Barshop Journal Club February 2010 (2)
Transcript of 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
Signaling that promote survival?
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
daf-2 alleles have weak and strong lifespan effects
m41G380E
m579R434Cm596G544S
e1370P1465S
MILD
SEVERE
daf-2 mutants: abnormal temperature response
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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
Dauer
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
15˚C
25˚C
daf-2
daf-2
daf-2 25˚C vsWT 25˚C
daf-2 15˚C vsWT 15˚C
Anti-survival InsulinsWT 15˚C vs WT 25˚C
daf-2 15˚C vsdaf-2 25˚C
daf-2 25˚C vsWT 25˚C
daf-2 15˚C vsWT 15˚C
15˚C
25˚C
daf-2
daf-2
Condition-specific Insulins
WT 15˚C vs WT 25˚C
daf-2 15˚C vsdaf-2 25˚C
daf-2 25˚C vsWT 25˚C
daf-2 15˚C vsWT 15˚C
15˚C
25˚C
daf-2
daf-2
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
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
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
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-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
Signaling that promote survival?
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?
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102030405060708090
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102030405060708090
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102030405060708090
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0 10 20 30 40 50 600
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40
60
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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
100
0 5 10 15 20 25 30 35 40 450
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0 5 10 15 20 25 30 35 40 45 500
102030405060708090
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daf-2(m41)
daf-2(m577)
0 5 10 15 20 25 30 35 40 45 500
102030405060708090
100
daf-2(m596)
daf-2(m579)
0 5 10 15 20 25 30 35 40 45 500
20
40
60
80
100
daf-2(e1370)
25°C eif4G RNAi
p<.0001 p= 0.6831
p= .0014
p= 0.4785
p= <.0001
p= 0.0012
wild type
(Takano, Sanchez, Larsen, unpublished)
• 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
100
0 10 20 30 40 50 600
102030405060708090
100
0 10 20 30 40 50 600
102030405060708090
1000 10 20 30 40 50 60
0102030405060708090
100
0 10 20 30 40 50 600
102030405060708090
100
0 10 20 30 40 50 600
102030405060708090
100
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
(Takano, Sanchez, Larsen, unpublished)
• 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
100
0 5 10 15 20 25 30 35 40 450
102030405060708090
100
daf-2(m41)
0 5 10 15 20 25 30 35 40 45 500
102030405060708090
100
daf-2(m577)
0 5 10 15 20 25 30 35 40 45 500
102030405060708090
100
daf-2(m596)
0 5 10 15 20 25 30 35 40 45 500
102030405060708090
100
daf-2(m579)
0 5 10 15 20 25 30 35 40 45 500
20
40
60
80
100
daf-2(e1370)
25°C eif3a RNAi
(Takano, Sanchez, Larsen, unpublished)
p<.0001 p<.0001
p<.0001
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
daf-2
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
dauer
Favorable
Unfavorable
HIGH FoodLOW PheromoneTemperature
How do we get dauers?
Dauer
Grow
Don’t Grow LOW FoodHIGH PheromoneHIGH Temperature
Favorable
Unfavorable
Dauer Mutant Screen
Dauer
Grow
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-5
daf-6
daf-7
daf-8
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