age-related impairment of the transcriptional response to oxidative stress tomas a. prolla ph.d...
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Age-related Impairment of the Transcriptional Response to
Oxidative Stress
Tomas A. Prolla Ph.DDept. of Genetics & Medical Genetics
University of Wisconsin-Madison
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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
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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).
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Electron Transport Chain
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Comparison of Isoprostane Levels in Young and Old Cardiac Tissue Before and 7 Hours After Injection of 50mg Paraquat/ Kg
Body Weight.
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* P<0.05 vs Control for that age group
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Free Radical-Induced Peroxidation of Arachidonic Acid
Roberts LJ 2nd, Salomon RG, Morrow JD, Brame CJ. 1999
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Gene Expression Profiles of All Measured Genes Following Paraquat Treatment in the Hearts of
Young and Aged Mice (9,977 Transcripts)
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Gene Expression Profiles of Only Present Genes Following Paraquat Treatment in Young and Old
Mice (5,523 Transcripts)
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Genes With P-value <0.01 (ANOVA) As Determined Separately For Each Age Group (459 Transcripts)
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2.6% 2.5%
5,580 present genes
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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
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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.
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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
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Metalothionein Gene Expression in Paraquat-Treated Mouse Hearts (All Ages)
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Metallothionein Staining in Young, Paraquat-Treated Mouse Hearts
Control 7 h Post-ParaquatA
nti
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Basal Levels of Expression of Oxidative Stress-Related Genes in Young, Middle Aged and Old Hearts
Young vs OldIdentifier Gene Young Middle Aged Old P-valueU49430 Ceruloplasmin 788 1164 1157 NSC
X03920 Glutathione peroxidase 1M 3150 3139 3689 NSCU13705 Glutathione peroxidase 3 24388 26482 24169 NSCD87896 Glutathione peroxidase 4 8477 8544 7109 < 0.01
X65021 Glutathione S-transferase, alpha 3Y 406 421 551 NSC
L06047 Glutathione S-transferase, alpha 4Y&M 1060 1051 736 NSCJ03952 Glutathione S-transferase, mu 1 20648 31976 34793 < 0.001J04696 Glutathione S-transferase, mu 2 8057 8229 6448 NSCJ03953 Glutathione S-transferase, mu 3 6037 7413 6599 NSCU24428 Glutathione S-transferase, mu 5 2456 1979 1954 NSCX53451 Glutathione S-transferase, pi 2 11968 13071 10368 < 0.05
X98055 Glutathione S-transferase, theta 1O 1246 1024 1302 NSC
AF054670 Heme oxygenase (decycling) 2Y&M 962 847 678 NSC
AI835051 Metallothionein 1Y,M&O 26268 22223 23274 < 0.05
K02236 Metallothionein 2Y,M&O 7347 5495 9097 NSCAB023564 Peroxiredoxin 1 26090 26184 22959 < 0.005AF032714 Peroxiredoxin 2 28718 29950 23412 < 0.005
U96746 Peroxiredoxin 4M 1164 984 992 NSC
AF093857 Peroxiredoxin 5M&O 5734 4486 4249 < 0.005
M35725 Superoxide dismutase 1, solubleO 11288 14856 7927 < 0.05L35528 superoxide dismutase 2, mitochondrial 9335 7905 7086 < 0.05U38261 Superoxide dismutase 3, extracellular 3201 4434 5238 < 0.001
Basal Expression (Raw)
NSC = No Significant Change
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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.
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Age-Associated Changes in Expression Profiles of MAPKK-Dependent IEGs in the Hearts of Mice After Induced
Oxidative Stress (9 Transcripts)
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*Wilcoxon Signed Ranks Test, P<0.05 for young mice vs old mice
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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)
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Cardiac GADD45 Gene Expression in All Ages of Mice Following Paraquat Treatment
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ConclusionsThere 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
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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
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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
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Aging in MuscleCardiac
-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