BUCK INSTITUTE FOR AGE RESEARCH
Grant: $485,000 - National Institutes of Health - Jul. 21, 2009
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Award Description: In the mitochondrial free radical theory of aging, mitochondrial reactive oxygen species (ROS) generation is an inevitable consequence of oxidative ATP production, and the primary cause of macromolecular damage. Some damage is not repaired, causing progressive failure of cellular machinery, aging-related diseases, and aging. There is much evidence in favor and against the theory. Most authors think mitochondria-generated oxidative stress is important, particularly in aging-related diseases, but is not the sole cause of aging. To examine the importance of mitochondrial ROS generation and test the theory critically, we need to identify the specific mitochondrial electron transport chain sites that are involved and measure their rates of ROS production, to allow their manipulation. These sites have been defined in isolated mitochondria using electron transport chain inhibitors, but we lack reliable measurements of their rates in the absence of such inhibitors, there is no consensus on their relative importance, and new approaches are needed. Our hypothesis is that at least one site in the mitochondrial electron transport chain (e.g. IF, IQ, IIIQo) produces significant ROS and is the main oxidative stress generator. In the mitochondrial free radical theory, ROS generation at this site would be the primary cause of aging and its diseases. The hypothesis will be investigated by a new approach: first define novel methods to assay rates non-invasively from each site in a model system (isolated skeletal muscle mitochondria), then measure the relative rates from each site under plausibly physiological conditions. To test the hypothesis we propose studies with the following specific aims. Aim 1. Establish and validate measurements of ROS production from different sites in the electron transport chain of isolated mitochondria in the absence of electron transport inhibitors. Our key innovation will be to calibrate endogenous reporter signals of ROS production at each specific site (defined with inhibitors), then use the reporters to measure ROS production at each site without inhibitors. Aim 2. Using these methods, determine the relative rate of ROS production by each site in mitochondria isolated from skeletal muscle as a model system. The long-term goals of this study are: (a) To understand fully the quantitative importance of different sites of ROS production by mitochondria, (b) To generalize the results to mitochondria from different tissues, oxidizing different substrates in different states, at different ages. (c) To develop methods that can subsequently be applied to the identification and measurement of the sites of ROS production by mitochondria within cells and organs, (d) To provide a solid foundation to facilitate critical testing of the mitochondrial free radical theory of aging, (e) To identify the specific sites to target to minimize deleterious mitochondrial ROS production and its consequences for aging-related diseases and aging.
Project Description: Establish and validate measurements of ROS production from different sites in the electron transport chain of isolated mitochondria in the absence of electron transport inhibitors. Our key innovation will be to calibrate endogenous reporter signals of ROS production at each specific site (defined with inhibitors), then use the reporters to measure ROS production at each site without inhibitors. Studies to define the indicators for complex I and complex II are underway and progressing satisfactorily.
Jobs Summary: Job Retention using Recovery Act funds: 2 Postdocs Casey Quinlan and Jason Treberg Description of the employment impact resulting using Recovery Act funds: Without ARRA funding these Postdocs would not have been able to be retained. (Total jobs reported: 2)
Project Status: Less Than 50% Completed
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