barshop journal club february 2010 (2)

To live and grow: Mechanisms of longevity via a

growth hormone pathway

Angela SanchezCSB

Larsen Lab

Goals for today: Feedback on current data

• New Data

• Critiques

• Key experiments for model testing

• HOLD QUESTIONS/COMMENTS for first portion

• Then free for all

daf-2 receptor integrates environmental signals

DAF-2 RECEPTOR

• Integrates environmental signals• Originally dauer formation• Mutations extend adult lifespan

Nutrients

Temperature

Pheromone

Growth Survival

Organismal Response

Main Question: Signaling of Longevity

DAF-2 RECEPTOR

LONGEVITY

Signaling that promote survival?

Comfortably mimic effects……without negative impact

daf-2 mutants: Positive and Negative Effects

DAF-2 RECEPTOR

• daf-2 affects many processes• Mutation negatively impacts

• Growth• Reproduction

Growth Survival

daf-2 mutation negative impacts% Growth to Adult % Brood Size Reduced Movement

Allele 15˚C 25.5C 15˚C 25˚C 15˚C 25.5C

WT 100 100 100 100 No No

Class I

M41 100 0 100 88 No No

Class 2

E1370 100 0 76 39 No Yes

M596 100 0 71 89 No Yes

m579 100 0 73 67 No Yes

(adapted from Gems 1998)

Capture the benefit…..avoid the risk

Identifying Mechanisms of Survival

1) Longevity Variable: COOL TEMPERATURE2) Sorting: ALLELIC SERIES

identify what is benefical or detrimental

Cool Temperature and daf-2 mutation:Similar effects

PHENOTYPE TEMPERATURE DAF-2Lifespan DOUBLES DOUBLES

Development DELAYED DELAYED

Oxygen Consumption

REDUCED REDUCED

Temperature daf-2 mutation

daf-2 alleles have weak and strong lifespan effects

m41G380E

m579R434Cm596G544S

e1370P1465S

SEVERE

daf-2 mutants: abnormal temperature response

0 10 20 30 40 50 600

daf-2(m41)

Ligand binding domain

15˚C vs 25˚C

daf-2 25˚C vs WT 25˚C daf-2 15˚C vs WT 15˚C daf-2 15˚C vs daf-2 25˚C

Wild type

15˚C 25˚C

Promoting Survival or Growth: daf-2 Ligands

DAF-2 RECEPTOR

LONGEVITY

39 daf-2 ligands

Growth

DAF-2 LIGAND

Sorting out daf-2 ligands• Which are expressed in adult?• Which are pro-survival?• Which are anti-survival?• Monitor mRNA levels 39 ligands

15˚C Wildtype PRO-SURVIVAL

25˚C Wildtype ANTI-SURVIVAL PRO-SURVIVAL

15˚C daf-2 vs WT

25˚C daf-2 vs WT

PRO-SURVIVAL

PRO-SURVIVAL ANTI-SURVIVAL

ANTI-SURVIVAL

Possible Function of These Peptide Growth Factors

Sorting out daf-2 ligands• Day 1 adults• Larval development at 15˚C • 24 hours at 15˚C or 25˚C • 3-4 Biological replicate• qRT-PCR duplicates– All 39 detectable– 1000 fold range

15˚C 25˚C

Wildtype Wildtype

M41 M41

E1370 E1370

M596 M596

m579 m579

Environment Effects

Gene Allele Effects

Gene X Environment Interaction Effects

• Environment Effects• Gene x Environment Interaction Effects• Gene Allele Effects

daf-2 ligands qRT-PCR Results: Questions we will answer

• Wild type 15˚C vs Wild type 25˚C Up at 15˚C

Up at 25˚C

daf-2 ligands qRT-PCR Results:Environment Effects

• Wild type 15˚C vs Wild type 25˚C

• daf-2 15˚C vs daf-2 25˚C m41 e1370

m596 m579

Up at 15˚C

Up at 25˚C

daf-2 ligands qRT-PCR Results: Gene x Environment Interaction Effects

41 1370

596 579

Increasing Lifespan

• Wild type 15˚C vs Wild type 25˚C

• daf-2 15˚C vs daf-2 25˚C m41 e1370

m596 m579

• daf-2 25˚C vs wild type 25˚C • daf-2 15˚C vs wild type 15˚C

Up at 15˚C

Up at 25˚C

Higher than WT

Lower than WT

daf-2 ligands qRT-PCR Results: Gene Allele Effects

41 1370

596 579

Increasing Lifespan

Genotype Changes

Pro-survival Insulins

WT 15˚C vs WT 25˚C

daf-2 15˚C vsdaf-2 25˚C

daf-2 25˚C vsWT 25˚C

Anti-survival InsulinsWT 15˚C vs WT 25˚C

Condition-specific Insulins

WT 15˚C vs WT 25˚C

Insulin Ligand Summary

PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC

TEMPERATURE REGULATED

6 daf-28, ins-28, ins-29, ins-35, ins-25, ins-20

6ins-19, ins-11, ins-16, ins-27, ins-34, ins-5

5ins-30, ins-24, ins-22, ins-23, ins-4

DAF-2 REGULATED

4ins-33, ins-6, ins-37, ins-21

9ins-7, ins-26, ins-34, ins-5, ins-2, ins-8, ins-3, ins-12, ins-36, ins-18, ins-15

2ins-9, ins-10

Testing anti-survival ins genes

• Inverse relationship between growth and survival• Growth assay• Lifespan

DAF-2 RECEPTOR

Growth Survival

DAF-2 REGULATED

2ins-9, ins-10

RNAi of ins genes: Growth Assay

• Adult hermaphrodite 24 hrs on RNAi plates• 2 hr egg lay to synchronize• Animals monitored for growth effects

ins-7 RNAi effects growth• ins-7– 100% embryonic lethality at 15˚C– Delayed variable growth at 25˚C• Results in death as early larvae• or dauer like arrest maybe death

• ins-7 unique cellular function• Other explanations

DAF-2 REGULATED

2ins-9, ins-10

Testing anti-survival ins-19 and ins-7Lifespan Analysis

• ins-17 RNAi – no significant change

• ins-19 RNAi – not significant at 15˚C– Significant at 25˚C • Trial one longer• Trial two shorter

• ins-7 RNAi – Not significant at either 15˚C or 25˚C– Unlike Murphy and Kenyon publications

daf-2 receptor levels?

Reduced by warm temperature in longest lived allele

Conclusions for discussion• All ins genes are made– with wide range of expression levels– Competition for the receptor occupancy

• ins gene expression is controlled by multiple factors• ins gene functions may differ in development and

adult longevity• Long life achieved by – enhancing survival patterns – reducing life limiting factors– RISKY

Main Questions: Signaling and Mechanisms of Longevity

DAF-2 RECEPTOR

LONGEVITY

Mechanisms of longevity?

Microarray: 25˚C vs 15˚C

mRNA mRNACompare

PROTEIN SYNTHESIS

LYSOSOMALMETABOLISM

• eIF4 G– Translation Initiation Factor– Inhibition Increases lifespan– Repressed at 25

• eIF3 A– Translation Initiation Factor

Temperature Regulated Translation Genes

Environment Effects

Gene Allele Effects

Gene X Environment Interaction Effects

Temperature Regulates Protein Synthesis

Is protein synthesis required for longevity at 15˚C?

Wild type?

daf-2 mutants?

0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

100 0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)

p= 0.2576 p= 0.9445

p= 0.938

p= 0.619

p= 0.3825

p= 0.0003

wild type

(Takano, Sanchez, Larsen, unpublished)

15°C eif4G RNAi

• Inhibition does not increase lifespan in wild type• Inhibition results in sickly/paralysis phenotype• Required for lifespan extension in– m579– e1370

eIF4 RNAi harmful at 15˚C

Temperature Regulates Protein Synthesis

Is protein synthesis decrease pro-survival or anti-survival at 25˚C?

In wild type?

In the daf-2 mutant background?

0 5 10 15 20 25 30 35 40 450

102030405060708090

1000 5 10 15 20 25 30 35 40 45 500

102030405060708090

0 5 10 15 20 25 30 35 40 450

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

daf-2(m41)

daf-2(m577)

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

daf-2(m596)

daf-2(m579)

0 5 10 15 20 25 30 35 40 45 500

daf-2(e1370)

25°C eif4G RNAi

p<.0001 p= 0.6831

p= .0014

p= 0.4785

p= <.0001

p= 0.0012

wild type

• Inhibition increases lifespan in wildtype• Effect enhanced in daf-2 mutants– Late in life liability

eIF4 RNAi extends lifespan at 25˚C

0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

102030405060708090

1000 10 20 30 40 50 60

0102030405060708090

0 10 20 30 40 50 600

102030405060708090

0 10 20 30 40 50 600

102030405060708090

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)

15°C eif3A RNAi

p= 0.0799 p= .4760

p= .0034

p= 0.2573

p= 0.0694

p= 0.838

wild type

• Inhibition does not increase lifespan in wildtype• Inhibition results in paralysis/hernia phenotype• Required for lifespan extension in– m579– m41

eIF3 RNAi harmful at 15˚C

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

0 5 10 15 20 25 30 35 40 450

102030405060708090

daf-2(m41)

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

daf-2(m577)

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

daf-2(m596)

0 5 10 15 20 25 30 35 40 45 500

102030405060708090

daf-2(m579)

0 5 10 15 20 25 30 35 40 45 500

daf-2(e1370)

25°C eif3a RNAi

p<.0001 p<.0001

p<.0001

wild type

• Inhibition increases lifespan in wildtype• Effect enhanced in daf-2 mutants

eIF3 RNAi extends lifespan at 25˚C

• Effect is CONDITION and GENOTYPE specific• Translation inhibition is harmful at 15˚C• Effect ENHANCED in daf-2 mutants • Not additive• daf-2 mutation overcomes life-limiting barrier

Conclusion: Be precise about context

Why is lifespan extension enhanced in daf-2 mutants?

PROTEIN SYNTHESIS

LYSOSOMALMETABOLISM

DAF-2 RECEPTOR

Temperature

A long long time ago……….. (1981)

in a lab far far away……… (missouri)

mutagenesis screen for dauer formation

daf-2 allelic variation

cxTi9878 e1369 m212 m631 mu150 sa197 sa231e1032 e1370 m41 m633 sa186 sa219 tm1236e1286 e1371 m577 m646 sa187 sa223 ttTi14461e1365 e1391 m579 m65 sa189 sa229 ttTi36575e1368 e979 m596 mg43 sa193 sa230 ttTi7618

• 35 alleles • Different severity for different phenotypes

daf-2 receptor: a historical perspective

•1981•Missouri•Mutagenesis screen•Dauer formation

What is a dauer?

• Alternative developmental stage• Thin elongated morphology• Lethargic• Super resistant

Favorable

Unfavorable

HIGH FoodLOW PheromoneTemperature

How do we get dauers?

Don’t Grow LOW FoodHIGH PheromoneHIGH Temperature

Favorable

Unfavorable

Dauer Mutant Screen

Don’t Grow

DAuer Formation Mutants“daf”

Dauer Constitutive

Dauer Defective

daf-1daf-2

daf-10

daf-12

daf-14daf-15

daf-16

daf-18

daf-19

daf-23

daf-28

daf-36 (makes daf-12 ligand)

daf-3daf-4

daf-9 (makes daf-12 ligand)

Epistatic Ordering of daf genesInsulin Pathway TGF-B Pathway

INCREASED LIFESPANDauer Hormone Synthesis

Sensory Mutants

Tools for deciphering daf-2

DAF-2 RECEPTOR

• 35 alleles • Different severity for different phenotypes• Need second longevity variable

TEMPERATURE

barshop journal club february 2010 (2)

Science

welcome to the barshop institute a world leader in aging...

journal club

barshop december 2011

journal club