Our long-term goal is to understand how mitochondrial DNA (mtDNA) mutations appear, propagate, and are distributed among the thousands of mtDNA molecules in a cell, a condition called heteroplasmy. While these mutations are associated with age-related diseases and the aging process, predicting the degree of heteroplasmy at which disease symptoms or age-related phenotypes will appear is practically impossible because the dynamics of heteroplasmy are not well understood. The central hypothesis of this application is that individual mitochondria, containing anywhere from 2 to 10 mtDNA copies, can be heteroplasmic, a condition resulting from the segregation of mtDNA molecules upon mitochondrial replication, and likely modified by the dynamic exchange of genomic material among mitochondria within a given cell. If this hypothesis is correct, a heteroplasmic mitochondrion will have both mutated and wild-type mtDNA, all the peptides encoded by the mitochondrial genome, and a normal mitochondrial membrane potential. We propose to develop the first bioanalytical technologies capable of investigating heteroplasmy in individual mitochondria. We will continue to use and improve upon an instrument based on capillary electrophoresis with laser-induced fluorescence detection (CE-LI F) to determine the properties of individual mitochondria that can then be collected and subjected to PCR amplification of their DNA. In addition, peptide profiles from mitochondria containing mutated mtDNA that will be determined by in situ matrix-assisted laser-desorption time-of-flight mass spectrometry will provide a more comprehensive characterization of heteroplasmy. As testing models, we will use NS-1 cells lines, two cybrid cell lines harboring 7522 and 4977 deletions, and rectus femoris and soleous muscles from Fisher 344 Rats, aged 6, 24, and 28 months. The NS-1 model will mainly be used to develop technologies and methods. The cybrid models have a defined degree of heteroplasmy (> 50 percent) and host deletions that omit the expression of the genes ND5, ND4, ND3, ND4L, COIII, A6, A8, and tRNAL, tRNAS, tRNAH, tRNAR, and tRNAG. In addition to these genes the cybrid hosting the 7522 base pair deletion further omits expression of the cytb, ND6, COIl (2), tRNAR, and tRNAK genes. These cybrid models will be used to study the progression of heteroplasmy in cell lines. The two muscle models will be used to study the progression of heteroplasmy along red ragged fibers that have been identified by (COX-, SDH++) phenotype. Our individual mitochondrial determinations will be the basis for monitoring (1) the progression of heteroplasmy after the formation and propagation of a cybrid clone, and (2) the degree of heteroplasmy along skeletal muscle fibers. The data resulting from the cybrid and muscle tissue models will be used to refine existing mathematical models that predict the clonal expansion of heteroplasmy. The determination of mtDNA mutations at the single mitochondrion level in the cybrid and muscle models will bring us closer to uncovering the intricacies of heteroplasmy and its implications in disease and aging.
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