Age-related Impairment of the Transcriptional Response to Oxidative Stress

1. Age-related Impairment of the Transcriptional Response to Oxidative Stress Tomas A. Prolla Ph.D Dept. of Genetics & Medical Genetics University of Wisconsin-Madison

2. 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

3. 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).

5. Electron Transport Chain

6. Comparison of Isoprostane Levels in Young and Old Cardiac Tissue Before and 7 Hours After Injection of 50mg Paraquat/ Kg Body Weight. * * * P<0.05 vs Control for that age group

7. Free Radical-Induced Peroxidationof Arachidonic Acid Roberts LJ 2nd, Salomon RG, Morrow JD, Brame CJ. 1999

8. Gene Expression Profiles of All Measured Genes Following Paraquat Treatment in the Hearts of Young and Aged Mice (9,977 Transcripts)

9. Gene Expression Profiles of Only Present Genes Following Paraquat Treatment in Young and Old Mice (5,523 Transcripts)

10. Genes With P-value <0.01 (ANOVA) As Determined Separately For Each Age Group (459 Transcripts)

11. 5,580 present genes 2.6% 2.5%

12. 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

13. 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 glucocorticoid-induced 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.

14. 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 Other Common Paraquat-Responsive Genes

15. Metalothionein Gene Expression in Paraquat-Treated Mouse Hearts (All Ages)

16. Metallothionein Staining in Young, Paraquat-Treated Mouse Hearts Control 7 h Post-Paraquat Anti-Mt Anti-Myosin

17. Basal Levels of Expression of Oxidative Stress-Related Genes in Young, Middle Aged and Old Hearts NSC = No Significant Change

18. 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.

19. Age-Associated Changes in Expression Profiles of MAPKK-Dependent IEGs in the Hearts of Mice After Induced Oxidative Stress (9 Transcripts) * *Wilcoxon Signed Ranks Test, P<0.05 for young mice vs old mice

20. 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 paraquat-responsive in the mouse hearts (ANOVA, P<0.01)

21. Cardiac GADD45 Gene Expression in All Ages of Mice Following Paraquat Treatment

22. 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

23. 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

24. 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

25. Cardiac -Postmitotic, high energy demanding cells -Evidence of increased oxidative damage in older animals -Congestive heart failure is the most frequent cause of hospitalization in >65 yr.old Skeletal -Postmitotic, high energy demanding cells -Evidence of increased oxidative damage in older animals -Loss of muscle mass (sarcopenia) is leading cause of frailty and disability in elderly Aging in Muscle