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Age-related Impairment of the Transcriptional Response to Oxidative Stress Tomas A. Prolla Ph.D Dept. of Genetics & Medical Genetics University of Wisconsin-Madison Hypothesis • The expression of many stress responsive genes is altered due to aging • This age-associated change in expression levels may contribute to the biological process of aging Molecular Evidence of Compromised Stress Response with Age • Stress signaling – Reduced levels of activated JNK and p38 signaling molecules 1 hour after genotoxic stress in aged rat livers (Suh Y, 2001) • Heat shock response – Reduced levels of HSP70 in aged liver (Hall et al.,2000) and myocardium (Locke and Tanguay, 1996) after heat stress. • Immediate early response – Diminished induction of proto-oncogenes in ischemic (Isoyama, 1996) and LPS-stimulated (Saito et al.,2001) aged rodent hearts. • DNA repair – Decreased expression of APE/Ref1 DNA repair enzyme in old rat brains after 6 hours of hyperoxia (Edwards et al., 1998). Electron Transport Chain Comparison of Isoprostane Levels in Young and Old Cardiac Tissue Before and 7 Hours After Injection of 50mg Paraquat/ Kg Body Weight. Control * 3 * Bound Isoprostane (ng/g) 2.5 2 1.5 1 0.5 0 Young * P<0.05 vs Control for that age group Old Mice 7 hrs. Paraquat Free Radical-Induced Peroxidation of Arachidonic Acid Roberts LJ 2nd, Salomon RG, Morrow JD, Brame CJ. 1999 Gene Expression Profiles of All Measured Genes Following Paraquat Treatment in the Hearts of Young and Aged Mice (9,977 Transcripts) Gene Expression Profiles of Only Present Genes Following Paraquat Treatment in Young and Old Mice (5,523 Transcripts) Genes With P-value <0.01 (ANOVA) As Determined Separately For Each Age Group (459 Transcripts) 5,580 present genes 2.6% 2.5% Common Paraquat-Responsive Genes (55 Transcripts) • 16 associated with stress, immune or inflammatory response • 11 associated with growth factor/hormonal response • 4 metabolic/catabolic • 3 involved with transcription regulation • 10 with miscellaneous function • 11 with unknown function FK506 binding protein 5 (fkbp5) • Fkbp5 had the highest level of induction for both young and old age groups. • Baughman et al. (1995) first isolated the gene based on its induction during glucocorticoidinduced apoptosis in murine thymoma cells. • Protein that binds to FK506, mediates calcineurin inhibition, interacts with the 90 kDa heat shock protein and may be a component of progesterone receptor complexes. Other Common Paraquat-Responsive Genes BCL2-like 1 (Bcl-XL) • Codes for an anti-apoptotic protein • Allows cells to maintain oxidative metabolism during stress by allowing continued transport of metabolites across the outer mitochondrial membrane • Highest normalized expression in all ages at 7 hours post-paraquat Pyruvate dehydrogenase kinase 4 (PDK4) • Key element involved in fuel selection • PDK4 inhibits pyruvate dehydrogenase and thus minimizes carbohydrate oxidation by preventing the flow of glycolytic products into the tricarboxylic acid cycle • Significantly higher normalized expression in old (10.4) than young (6.5) and middle aged (4.4) 7 hours post-paraquat Metalothionein Gene Expression in Paraquat-Treated Mouse Hearts (All Ages) Metallothionein Staining in Young, Paraquat-Treated Mouse Hearts Anti-Myosin Anti-Mt Control 7 h Post-Paraquat Basal Levels of Expression of Oxidative Stress-Related Genes in Young, Middle Aged and Old Hearts Identifier U49430 X03920 U13705 D87896 X65021 L06047 J03952 J04696 J03953 U24428 X53451 X98055 AF054670 AI835051 K02236 AB023564 AF032714 U96746 AF093857 M35725 L35528 U38261 Gene Ceruloplasmin Glutathione peroxidase 1M Glutathione peroxidase 3 Glutathione peroxidase 4 Glutathione S-transferase, alpha 3Y Glutathione S-transferase, alpha 4Y &M Glutathione S-transferase, mu 1 Glutathione S-transferase, mu 2 Glutathione S-transferase, mu 3 Glutathione S-transferase, mu 5 Glutathione S-transferase, pi 2 Glutathione S-transferase, theta 1O Heme oxygenase (decycling) 2Y &M Metallothionein 1Y ,M&O Metallothionein 2Y ,M&O Peroxiredoxin 1 Peroxiredoxin 2 Peroxiredoxin 4M Peroxiredoxin 5M&O Superoxide dismutase 1, solubleO superoxide dismutase 2, mitochondrial Superoxide dismutase 3, extracellular Basal Expression (Raw) Young vs Old Young Middle Aged Old P-value 788 1164 1157 NSC 3150 3139 3689 NSC 24388 26482 24169 NSC 8477 8544 7109 < 0.01 406 421 551 NSC 1060 1051 736 NSC 20648 31976 34793 < 0.001 8057 8229 6448 NSC 6037 7413 6599 NSC 2456 1979 1954 NSC 11968 13071 10368 < 0.05 1246 1024 1302 NSC 962 847 678 NSC 26268 22223 23274 < 0.05 7347 5495 9097 NSC 26090 26184 22959 < 0.005 28718 29950 23412 < 0.005 1164 984 992 NSC 5734 4486 4249 < 0.005 11288 14856 7927 < 0.05 9335 7905 7086 < 0.05 3201 4434 5238 < 0.001 NSC = No Significant Change Immediate Early Response Genes - Typically transcription factors and cell signaling molecules. - After cell stimulation (e.g., with a mitogen or cell stressor), upregulation of IEG mRNA is rapid (occurring within minutes) and transient. - IEG expression represents the first round of gene expression after cell stimulation. Age-Associated Changes in Expression Profiles of MAPKKDependent IEGs in the Hearts of Mice After Induced Oxidative Stress (9 Transcripts) Young Middle Aged Old 4 * Average Normalized Expression 3.5 * * 3 2.5 2 1.5 1 0.5 0 0 1 2 3 4 Tim e (hours) *Wilcoxon Signed Ranks Test, P<0.05 for young mice vs old mice 5 6 7 GADD45 Genes • GADD45 was initially identified as a gene whose transcription rapidly increases in cells treated with DNA-damage causing agents. • Takekawa and Saito previously isolated three GADD45-like cDNAs (GADD45α, GADD45β, and GADD45γ) that encode for three similar proteins that bind to MAP3K4. – MAP3K4 mediates activation of both p38 and JNK pathways in response to environmental stresses • All 3 isoforms of GADD45 in the young, 2 isoforms in the middle aged (alpha and gamma) and no isoforms in the old were considered paraquatresponsive in the mouse hearts (ANOVA, P<0.01) Cardiac GADD45 Gene Expression in All Ages of Mice Following Paraquat Treatment Aged 4.5 Middle Aged Young Average Normalized Expression 4 3.5 3 2.5 2 1.5 1 0.5 0 0 1 2 3 Tim e (hours) 4 5 6 7 Conclusions There are age-associated changes in the transcriptional response to paraquat in the mouse heart – Induction levels for stress-responsive genes change due to aging in mice • Only 55 out of a total of 459 induced genes filtered are common to both age groups – Time course of induction for classes of genes is altered as a result of the aging process • Delayed induction of MAPKK-dependent IEG genes in aged hearts – Evidence of altered stress-signaling due to age • Only young show induction of GADD45 genes, MAP3K6 and Junb Future Directions • Same type of microarray study of stress response in paraquat-treated, young and old skeletal muscle – Determination of tissue specific and shared, age-associated effects on the cellular response to paraquat in both heart and muscle • Identify molecular basis for the age-related transcriptional impairment in the stress response – Examine other tissue types to determine whether observed defects represent a global change in the stress response as a result of the aging process – Further validation of the microarray data The Prolla Laboratory C.K Lee L. Motta T. Kayo K. Jolivvette G. Kujoth K. Higami M. Edwards S. Park R. Puthagunta Collaborators Richard Weindruch David Allison Aging in Muscle Cardiac Skeletal -Postmitotic, high energy -Postmitotic, high energy demanding cells demanding cells -Evidence of increased -Evidence of increased oxidative damage in oxidative damage in older animals older animals -Congestive heart failure -Loss of muscle mass is the most frequent (sarcopenia) is leading cause of hospitalization cause of frailty and in >65 yr.old disability in elderly