Age-related Impairment of the Transcriptional Response to

Oxidative Stress

Tomas A. Prolla Ph.DDept. 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.

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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)

2.6% 2.5%

5,580 present genes

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 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.

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

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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

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 MAPKK-Dependent IEGs in the Hearts of Mice After Induced

Oxidative Stress (9 Transcripts)

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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)

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

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 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