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Transcript
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