error catastrophe? none detected. · heritable even in a population of isogenic animals. models: 1)...

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.