Barshop December 2011
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This document summarizes a seminar presentation on the role of proteostasis and the unfolded protein response (UPR) pathway in longevity in C. elegans. Key findings include: 1) Inhibition of pro-aging branches of the UPR, including hsp-3 and hsp-4, increases lifespan in C. elegans, similarly to reduced temperature or mutation in the insulin/IGF receptor gene daf-2. 2) Both reduced temperature and daf-2 mutation promote longevity by reducing levels of hsp-3 and hsp-4, representing a shared mechanism. 3) Elements of the UPR pathway, including pek-1 and atf-6, act in a
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Barshop December 2011
- 1. Proteostasis: A Key Node in Longevity Barshop Seminar December 14th 2011 Angela Sanchez Larsen Lab
- 2. C.elegansas a model systemfor aging • Short-lived • Inexpensive • Environmental • Genetic
- 3. Cool Temperature Doubles Lifespan in C. elegans
- 4. Cool temperature correlates with longevity Caloric Restriction (Lane 1996)
- 5. Cool temperature correlates with longevity Ames Dwarf (Hunter 1999)
- 6. Cool temperature correlates with longevity Long-lived human (Roth 2002)
- 7. Why does cool temperature improve health and survival? Underlying mechanism not understood
- 8. Cool Temperatures Double Lifespan in C. elegans Can we identify mechanisms that underlie cool longevity?
- 9. Classic Genetic Approach for Mutants Screen for mutants that fail to be normal (WT) Are there any known mutants that have an abnormal longevity response to ambient temperature? Mutants that fail to increase lifespan due to cool temperature
- 10. 0 20 40 60 80 Maximal Adult Life Span (DAYS) daf-2 allele 15˚C 22.5˚C Longevity is abnormal in daf-2 mutants Fail to respond to temperature No increase in lifespan due to ambient temperatures
- 11. Shared Phenotypes Between Cool Temperature and daf-2 mutants Temperature affects – life span – development – metabolism – reproduction daf-2 affects – life span – development – metabolism – reproduction daf-2 mutants phenocopy cool temperature Suggests its involved in response to temperature
- 12. Understanding cool mediated longevity Microarray • Affymetrix C. elegans array • Genome wide snapshot • UNBIASED • Temperature response • Six age-synchronous alleles • Look for differences in temperature and genotype regulation allow identification of key adult lifespan processes
- 13. daf-2: Mild and Severe Alleles Selected • 40 independently isolated alleles • Based on 15 phenotypes – 2 mild missense mutation – 3 severe missense mutation m41 G380E m579 R434C m596 G544S m577 C1042Y e1370 P1465S N- -CLigand binding Tyrosine kinase
- 14. 0 20 40 60 80 Maximal Adult Life Span (DAYS) daf-2 allele 15˚C 22.5˚C Cool temperature longevity attenuated in daf-2 mutants All daf-2 alleles have an aberrant temperature response
- 15. Number of Temperature Regulated Genes (Curtis, Tavare’, Takano S. Larsen P. et al.) 179 703 461 1618 645 828 280 779 441 1792 810 1048 0 500 1000 1500 2000 2500 3000 3500 4000 N2 m41 m577 e1370 m596 m579 daf-2 allele <50% Shared between daf-2 alleles
- 16. Proteostasis Genes Regulated by Temperature and daf-2 (Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished) • ER Resident/UPR • Golgi • Translation • Lysosomal proteases
- 17. BiP Canonical Unfolded Protein Response IRE-1PEK-1 BiP Translation eIF2 Transcription of UPR Genes ATF-5 Misfolded Protein BiP Site 2 Protease XB P-1 CHOP * * * *
- 18. 0 1 2 3 4 5 6 N2 25 m41 25 m577 25 m596 25 m579 25 e1370 25 xbp-1 15C xbp-1 25C ** ** ** ** ** ** Temperature Regulation of xbp-1 in Wild Type and daf-2 Levels increased in all genotypes NOT WHAT WE ARE LOOKING FOR MicroarrayExpressionLevels WT m41 m577 m596 m579 e1370 xbp-1 (15˚C) xbp-1 (25˚C) daf-2 allele
- 19. 0 1 2 3 4 5 6 N2 25 m41 25 m577 25 m596 25 m579 25 e1370 25 hsp-3 15C hsp-3 25C * ** Insignificant response to temperature multiple alleles MicroarrayExpressionLevels hsp-3 (15˚C) hsp-3 (25˚C) daf-2 allele WT m41 m577 m596 m579 e1370 Temperature Regulation of hsp-3
- 20. 0 2 4 6 N2 25 m41 25 m577 25 m596 25 m579 25 e1370 25 hsp-3 25C + + Reduced levels in some mutant alleles Lower levels correlate with longevity BINGO! Genotype Regulation of hsp-3 MicroarrayExpressionLevels hsp-3 (25˚C) daf-2 allele WT m41 m577 m596 m579 e1370
- 21. Canonical Unfolded Protein Response IRE-1PEK-1 BiP ATF-5 Site 2 Protease XBP-1 CHOP Simultaneous Presence of PRO- LONGEVITY and PRO-AGING BRANCHES of UPR
- 22. BiP What is the role of UPR genes in longevity? IRE-1PEK-1 Translation eIF2 ATF-5 Site 2 Protease CHOP hsp-3/4 hsp-3/4 hsp-3/4 RNAi of wild type animals
- 23. Two BiP Isoforms in C. elegans: hsp-3 and hsp-4 IRE-1PEK-1 hsp-4hsp-3 Heatshock InducibleConstitutive/Non-Heatshock BiP
- 24. C.eleganshas two Bip homologues hsp-3and hsp-4 •93% Identical 607/658 amino acids •hsp-4 is heatshock-inducible (>33°C) •hsp-3 constitutive
- 25. hsp-3 and hsp-4 RNAi increase lifespan at 25˚C in wild type
- 26. Inhibition of UPR genes increases lifespan at 25˚C in wild type%Survival Adult Age (days) Knockdown increases lifespan under warm conditions
- 27. Inhibition of Pro-Aging UPR and Cool Temperature: Same Mechanism of Longevity? Can we identify signaling pathways underlie cool longevity?
- 28. Inhibition of Pro-Aging UPR and Cool Temperature: Same Mechanism of Longevity? daf-2 like response No increase in lifespan due to cool temperature 0 20 40 60 80 Maximal Adult Life Span (DAYS) daf-2 allele 15˚C 22.5˚C
- 29. IRE-1PEK-1 Translation eIF2 Site 2 Protease CHOP ATF-5 BiP BiP BiP Inhibition of Pro-Aging UPR and Cool Temperature: Same Mechanism of Longevity?
- 30. RNAi of UPR genes does not alter lifespan at 15˚C Cool temperature promotes longevity by INHIBITION OF PRO-AGING UPR
- 31. Cool Temperature hsp-4 hsp-3 PRO-AGING PRO-AGING hsp-4 hsp-4 and potentially hsp-3 inhibit long-term healthy survival at 25°C
- 32. Temperature Cool Temperature and daf-2: Same Mechanism of Longevity? GeneticEnvironment daf-2 gene INHIBITION OF PRO-AGING hsp-3/4 SHARED MECHANISM?
- 33. hsp-3 and hsp-4 RNAi in mild daf-2 mutants
- 34. daf-2 and cool temperature promote longevity by reducing levels of hsp-4 and hsp-3 Represents a shared mechanism of longevity between daf-2 and temperature CONCLUSIONS
- 35. hsp-4 hsp-3 PRO-AGING PRO-AGING hsp-4 DAF-2 Cool TemperatureWarm Temperature
- 36. hsp-4 hsp-3 PRO-AGING PRO-AGING + + hsp-4 DAF-2 Cool TemperatureWarm Temperature Is this going to matter in humans and mammals? PRO-LONGEVITY
- 37. Cool temperature correlates with longevity Long-lived human (Roth 2002)
- 38. Reduced Bip Levels in Offspring of Long-lived Individuals Reduced Bip in other longevity promoting interventions?
- 39. Cool temperature correlates with longevity Caloric Restriction (Lane 1996)
- 40. Reduced Bip levels in Dietary Restricted Mice
- 41. Cool temperature correlates with longevity Ames Dwarf (Hunter 1999)
- 42. Reduced Bip levels in Growth Hormone Deficient Mice
- 43. Why does cool temperature improve health and survival? Lower Bip levels track with longevity FUNDAMENTAL MECHANISM OF LONGEVITY?
- 44. Acknowledgements Committee MembersLarsen Lab CollaboratorsFunding Dr. Shelly Buffenstein Dr. Ellen Kraig Dr. Merry Lindsey Dr. Jim Lechleiter Dr. Syuichi Takano Wendy Lee Dr. Hal Boylston Dr. Pamela Larsen NIA Training Grant Glenn Medical Foundation Ellison Medical Foundation Dr. Simon Tavaré Dr. Christina Curtis
- 45. Pro-Aging Branches of the Unfolded Protein Response IRE-1PEK-1 Site 2 Protease CHOPATF-5 BiPBEFORE AFTER ?
- 46. Pro-Aging Branches of the Unfolded Protein Response IRE-1PEK-1 Site 2 Protease CHOPATF-5 BiP ?
- 47. pek-1, ire-1, atf-6 deletion mutants ire-1 (C41C4.4) ok799 Deletion atf-6 (F45E6.2) ok551 Deletion pek-1 (F46C3.1) ok275 Deletion
- 48. Other heat shock proteins show SAME EXPRESSION PATTERN IRE-1PEK-1 hsp-4hsp-3 Heatshock InducibleHeatshock Constitutive % ΔWTTemp % Δm41 Temp % Δ m577 Temp % Δ m596 Temp % Δ m579 Temp % Δ e1370 Temp xbp-1 X-box binding protein 16.45 ** 15.22 ** 19.30 ** 15.99 ** 21.34 ** 20.02 ** atf-5 Activating transcription factor 4/5 24.20 * 13.52 19.22 29.31 ** 24.58 ** 21.63 ** hsp-3 Bip homologue (cognate) 13.35 * 4.87 7.82 9.51 16.11 ** -5.81 hsp-4 Bip homologue (heat inducible) 10.71 9.02 6.49 3.44 2.37 -5.54 Y56A3A.2 Site-2 protease -27.85 ** -45.40 ** 39.26 ** -37.08 ** -53.63 ** -49.22 ** CHOP CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP), a-9.23 -20.14 ** -11.16 -23.28 ** -25.65 ** -29.19 ** Temperature Regulation of hsp genes in Wild Type and daf-2 hsp-? hsp-? hsp-? SIMILAR REGULATION OF LONGEVITY OUTSIDE THE ER?
- 49. ire-1 deletion reduces lifespan at 25˚C ire-1 is reduces for wild type life span
- 50. pek-1 and atf-6 deletion increases lifespan at 25˚C pek-1 and atf-6 are PRO-AGING
- 51. Inhibition of UPR genes increase lifespan IRE-1PEK-1 Site 2 Protease CHOP hsp-3/4 hsp-3/4 hsp-3/4 ATF-5 RNAi in UPR deletion mutants
- 52. hsp-3 and hsp-4 RNAi rescues ire-1 lifespan hsp-3 and hsp-4 longevity DOWNSTREAM of ire-1
- 53. hsp-3and hsp-4 RNAi rescues ire-1 to hsp-3/4 RNAi levels hsp-3 and hsp-4 RNAi longevity is INDEPENDENT of ire-1
- 55. pek-1 deletion increases lifespan and is hsp-3 dependent hsp-4 RNAi promotes longevity by pek-1 inhibition
- 56. pek-1 deletion increases lifespan and is hsp-3 dependent hsp-4 RNAi promotes longevity by pek-1 inhibition
- 57. atf-6 deletion increases lifespan and is hsp-3 independent Different response to hsp-3 RNAi in atf-6 and pek-1 deletion mutants
- 58. atf-6 deletion increases lifespan and is hsp-3 independent hsp-4 RNAi promotes longevity by inhibition of atf-6
- 59. hsp-3 interacts with pek-1 to regulate longevity atf-6 and hsp-3 inhibition have an additive affect on lifespan pek-1 mutant longevity partially dependent on hsp-3
- 60. hsp-4 RNAi promotes longevity by inhibition of atf-6
- 61. Links between Unfolded Protein Response and Insulin
- 62. ATPADP
- 64. Pro-Aging Branches of the Unfolded Protein Response
- 65. daf-2: Candidate for cool temperature mediated longevity • Homologous to human insulin/IGF receptor • Found to have increased lifespan Wild type daf-2(m41)
- 66. BiP What is the role of UPR genes in longevity? IRE-1PEK-1 Translation eIF2 ATF-5 Site 2 Protease CHOP hsp-3/4 hsp-3/4 hsp-3/4 RNAi of wild type animals
- 67. 0 1 2 3 4 5 6 N2 25 m41 25 m577 25 m596 25 m579 25 e1370 25 atf-5 15C atf-5 25C * ** ** ** Insignificant response to temperature in mild alleles % ΔWTTemp % Δm41 Temp % Δ m577 Temp % Δ m596 Temp % Δ m579 Temp % Δ e1370 Temp xbp-1 X-box binding protein 16.45 ** 15.22 ** 19.30 ** 15.99 ** 21.34 ** 20.02 ** atf-5 Activating transcription factor 4/5 24.20 * 13.52 19.22 29.31 ** 24.58 ** 21.63 ** hsp-3 Bip homologue (cognate) 13.35 * 4.87 7.82 9.51 16.11 ** -5.81 hsp-4 Bip homologue (heat inducible) 10.71 9.02 6.49 3.44 2.37 -5.54 Y56A3A.2 Site-2 protease -27.85 ** -45.40 ** 39.26 ** -37.08 ** -53.63 ** -49.22 ** Temperature Regulation of atf-5 in Wild Type and daf-2 MicroarrayExpressionLevels WT m41 m577 m596 m579 e1370 atf-5 (15˚C) atf-5 (25˚C) daf-2 allele
- 68. 0 1 2 N2 25 m41 25 m577 25 m596 25 m579 25 e1370 25 site-2 protease 15C site-2 protease 25C ** ** ** ** ** **++ + + + ++ ++ Longest lived have lower levels % ΔWTTemp % Δm41 Temp % Δ m577 Temp % Δ m596 Temp % Δ m579 Temp % Δ e1370 Temp xbp-1 X-box binding protein 16.45 ** 15.22 ** 19.30 ** 15.99 ** 21.34 ** 20.02 ** atf-5 Activating transcription factor 4/5 24.20 * 13.52 19.22 29.31 ** 24.58 ** 21.63 ** hsp-3 Bip homologue (cognate) 13.35 * 4.87 7.82 9.51 16.11 ** -5.81 hsp-4 Bip homologue (heat inducible) 10.71 9.02 6.49 3.44 2.37 -5.54 Y56A3A.2 Site-2 protease -27.85 ** -45.40 ** 39.26 ** -37.08 ** -53.63 ** -49.22 ** CHOP CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP), a-9.23 -20.14 ** -11.16 -23.28 ** -25.65 ** -29.19 ** Temperature and Genotype Regulation of S2P MicroarrayExpressionLevels S2P (15˚C) S2P (25˚C) daf-2 allele WT m41 m577 m596 m579 e1370
- 69. 0 1 2 3 4 N2 25 m41 25 m577 25 m596 25 m579 25 e1370 25 CHOP 15C CHOP 25C ** ** ** ** ** ++ ++ ++ Lower levels correlate with longevity % ΔWTTemp % Δm41 Temp % Δ m577 Temp % Δ m596 Temp % Δ m579 Temp % Δ e1370 Temp xbp-1 X-box binding protein 16.45 ** 15.22 ** 19.30 ** 15.99 ** 21.34 ** 20.02 ** atf-5 Activating transcription factor 4/5 24.20 * 13.52 19.22 29.31 ** 24.58 ** 21.63 ** hsp-3 Bip homologue (cognate) 13.35 * 4.87 7.82 9.51 16.11 ** -5.81 hsp-4 Bip homologue (heat inducible) 10.71 9.02 6.49 3.44 2.37 -5.54 Y56A3A.2 Site-2 protease -27.85 ** -45.40 ** 39.26 ** -37.08 ** -53.63 ** -49.22 ** CHOP CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP), a-9.23 -20.14 ** -11.16 -23.28 ** -25.65 ** -29.19 ** MicroarrayExpressionLevels WT m41 m577 m596 m579 e1370 CHOP (15˚C) CHOP (25˚C) daf-2 allele Temperature and Genotype Regulation of CHOP
Editor's Notes
- 452 823 1 1.67
- 452 823 1 1.67
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- Implies manipulations affect the same processes
- Daf-2 gene model here? Put where mutations are. Include only alleles in experiments
- This doesn’t support the idea that everything is lower and slower at 15
- More than 20% here. What is the molecular mechanism of cool longevity. MAKE OWN TEMPERATURE GRAPH TO MATCH FORMAT OF DAF-2 GRAPH. INCLUDE ALL DATA POINTS. Point out early death in daf-2 that wt 15C is better. 15C may bring health benefits mention this . Is this respose conserved?
- More than 20% here. What is the molecular mechanism of cool longevity. MAKE OWN TEMPERATURE GRAPH TO MATCH FORMAT OF DAF-2 GRAPH. INCLUDE ALL DATA POINTS. Point out early death in daf-2 that wt 15C is better. 15C may bring health benefits mention this . Is this respose conserved?
- More than 20% here. What is the molecular mechanism of cool longevity. MAKE OWN TEMPERATURE GRAPH TO MATCH FORMAT OF DAF-2 GRAPH. INCLUDE ALL DATA POINTS. Point out early death in daf-2 that wt 15C is better. 15C may bring health benefits mention this . Is this respose conserved?
- You have an unbiased data set, but the experimental design allows you to parse results and understand more deeply. You can say why what is important in your conclusions. You do not have to wonder what if a different dose/severity of DR was used- we have two mild and three severe alleles. The multiple alleles all backcrossed to the same wild type allows you to accurately associate molecular changes with share phenotypes of known magnitude difference from WT and pleiotropic effects of the alleles with their unique profile of mutantness. Then put it together in general concepts to humans, how association of specific SNPs or polymorphisms or alleles with longevity or health or disease makes sense. E.g. which is Hispanic-like prone to diabetes, smaller stature, potential longevity.....How is a centenarian possible? analogy to daf-2 mutations opening an maximal life span option that is not possible for WT. Over 100 or over 110 years old are rare- absolutely what we that really know extent of pleiotropies of daf-2 mutants.
- More than 20% here. What is the molecular mechanism of cool longevity. MAKE OWN TEMPERATURE GRAPH TO MATCH FORMAT OF DAF-2 GRAPH. INCLUDE ALL DATA POINTS. Point out early death in daf-2 that wt 15C is better. 15C may bring health benefits mention this . Is this respose conserved?