Project 1: Gene expression analysis of skeletal muscle from aged animals.
This project will involve performing a microarray analysis of aged skeletal muscle tissue. We will collect tissues from animals and perform an array comparing gene expression profiles in 6, 12, 18, and 24 month old mice. The hope is that we will identify an aging specific signature of gene expression in older animals compared to younger animals. Genes that show a differential expression profile will be further analyzed using cell culture and well established gene expression techniques, such as transfections and reporter assays. A long-term goal of this project will be to study animals with targeted disruption of the most promising gene(s) identified from the array that also show potential to be important aging effectors.
Project 2: Analysis of the role of cellular stresses, including Reactive Oxygen Species (ROS), in aged tissue using skeletal muscle as a model organ.
In addition to gene expression analysis of aged skeletal muscle tissue, the lab will also undertake a project to analyze the more specific role of ROS and other cellular stresses in the aging process. Preliminary evidence, from microarray data generated from MuRF1 null and wild-type mice following denervation, suggests that loss of muscle innervation leads to changes in expression of a collection of genes that play an important role in regulating ROS levels in the cell. Based on these observations, we will conduct research into the role of ROS in the aging process and determine if environmental and heritable differences may impact this role. It is known that as muscle ages, it shows a reduction in the number of neuromuscular junctions resulting in a decrease in innervation of the muscle. Based on preliminary results from the microarray mentioned above, it is reasonable to hypothesize that a reduction in innervation might lead to an increase in ROS, resulting in an increase in cellular stress and ultimately macromolecular, cellular and tissue damage. We will strive to employee a combinatorial approach to studying ROS by using biochemical techniques, tissue culture and animal models. In tissue culture, ROS stress can be mimicked by addition of hydrogen peroxide to cells, which will then be analyzed by determining transcriptional (via qPCR or Northern Blot analysis) and translational (via Western Blot analysis) responses of known ROS regulated genes. Identified transcriptional changes in expression of ROS responsive genes will then be further analyzed by use of reporter gene techniques in cell culture. A more long range goal of this research will be to use the SOD1null mice, which have increased oxidative stress, and monitor both gene expression and epigenetic events induced by ROSas the animals age. This would be done by performing microarray and ChIP-CHIP or ChIP-Seq techniques to analyze changes in gene expression, DNA methylation and/or histone acetylation in SOD1 null mice compared to wild type, age-matched litter mates.
Project 3: Analysis of chromatin remodeling in aged skeletal muscle tissue.
This project will focus on the global epigenetic changes that appear to occur naturally in aged individuals. In particular, the fact that HDAC4, a class II histone deacetylase, shows increased expression in skeletal muscle of aged rodents and humans suggests that remodeling of the chromatin structure might be one important factor that results in loss of plasticity of this tissue in older individuals. Furthermore, previous studies have shown that HDAC4 expression increases significantly during denervation and is accompanied by a rapid translocation to the nucleus. In light of these findings, it is not unreasonable to speculate that in aged muscle, in which innervation is reduced and HDAC4 levels are increased, HDAC4 may play a role in global chromatin remodeling and alterations in gene expression. Therefore, I am particularly interested in identifying regulatory regions of genes that are either bound by HDAC4 and/or have undergone significant changes in histoneacetylation in aged muscle when compared to tissue from younger animals. This work will again entail the use of several advanced molecular techniques including ChIP-CHIP or ChIP-Seq analysis of skeletal muscle tissue in hopes of identifying global changes in DNA that occur during the aging process. This powerful and relatively new technique would allow us to analyze samples from aged rodents and potentially identify regulatory regions that might be important in the progression of aging. A long term objective of this project will be to eventually analyze identified regions of the genome using cell culture, advanced molecular techniques and animal models in hopes of elucidating the importance of these regions in the aging process.
