucleoside reverse transcriptase inhibitors (NRTI) for HIV/AIDS. Survival with HIV/AIDS improved because of pyrimidine NRTIs like zidovudine (AZT) and stavudine (d4T), but NRTI mitochondrial toxicity limits therapy and manifests as mtDNA depletion and organ dysfunction. The """"""""DNA pol gamma hypothesis"""""""" underscores NRTI phosphorylation (to active moieties) and inhibition of DNA pol gamma (the mtDNA replicase) by NRTI triphosphates (-TP) in the mechanism of acquired mtDNA depletion. Cellular kinases control mitochondrial nucleotide and NRTI phosphorylations. Pyrimidine NRTIs compete with dThd for phosphorylation to monophosphates (MP) by thymidine kinase (cytoplasmic TK1 and mitochondrial TK2 isoforms) and to diphosphates (DP) by thymidylate kinase (TMPK). The working hypothesis states: Transgenic (TG) over-expression of wild type (WT) TK2 or WT TMPK promotes mtDNA replication by increasing the mitochondrial dTTP mass in parallel with increased enzyme abundance. TGs with TMPK mutants (""""""""gain of function"""""""") are active with dTMP phosphorylation, but exhibit enhanced activity with AZTMP. In absence of AZT, these TGs each increases dTTP abundance. With AZT therapy, AZT-DP abundance increases dramatically and is imported into mitochondria for phosphorylation to AZT-TP. Conversely, TGs harboring specific TK2 point mutations decrease TK2 activity (""""""""loss of function""""""""). These latter TGs deplete mtDNA by limiting mitochondrial dTMP for down- stream intra-mitochondrial processing. AZT treatment here enhances mtDNA depletion by AZT's competition with dThd for phosphorylation by TK2. Mitochondrial AZT-TP competes with dTTP for incorporation into mtDNA by DNA pol gamma. This inhibits mtDNA replication, causes mtDNA depletion and mutation, mitochondrial dysfunction, and organ dysfunction.
AIM 1 defines molecular, pathological and physiological events in mtDNA depletion from NRTIs (like AZT) using TGs with cardiac targeted, conditional, overexpression of WT TMPK, chimeric TMPK, and mutant TMPK.
AIM 2 defines molecular, pathological, and physiological events in mtDNA depletion from NRTIs (like AZT) using TGs with cardiac targeted, conditional, overexpression of WT TK2 and mutant TK2. Experiments offer insights into improving therapy with antiretrovirals used in AIDS. ? ? ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL079867-01A2
Application #
7161975
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Reid, Diane M
Project Start
2006-09-01
Project End
2011-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
1
Fiscal Year
2006
Total Cost
$385,050
Indirect Cost
Name
Emory University
Department
Pathology
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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Kushnir, Vitaly A; Lewis, William (2011) Human immunodeficiency virus/acquired immunodeficiency syndrome and infertility: emerging problems in the era of highly active antiretrovirals. Fertil Steril 96:546-53
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Kohler, James J; Hosseini, Seyed H; Green, Elgin et al. (2010) Absence of mitochondrial toxicity in hearts of transgenic mice treated with abacavir. Cardiovasc Toxicol 10:146-51
Kohler, James J; Hosseini, Seyed H; Cucoranu, Ioan et al. (2010) Transgenic cardiac-targeted overexpression of human thymidylate kinase. Lab Invest 90:383-90
Kohler, James J; Hosseini, Seyed H; Hoying-Brandt, Amy et al. (2009) Tenofovir renal toxicity targets mitochondria of renal proximal tubules. Lab Invest 89:513-9
Kohler, James J; Hosseini, Seyed H; Cucoranu, Ioan et al. (2009) Murine cardiac mtDNA: effects of transgenic manipulation of nucleoside phosphorylation. Lab Invest 89:122-30
Kohler, James J; Cucoranu, Ioan; Fields, Earl et al. (2009) Transgenic mitochondrial superoxide dismutase and mitochondrially targeted catalase prevent antiretroviral-induced oxidative stress and cardiomyopathy. Lab Invest 89:782-90
Kohler, James J; Hosseini, Seyed H; Lewis, William (2008) Mitochondrial DNA impairment in nucleoside reverse transcriptase inhibitor-associated cardiomyopathy. Chem Res Toxicol 21:990-6

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