Metabolism of nutrients in mitochondria is the major source of energy for eukaryotic cells. Consequently, mitochondrial dysfunction can impair cellular and organismal homeostasis. The effects of mitochondrial dysfunction are exemplified by heritable genetic diseases and perhaps, normal aging. Mitochondria require the expression and translocation of nuclear proteins, as well as proteins encoded by mitochondrial DNA. Therefore, mutations that lead to dysfunction can occur in nuclear or mitochondrial DNA. The rate of mutation in primate mitochondrial DNA has been shown to be 5 to 10 times higher than in nuclear DNA. There are several factors that may contribute to the increased mutation rate detected in mitochondrial DNA including: 1) mitochondria utilize 90% of the cells 02 and generate many reactive oxygen species that can lead to mutations, 2) mitochondrial DNA is not associated with histones, and 3) mitochondrial DNA repair mechanisms are more limited in scope than nuclear DNA repair pathways. The primary objective of research described in this proposal is to determine if DNA repair in mitochondria can be increased by targeting DNA repair proteins to the mitochondrial matrix. This is the first step in positioning our laboratory to directly test the hypothesis that accumulation of mitochondrial mutations contribute to the aging process.
The specific aims of the project are: 1) to generate and characterize stably transfected mammalian neuronal cell lines containing coding sequences for the luciferase reporter gene with or without mitochondrial matrix presequences, 2) to generate and characterize stably transfected mammalian neuronal cell lines containing DNA repair genes with or without mitochondrial matrix presequences, and 3) to generate and characterize transgenic mice containing transgenes that code for neuron-specific expression of DNA repair genes with or without mitochondrial matrix presequences. The proposed studies will determine whether mitochondrial matrix presequences can be fused to direct translocation of a non-mitochondrial protein to the mitochondrial matrix in mammalian cells. If successful, the next step will be to demonstrate that DNA repair proteins can be targeted to the mitochondrial matrix and result in elevated levels of repair for the specific lesions they recognize. Finally, transgenic mice will be generated using transgenes that contain mitochondrial matrix presequences to demonstrate that functional DNA repair proteins can be targeted to the matrix and effect increased DNA repair. Completion of this study will effectively position our laboratory to test the hypothesis that mitochondrial mutations contribute to aging.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
1R01AG013560-01
Application #
2055559
Study Section
Special Emphasis Panel (ZRG4-NTN (06))
Project Start
1995-07-20
Project End
2000-06-30
Budget Start
1995-07-20
Budget End
1996-06-30
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Street, K A; Xu, G; Hall, K L et al. (2005) Rat synapsin 1 promoter mediated transgene expression in testicular cell types. DNA Cell Biol 24:133-40
Intano, Gabriel W; Cho, Eun Ju; McMahan, C Alex et al. (2003) Age-related base excision repair activity in mouse brain and liver nuclear extracts. J Gerontol A Biol Sci Med Sci 58:205-11
Intano, Gabriel W; McMahan, C Alex; McCarrey, John R et al. (2002) Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice. Mol Cell Biol 22:2410-8
Intano, G W; McMahan, C A; Walter, R B et al. (2001) Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity. Nucleic Acids Res 29:1366-72
Walter, C A; Zhou, Z Q; Manguino, D et al. (2001) Health span and life span in transgenic mice with modulated DNA repair. Ann N Y Acad Sci 928:132-40
Walter, C A; Intano, G W; McCarrey, J R et al. (1998) Mutation frequency declines during spermatogenesis in young mice but increases in old mice. Proc Natl Acad Sci U S A 95:10015-9
Walter, C A; Grabowski, D T; Street, K A et al. (1997) Analysis and modulation of DNA repair in aging. Mech Ageing Dev 98:203-22