Barshop Journal Club February 2010 (2)
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This document summarizes a presentation on the role of the DAF-2 receptor pathway in regulating longevity in C. elegans. Key points include: 1) The DAF-2 receptor integrates environmental signals like temperature and regulates processes involved in growth and survival. 2) Mutations in daf-2 can both positively and negatively impact longevity, growth, and reproduction depending on temperature. 3) Experiments examining expression levels of daf-2 ligands identified pro-survival, anti-survival, and condition-specific ligands. 4) Further experiments showed that inhibiting protein synthesis extends lifespan at 25°C but is harmful at 15°C, and this effect is dependent on daf
Barshop Journal Club February 2010 (2)
- 1. To live and grow: Mechanisms of longevity via a growth hormone pathway Angela Sanchez CSB Larsen Lab
- 2. 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
- 3. 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
- 4. Main Question: Signaling of Longevity DAF-2 RECEPTOR LONGEVITY Signaling that promote survival? Comfortably mimic effects……without negative impact
- 5. daf-2 mutants: Positive and Negative Effects DAF-2 RECEPTOR • daf-2 affects many processes • Mutation negatively impacts • Growth • Reproduction Growth Survival
- 6. 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
- 7. Identifying Mechanisms of Survival 1) Longevity Variable: COOL TEMPERATURE 2) Sorting: ALLELIC SERIES Signaling that promote survival? identify what is benefical or detrimental
- 8. Cool Temperature and daf-2 mutation:Similar effects PHENOTYPE TEMPERATURE DAF-2 Lifespan DOUBLES DOUBLES Development DELAYED DELAYED Oxygen Consumption REDUCED REDUCED Temperature daf-2 mutation
- 9. DAF-2 daf-2 alleles have weak and strong lifespan effects m41 G380E m579 R434C m596 G544S e1370 P1465S MILD SEVERE
- 10. daf-2 mutants: abnormal temperature response 0 20 40 60 80 100 0 20 40 60 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˚C25˚C
- 11. Promoting Survival or Growth: daf-2 Ligands DAF-2 RECEPTOR LONGEVITY 39 daf-2 ligands Growth DAF-2 LIGAND Dauer
- 12. 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
- 13. 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
- 14. 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
- 15. • 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
- 16. • 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 Increasing Lifespan
- 17. • 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 Increasing Lifespan Genotype Changes
- 18. Pro-survival Insulins WT 15˚C vs WT 25˚C daf-2 15˚C vs daf-2 25˚C 15˚C 25˚C daf-2 daf-2 daf-2 25˚C vs WT 25˚C daf-2 15˚C vs WT 15˚C
- 19. Anti-survival Insulins WT 15˚C vs WT 25˚C daf-2 15˚C vs daf-2 25˚C daf-2 25˚C vs WT 25˚C daf-2 15˚C vs WT 15˚C 15˚C 25˚C daf-2 daf-2
- 20. Condition-specific Insulins WT 15˚C vs WT 25˚C daf-2 15˚C vs daf-2 25˚C daf-2 25˚C vs WT 25˚C daf-2 15˚C vs WT 15˚C 15˚C 25˚C daf-2 daf-2
- 21. Insulin Ligand Summary PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC TEMPERATURE REGULATED 6 daf-28, ins-28, ins- 29, ins-35, ins-25, ins-20 6 ins-19, ins-11, ins-16, ins-27, ins-34, ins-5 5 ins-30, ins-24, ins-22, ins-23, ins-4 DAF-2 REGULATED 4 ins-33, ins-6, ins-37, ins-21 9 ins-7, ins-26, ins-34, ins-5, ins-2, ins-8, ins- 3, ins-12, ins-36, ins- 18, ins-15 2 ins-9, ins-10
- 22. Testing anti-survival ins genes • Inverse relationship between growth and survival • Growth assay • Lifespan DAF-2 RECEPTOR Growth Survival
- 23. Insulin Ligand Summary PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC TEMPERATURE REGULATED 6 daf-28, ins-28, ins- 29, ins-35, ins-25, ins-20 6 ins-19, ins-11, ins-16, ins-27, ins-34, ins-5 5 ins-30, ins-24, ins-22, ins-23, ins-4 DAF-2 REGULATED 4 ins-33, ins-6, ins-37, ins-21 9 ins-7, ins-26, ins-34, ins- 5, ins-2, ins-8, ins-3, ins- 12, ins-36, ins-18, ins-15 2 ins-9, ins-10
- 24. RNAi of ins genes: Growth Assay • Adult hermaphrodite 24 hrs on RNAi plates • 2 hr egg lay to synchronize • Animals monitored for growth effects
- 25. 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
- 26. Insulin Ligand Summary PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC TEMPERATURE REGULATED 6 daf-28, ins-28, ins- 29, ins-35, ins-25, ins-20 6 ins-19, ins-11, ins-16, ins-27, ins-34, ins-5 5 ins-30, ins-24, ins-22, ins-23, ins-4 DAF-2 REGULATED 4 ins-33, ins-6, ins-37, ins-21 9 ins-7, ins-26, ins-34, ins-5, ins-2, ins-8, ins- 3, ins-12, ins-36, ins- 18, ins-15 2 ins-9, ins-10
- 27. Testing anti-survival ins-19 and ins-7 Lifespan 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
- 28. daf-2 receptor levels? Reduced by warm temperature in longest lived allele
- 29. 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
- 30. Main Questions: Signaling and Mechanisms of Longevity DAF-2 RECEPTOR LONGEVITY Signaling that promote survival? Mechanisms of longevity?
- 31. Microarray: 25˚C vs 15˚C mRNA mRNA Compare PROTEIN SYNTHESIS LYSOSOMALMETABOLISM
- 32. • 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
- 33. Temperature Regulates Protein Synthesis Is protein synthesis required for longevity at 15˚C? Wild type? daf-2 mutants?
- 34. 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 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
- 35. • 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
- 36. 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?
- 37. 0 20 40 60 80 100 0 10 20 30 40 50 0 20 40 60 80 100 0 10 20 30 40 50 0 20 40 60 80 100 0 10 20 30 40 50 0 20 40 60 80 100 0 10 20 30 40 50 daf-2(m41) daf-2(m577) 0 20 40 60 80 100 0 10 20 30 40 50 daf-2(m596) daf-2(m579) 0 20 40 60 80 100 0 10 20 30 40 50 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)
- 38. • Inhibition increases lifespan in wildtype • Effect enhanced in daf-2 mutants – Late in life liability eIF4 RNAi extends lifespan at 25˚C
- 39. 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 0 10 20 30 40 50 60 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)
- 40. • 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
- 41. 0 20 40 60 80 100 0 10 20 30 40 50 0 20 40 60 80 100 0 10 20 30 40 50 daf-2(m41) 0 20 40 60 80 100 0 10 20 30 40 50 daf-2(m577) 0 20 40 60 80 100 0 10 20 30 40 50 daf-2(m596) 0 20 40 60 80 100 0 10 20 30 40 50 daf-2(m579) 0 20 40 60 80 100 0 10 20 30 40 50 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
- 42. • Inhibition increases lifespan in wildtype • Effect enhanced in daf-2 mutants eIF3 RNAi extends lifespan at 25˚C
- 43. • 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
- 44. Why is lifespan extension enhanced in daf-2 mutants? PROTEIN SYNTHESIS LYSOSOMALMETABOLISM DAF-2 RECEPTOR Temperature
- 46. A long long time ago……….. (1981) in a lab far far away……… (missouri) mutagenesis screen for dauer formation
- 47. daf-2 allelic variation daf-2 cxTi9878 e1369 m212 m631 mu150 sa197 sa231 e1032 e1370 m41 m633 sa186 sa219 tm1236 e1286 e1371 m577 m646 sa187 sa223 ttTi14461 e1365 e1391 m579 m65 sa189 sa229 ttTi36575 e1368 e979 m596 mg43 sa193 sa230 ttTi7618 • 35 alleles • Different severity for different phenotypes
- 48. daf-2 receptor: a historical perspective •1981 •Missouri •Mutagenesis screen •Dauer formation
- 49. What is a dauer? • Alternative developmental stage • Thin elongated morphology • Lethargic • Super resistant dauer
- 50. Favorable Unfavorable HIGH Food LOW Pheromone Temperature How do we get dauers? Dauer Grow Don’t Grow LOW Food HIGH Pheromone HIGH Temperature
- 51. Favorable Unfavorable Dauer Mutant Screen Dauer Grow Don’t Grow DAuer Formation Mutants “daf” Dauer Constitutive Dauer Defective
- 52. 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-3 daf-4 daf-5 daf-6 daf-7 daf-8 daf-9 (makes daf-12 ligand) Epistatic Ordering of daf genes Insulin Pathway TGF-B Pathway INCREASED LIFESPAN Dauer Hormone Synthesis Sensory Mutants
- 53. Tools for deciphering daf-2 DAF-2 RECEPTOR • 35 alleles • Different severity for different phenotypes • Need second longevity variable TEMPERATURE
Editor's Notes
- #4: Predisposition to live long
- #5: The talk is broken into two parts. The first part will focus on signaling that will promote either survival or grwth and the second will focus on mechanisms of longevity mediated by daf-2 receptor
- #6: Integrates enviornmental signals such as nutrient availability, temperture to decide if it should grow or hunker down to survive etc originally isolated for dauer formation which is a resistant stage, and mutations in this receptor extend lifespan
- #7: Mention that these are independently isolated alleles. Capture benefit and treat risk
- #8: Homology to the insulin receptor blah blah done. Focus in on daf-2 receptor
- #9: Why so interesetd in temperature? Well as it happens temperature and daf-2 have an awful lot in common. In polkiothermic animals are known to double lifespan – as are daf-2 mutations. When we look at other phenotypes like development – it is slowed both at cool temperatures.
- #10: Daf-2 mutations are sequenced. Point out that these are alleleles. Mutation known and causes an amino acid change these are missense alleles.
- #11: Change this to make mutants more obvious
- #12: Homology to the insulin receptor blah blah done. Focus in on daf-2 receptor. MENTION AGONISTS OR ANTAGONISTS
- #13: Arrows going up or down
- #15: Predisposition not deterministic
- #22: 34-18
- #29: qRTPCR of daf-2 mRNA 15˚C 25˚C mutants
- #31: The talk is broken into two parts. The first part will focus on signaling that will promote either survival or grwth and the second will focus on mechanisms of longevity mediated by daf-2 receptor
- #40: FIX LABELS
- #47: 43-18
- #48: Homology to the insulin receptor blah blah done. Focus in on daf-2 receptor
- #51: Explain dauers. Factors determing. Screened for mutants
- #52: Explain dauers. Factors determing. Screened for mutants
- #53: Ordered into pathways. One branch increases lifespan the other does not
- #54: In order to figure out how daf-2 works we are very lucky that the c elegans community affords us many tools. There are 35 isolated daf-2 mutants and what can be gleaned from this is that daf-2 regulates a variety of processes and these mutants effect each of these processes differently. We can exploit this fact to help us decipher which signaling may be important for the process – survival that we are interested in. But how are we to tell which signaling is good for the trait we’re interested in or which is a block. We need a second longevity variable, one that feeds into this receptor. And for that we are choosing temperature