In order to increase understanding of the molecular basis of aging, we have generated a mouse model (PolgD257A) that should display increased spontaneous mutation rates in mitochondrial DNA (mtDNA). This animal model was generated by introducing a specific mutation in the exonuclease domain of mouse DNA Polymerase Gamma (POLG) in ES cells. Mice carrying a """"""""mutator"""""""" phenotype in mtDNA will allow investigators to elucidate one of the central unresolved issues in aging research, the contribution of mitochondrial mutations to the aging process. The central hypothesis to be tested is that mutations in mtDNA, known to be associated with aging in many postmitotic tissues, play a causal role in the aging process.
Specific Aim 1. Characterization of skeletal muscle in mitochondrial mutator mice. Mice carrying the PolgD257A mutation are viable and display no obvious developmental defects. We propose to determine the mutational spectrum in mtDNA of these animals in order to estimate mutational frequency in vivo. We also propose to determine the level of electron transport system (ETS) abnormalities, as determined by loss of cytochrome c oxidase (COX) activity along individual muscle fibers of 5-month, 15-month and 30-month PolgD257A and wild-type control animals. Isolated mitochondria will also be characterized for age-related biochemical abnormalities, including alterations in metabolic potential and OS markers.
Specific Aim 2. Gene expression profiling in skeletal muscle. We propose to characterize the gene expression profile of skeletal muscle of PolgD257A mice over the adult lifespan in order to determine if mitochondrial mutations accelerate the aging process at the molecular level. Specifically, mice will be studied at 5 months, 15 months and 30 months of age. These experiments will involve both wild-type and PolgD257A mice and utilize high density oligonucleotide arrays, which provide data on over 12,000 genes and ESTs.
Specific Aim 3. Survival and Disease Patterns in PolgD257A Mice. We propose to determine the survival and disease patterns of PolgD257A mice. Animals will be backcrossed to the B6 (C57BL/6 mice) genetic background, which our laboratory has extensively characterized for disease patterns and mortality. Survival patterns, including survival curves and calculation of mortality rates, will be performed for PolgD257A mice and wild-type controls. Animals will be observed for the development of neoplastic, neurologic and cardiac phenotypes and characterized at the histopathology level accordingly.
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