error catastrophe? none detected. · heritable even in a population of isogenic animals. models: 1)...
TRANSCRIPT
Questions about Aging• Can We Slow It? WE SURE CAN!
• Can We Produce Drugs To Do This? YES!• Is Aging an Extension of Development? NO!• How Much Expression Variation?
Protein: No New Proteins (~1800 seen)RNA: 163 Vary Significantly (Differentially)
with AgeWhole Transcriptome Decreases 3XWhole Translational System Decreases 12X
• Error Catastrophe?None Detected.
Questions about Aging• Can We Slow It? WE SURE CAN!
• Can We Produce Drugs To Do This?• Is Aging an Extension of Development? NO• How Much Expression Variation? Not much• Error Catastrophe? None Detected• Is Aging Regulated?
AGE-1
PD
K-1
P PP P
DAF-18PP
P P
AKT-1/2
DAF-16
DAF-2
Target gene
nucleus
Good Times
AGE-1
PD
K-1
P P
DAF-18PP
P
AKT-1/2
DAF-2
Stress
CBP-1
Target gene
DAF-16CBP-1
nucleus
P14-3-3 14-3-3
R13H8
daf-16a1
daf-16a2
daf-16b
daf-16::gfp
A B C
D
E
10/29/02 Henderson & J, 2001
Starvation causes nuclear localization of DAF-16::GFP
0hr 1hr
1hr 15min 1hrrecovery
Henderson & J, 2001
DAF-16::GFP responds to stress in adult animals
Well fed
Starvation
Heat
Juglone
+E. coli
-E. coli 6hr
35°C 2hr
200 µM juglone
Henderson & J, 2001
20%
Higher DAF-16::GFP Increases Life Span
0 2 4 6 8 10 12 14 16 18 20 22 240.0
0.2
0.4
0.6
0.8
1.0
pRF4 (control)zIs daf-16::GFP
Days
Surv
ivin
g fr
actio
n
Henderson & J, 20013/25/06
Reduction of daf-16 by RNAi reduces life span
0 2 4 6 8 10 12 14 16 18 20 22 240.0
0.2
0.4
0.6
0.8
1.0
pRF4 daf pRF4 vec
Days
Frac
tion
surv
ivin
g
20%
Henderson & J, 20013/25/06
RepairReproduction
Allocation of resources may alter lifespan
After Kirkwood
RepairReproduction
Allocation of resources may alter lifespan
Changes in subcellular localization of DAF-16 may allow for rapid reallocation of resources in response to a changing environment.
Changes in subcellular localization of DAF-16 may allow for rapid reallocation of resources in response to a changing environment.
After Kirkwood
Questions about Aging• Can We Slow It? WE SURE CAN!
• Can We Produce Drugs To Do This?• Is Aging an Extension of Development? NO• How Much Expression Variation? Not much• Error Catastrophe? None Detected• Is Aging Regulated? No; Living Is Regulated.
Upregulated by DAF-16 (select 40)•Xenobiotic Detoxification (10)•Alpha crystallin like: hsp-16.1, .2, .11, .49, hsp-12.1, sip-1 (6)•Oxidative Stress: ctl-1, -2, sod-3, mtl-1•Bacterial Response: lys-7, saposins (3)•Insulin: ins-18
Downregulated by DAF-16 (19)•Vitellogenin: vit-2, -5•Insulin, ins-7↓•old-1
DAF-16 Regulates Response to Toxins
Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer
J, Li H, Kenyon C. 2003 Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature, 424:277-83.
3/25/06
Favorable conditions•IGF-1↑•DAF-16 retained in cytoplasm•Reproduction and growth favored•Fewer resources are put into maintenance and repair.•Life span is shorter.
Unfavorable conditions:•IGF-1↓•DAF-16 transported to nucleus.•Cellular repair and maintenance genes induced•Reproduction and growth genes repressed•Life span is longer.
DAF-16 Is a “GERONTOSTAT”
Questions about Aging
• Can We Slow It? WE SURE CAN!
• Can We Produce Drugs To Do This?• Is Aging an Extension of Development?• What Causes Stochastic Variation in
Life Span?
Ages at Death of Wild-type and age-1 Worms
0
5
10
15
20
25
30
35
0 10 20 30 40 50
Days
No
dyin
g
Wild type age-1
Kirkwood & Finch Nature 2002 (data from T. Johnson)
Green Fluorescent Protein Is a Surrogate (“Reporter”) of HSP-16
hsp-16::GFPtransform
induce
GP73
5/20/02
Isogenic population
• hsp-16.2 encodes a 16-kD heat shock protein (HSP)• Member of the hsp16/hsp20/alphaB-crystallin (HSP16)
family of heat shock proteins; 6 near identical homologs• hsp-16.2 expression, strongest in intestine and pharynx• Under Daf-16 & HSF-1 • Induced in response to heat shock, hypoxia, or other
environmental stresses • Mount 36; other HSPs• Up regulated in Age mutants• Over expression has modest life extension• Interacts with intracellular human beta amyloid peptide, a
primary component of plaques in Alzheimer's disease• HSP-16.2 is likely to function as a passive ligand temporarily
preventing unfolded proteins from aggregating
HSP-16.2
Induction Methods
Young Adulthours
Not All Worms Are Created Equal
Nature Genetics, 2005
GFP Is Normally Distributed
GFP Level (Arbitrary Units)
2 hour induction, 19 hour sort, 60,000 worms
Optical Analysis During Flow Sorting
5/20/02
COPAS Worm SorterRed laser: to measure both size and density of worms.
Green laser: to measure green Fluorescence level of worms.
Parameters are real time analyzed. Those worms meeting the criteria are collected in microwells.
Questions about Aging
• What Causes Stochastic Variation?
Does HSP-16 Expression Correlate with Differential Thermotolerance?With Differential Lifespan?
Variance Increases with Time after Sort
Sorting for Differential Brightness
Brighter Worms Are Longer Lived
(P < 0.0001)
0 5 10 15 20 25 30 35-5
0
5
10
15
TJ375, p<0.05TJ375, p>0.05TJ550, p<0.05
Sort Time (hours)
Hig
h - L
ow (d
ays)
Differential Longevity Bright - Dim: All Individual Experiments
(two-hour heat shock)
(P = 8.0 x 10-29)
Combined Longevity
Conclusions:• Isogenic worms are not the same
- Variable response to stress- Variable life span
• Response to stress is a predictor of life span• Not due to genetic changes• Probably not due to HSP-16 alone? • “Physiologic State” on first day of adulthood
predicts life expectancy
5/20/02
NATURE GENETICS August, 2005
Questions about Aging
• What Causes Stochastic Variation?Can We Explain It?Is Any of It Heritable?Are There Other Genes Differentially
Expressed?What Causes Differential Survival?
0
20
40
60
80
100
120
140
160
180
hsp-16.1
hsp-16.2
hsp-16.41
hsp-16.48
Gen
e Ex
pres
sion
, % o
f Dim
DimBright
Induction of hsp-16 class in Bright worms
*** **
* 0.05 < p-value < 0.1** p-value < 0.05
The effect on the expression of other hsp
0
20
40
60
80
100
120
140
160
180
hsp-1 hsp-3 hsp-4 hsp-6 hsp-6 hsp-12.1
hsp-12.2
hsp-43 hsp-60 hsp-70
Gen
e Ex
pres
sion
, % o
f Dim
DimBright
** p-value < 0.05
**
0
2040
60
80100
120
140160
180
ctl-2 ctl-1,3 sod-1 sod-2 sod-4
Gen
e Ex
pres
sion
, % o
f Dim
DimBright
The effect on the expression of antioxidant genes
0
20
4060
80
100
120140
160
180
gst-1 gst-3 gst-4 gst-5 gst-7 gst-10 gst-13 gst-20 gst-22 gst-24 gst-27 gst-36 gst-38 gst-39 gst-40 gst-41 gst-42 gst-43
Gen
e Ex
pres
sion
, % o
f Dim
DimBright
The effect on the expression of gst
Dim All Bright24 hr egg lay
Progeny of Dim
1 hourheat
SorterMeasure GFP
Inheritance of Brightness
Maturation
24 hr egg lay
Progeny of Bright
SorterMeasure GFP
Maturation
24 hr egg lay
Progeny of All
SorterMeasure GFP
Maturation
Expectation: All groups of progeny should have similar GFP expression
1 hourheat
1 hourheat
0
5
10
15
20
25
030
060
090
012
0015
0018
0021
00
GFP Expression
Freq
uenc
y (%
)
F1-AllF1-DimF1-MediumF1-Bright
GFP Expression of Progeny
GFP Expression of F1 (Progeny)Experiment 1
0.00
0.05
0.10
0.15
0.20
0 500 1000 1500 2000 2500
GFP Score
% o
f Ani
mal
s
F1 of AllF1 of DimF1 of Bright
Mean ± SD ~nF1 of All 847 ± 273 6300F1 of Dim 835 ± 271 3300F1 of Bright 1167 ± 371 3100
Dim vs. Bright: p << 10-17 2א) test)
Inheritance Summary
Degree of expression of GFP driven by hsp-16.2 isheritable even in a population of isogenic animals.
Models:1) Duplications/deletions of the hsp-16.2::GFP transgene
occur rapidly creating subpopulations withdifferent copy numbers
2) Difference in promoter sequence variation3) Differences in epigenetic state
Anti Histone 3
Classical (simple) definition of the transcription
5’ 3’
Promoter Gene
Binding Motif
TFTranscription (mRNA)
Question
• DNA sequence is completely same in all cells. But the proteins contained are different in each cell. How the transcription is regulated?
5’ 3’
Promoter Gene
Binding Motif
TFTranscription
Answers• Transcription factor modification (especially,
phosphorylation)• DNA modification (especially, methylation)• Chromatin remodeling• Others (Micro RNA, mRNA modificatiions, etc.)• Abnormal (DNA mutation <- Cancer etc.)
5’ 3’
Promoter Gene
Binding Motif
TFTranscriptionp
MeMe Me Me
Chromatin Remodeling
Transcription Active Transcription Inactive
Chromatin Unwinding,
HistoneEviction
NucleosomeAssembly
Histone “Code”
Genes Dev. 2004; 18: 2315-2335
Demography Group
Demography Group
Shane Rea Jim Cypser Deqing Wu
Questions about Aging• Is Aging an Extension of Development?
NO• How Much Expression Variation?
VERY LITTLE (>1% RNA)• Error Catastrophe?
NO EVIDENCE• Is Aging Regulated?
NO. LIFE IS SELECTED FOR!• Can We Block Aging?
NO. WE CAN MODIFY IT.