Agerelated 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).

4
(No Transcript)
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.


Plt0.05 vs Control for that age group
7
Free Radical-Induced Peroxidation of 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 lt0.01 (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-induc
  ed 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
Other Common Paraquat-Responsive Genes

• 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

• 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

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, Plt0.05 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 (GADD45a, GADD45ß, and GADD45?)
  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,
  Plt0.01)

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
Aging in Muscle

• 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 gt65 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