2008 AFAR Research Grant: Protein Phosphatases in the Regulation of ATM-p53 Signaling Pathway and Cell Senescence

Please give a brief summary of your AFAR research project.
Recent research on aging suggests that the inevitable generation of oxygen free radicals from aerobic cellular respiration causes oxidative damage to proteins, lipids, and DNA, and the buildup of these damaged macromolecules has been implicated in a wide variety of diseases and age-related disorders, including stroke and heart disease, ALS, Parkinson's and Alzheimer's diseases, and cataracts. The peptide Methionine sulfoxide reductase (Msr) is a "repair enzyme" for oxidatively damaged proteins and amino acids, which may restore biological activity to damaged proteins and thus reduce the age-related accumulation of such errors. The reversible oxidation/reduction of Methionine residues in proteins may also be one of the prime mechanisms by which cells scavenge reactive oxygen species (ROS) before they damage cellular constituents, and increased cell death and ROS damage occur when Msr levels are reduced in several aging models. This research project will examine the control and functions of Msr in a novel animal model, the freshwater turtle, which is known both to be long-lived and is also the only known vertebrate in which Msr is naturally regulated by fluctuating oxygen levels. My hypothesis is that Msr protects brain cells against both free radical generation and damage, and the discovery of the molecular mechanisms by which this protection occurs will elucidate the links between MsrA, oxidative damage, and ageing.

What problems are you addressing and what specific questions will your research seek to answer?
Free radicals generated in excess of cellular defense mechanisms (antioxidants) induce cellular damage, and aging and senescence may thus result from the accumulation of damaged molecules with time. Increased cell death and ROS damage are observed when Msr levels are reduced in several aging models, while conversely increased Msr levels protect cells against free radical damage. My lab has recently shown that the freshwater turtle is able to suppress ROS production under conditions that kill mammalian neurons; turtles are also long-lived and, interestingly, Msr is naturally induced by low oxygen levels. This is the first report in any system - eukaryotic or prokaryotic - of the induction and subsequent down-regulation of MsrA transcription and protein levels regulated by oxygen supply, which makes T. scripta radically different from other animal models, and provides a unique opportunity to investigate the function and regulation of this peptide which is likely to play a critical role in aging. This research will study the role of MsrA in neuronal resistance to oxidative stress, cellular damage, and mitochondrial dysfunction, and thus its role in aging without senescence, utilizing in vivo studies as well as siRNA technology to alter Msr levels in vitro.

What aspects of your project are most interesting from a scientific point of view?
The most interesting aspect of this research from a scientific point of view is that the freshwater turtle is the only known vertebrate animal in which the enzyme Methionine sulfoxide reductase is naturally up- and downregulated in response to changing oxygen levels. This makes this animal a unique model in which to examine the regulation and function of a compound that appears to be critically important in staving off age-related diseases. The turtle, as an animal able to withstand repeated periods of anoxia and subsequent reoxygenation without cellular damage, provides a unique model for such studies. By contrast, anoxia and reoxygenation (or ischemia and reperfusion) in the mammal engender both pathological and adaptive responses simultaneously, making it difficult to distinguish survival mechanisms within the cell from those that trigger cell death.

What are the implications of your research for age-related diseases and disorders?
Recent research on aging suggests that the inevitable generation of oxygen free radicals from aerobic cellular respiration causes oxidative damage to proteins, lipids, and DNA, and the buildup of these damaged macromolecules has been implicated in a wide variety of diseases and age-related disorders, including stroke and heart disease, ALS, Parkinson's and Alzheimer's diseases, and cataracts. The peptide Methionine sulfoxide reductase (Msr) is a "repair enzyme" for oxidatively damaged proteins and amino acids, which may restore biological activity to damaged proteins and thus reduce the age-related accumulation of such errors. Low Msr levels are associated with increased susceptibility to oxidative damage and shortened lifespans in animals, whereas high levels protect cells and reduce oxidative damage. This research will utilize an animal model that uniquely combines three critical aspects related to the "free radical theory" of aging: the turtle does not "age" as we know it, it is able to suppress free radical generation and damage, and it naturally expresses high levels of Msr. Thus this novel animal model will allow us to examine the molecular mechanisms by which Msr protects cells and reduces free radical damage.

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