Proteostasis: A Key Node in Longevity
Barshop SeminarDecember 14th 2011
Angela SanchezLarsen Lab
C. elegans as a model system for aging
• Short-lived
• Inexpensive
• Environmental
• Genetic
Cool Temperature Doubles Lifespan in C. elegans
Cool temperature correlates with longevity
Caloric Restriction
(Lane 1996)
Cool temperature correlates with longevity
Ames Dwarf
(Hunter 1999)
Cool temperature correlates with longevity
Long-lived human
(Roth 2002)
Why does cool temperature improve health and survival?
Underlying mechanism not understood
Cool Temperatures Double Lifespan in C. elegans
Can we identify mechanisms that underlie cool longevity?
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
WTm41
e1370m596
m5790
20
40
60
80
15˚C22.5˚C
daf-2 allele
Maximal Adult
Life Span(DAYS)
Longevity is abnormal in daf-2 mutants
Fail to respond to temperature
No increase in lifespan due to ambient temperatures
Shared Phenotypes BetweenCool 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 temperatureSuggests its involved in response to temperature
Understanding cool mediated longevityMicroarray
• 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
daf-2: Mild and Severe Alleles Selected
• 40 independently isolated alleles• Based on 15 phenotypes– 2 mild missense mutation– 3 severe missense mutation
m41G380E
m579R434C
m596G544S
m577C1042Y
e1370P1465S
N- -CLigand binding Tyrosine kinase
WTm41
e1370m596
m5790
20
40
60
80
15˚C22.5˚C
daf-2 allele
Maximal Adult
Life Span(DAYS)
Cool temperature longevity attenuated in daf-2 mutants
All daf-2 alleles have an aberrant temperature response
Number of Temperature Regulated Genes
(Curtis, Tavare’, Takano S. Larsen P. et al.)
N2 m41 m577 e1370 m596 m5790
500
1000
1500
2000
2500
3000
3500
4000
179
703461
1618
645828
280
779
441
1792
810
1048
daf-2 allele
<50% Shared between daf-2 alleles
Proteostasis Genes Regulated by Temperature and daf-2
(Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)
• ER Resident/UPR
• Golgi
• Translation
• Lysosomal proteases
BiP
Canonical Unfolded Protein Response
IRE-1PEK-1
BiP
Translation
eIF2
Transcription of UPR Genes
ATF-5
Misfolded Protein
ATF-6BiP
Site 2Protease
XB P-1 CHOP
*
*
*
*
N2 15 m41 15 m577 15 m596 15 m579 15 e1370 150
1
2
3
4
5
6
xbp-1 15Cxbp-1 25C
** ** ** ** ** **
Temperature Regulation of xbp-1 in Wild Type and daf-2
Levels increased in all genotypesNOT WHAT WE ARE LOOKING FOR
Mic
roar
ray
Exp
ress
ion
Leve
ls
WT m41 m577 m596 m579 e1370
xbp-1 (15˚C)xbp-1 (25˚C)
daf-2 allele
N2 15 m41 15 m577 15 m596 15 m579 15 e1370 150
1
2
3
4
5
6
hsp-3 15Chsp-3 25C
***
Insignificant response to temperature multiple alleles
Mic
roar
ray
Exp
ress
ion
Leve
ls
hsp-3 (15˚C)hsp-3 (25˚C)
daf-2 allele
WT m41 m577 m596 m579 e1370
Temperature Regulation of hsp-3
N2 25 m41 25 m577 25 m596 25 m579 25 e1370 250
2
4
6
hsp-3 25C
++
Reduced levels in some mutant allelesLower levels correlate with longevity
BINGO!
Genotype Regulation of hsp-3M
icro
arra
y E
xpre
ssio
n Le
vels
hsp-3 (25˚C)
daf-2 allele
WT m41 m577 m596 m579 e1370
Canonical Unfolded Protein Response
IRE-1PEK-1
BiP
ATF-5
ATF-6
Site 2Protease
XB P-1 CHOP
Simultaneous Presence of PRO- LONGEVITY and PRO-AGING
BRANCHES of UPR
BiP
What is the role of UPR genes in longevity?
IRE-1PEK-1
Translation
eIF2
ATF-5
ATF-6
Site 2Protease
CHOP
hsp-3/4 hsp-3/4 hsp-3/4
RNAi of wild type animals
Two BiP Isoforms in C. elegans: hsp-3 and hsp-4
IRE-1PEK-1 ATF-6
hsp-4hsp-3
Heatshock InducibleConstitutive/Non-Heatshock
BiP
C. elegans has two Bip homologues hsp-3 and hsp-4
•93% Identical 607/658 amino acids•hsp-4 is heatshock-inducible (>33°C)•hsp-3 constitutive
hsp-3 and hsp-4 RNAi increase lifespan at 25˚C in wild type
Inhibition of UPR genes increases lifespan at 25˚C in wild type%
Sur
viva
l
Adult Age (days)
Knockdown increases lifespan under warm conditions
Inhibition of Pro-Aging UPR and Cool Temperature:Same Mechanism of Longevity?
Can we identify signaling pathways underlie cool longevity?
Inhibition of Pro-Aging UPR and Cool Temperature:Same Mechanism of Longevity?
daf-2 like responseNo increase in lifespan due to cool temperature
WTm41
e1370m596
m5790
20
40
60
80
15˚C22.5˚C
daf-2 allele
Maximal Adult
Life Span(DAYS)
IRE-1PEK-1
Translation
eIF2ATF-6
Site 2Protease
CHOPATF-5
BiP BiP BiP
Inhibition of Pro-Aging UPR and Cool Temperature:Same Mechanism of Longevity?
RNAi of UPR genes does not alter lifespan at 15˚C
Cool temperature promotes longevity byINHIBITION OF PRO-AGING UPR
Cool Temperature
hsp-4 hsp-3
PRO-AGING PRO-AGINGhsp-4
hsp-4 and potentially hsp-3 inhibit long-term healthy survival at 25°C
Temperature
Cool Temperature and daf-2:Same Mechanism of Longevity?
GeneticEnvironment
daf-2 gene
INHIBITION OF PRO-AGING hsp-3/4 SHARED MECHANISM?
hsp-3 and hsp-4 RNAi in mild daf-2 mutants
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
hsp-4 hsp-3
PRO-AGING PRO-AGING
hsp-4
DAF-2
Cool TemperatureWarm Temperature
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
Cool temperature correlates with longevity
Long-lived human
(Roth 2002)
Reduced Bip Levels in Offspring of Long-lived Individuals
Reduced Bip in other longevity promoting interventions?
Cool temperature correlates with longevity
Caloric Restriction
(Lane 1996)
Reduced Bip levels in Dietary Restricted Mice
Cool temperature correlates with longevity
Ames Dwarf
(Hunter 1999)
Reduced Bip levels in Growth Hormone Deficient Mice
Why does cool temperature improve health and survival?
Lower Bip levels track with longevity
FUNDAMENTAL MECHANISM OF LONGEVITY?
AcknowledgementsCommittee 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 GrantGlenn Medical FoundationEllison Medical Foundation
Dr. Simon Tavaré
Dr. Christina Curtis
Pro-Aging Branches of the Unfolded Protein Response
IRE-1PEK-1 ATF-6
Site 2Protease
CHOPATF-5
BiPBEFORE
AFTER
?
Pro-Aging Branches of the Unfolded Protein Response
IRE-1PEK-1 ATF-6
Site 2Protease
CHOPATF-5
BiP
?
pek-1, ire-1, atf-6 deletion mutantsire-1 (C41C4.4)
ok799Deletion
atf-6 (F45E6.2)
ok551Deletion
pek-1 (F46C3.1)
ok275Deletion
Other heat shock proteins show SAME EXPRESSION PATTERN
IRE-1PEK-1 ATF-6
hsp-4hsp-3
Heatshock InducibleHeatshock Constitutive
% Δ WT Temp % Δ m41 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 -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?
ire-1 deletion reduces lifespan at 25˚C
ire-1 is reduces for wild type life span
pek-1 and atf-6 deletion increases lifespan at 25˚C
pek-1 and atf-6 are PRO-AGING
Inhibition of UPR genes increase lifespan
IRE-1PEK-1 ATF-6
Site 2Protease
CHOP
hsp-3/4 hsp-3/4 hsp-3/4
ATF-5
RNAi in UPR deletion mutants
hsp-3 and hsp-4 RNAi rescues ire-1 lifespan
hsp-3 and hsp-4 longevityDOWNSTREAM of ire-1
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
IRE-1PEK-1 ATF-6
hsp-4 hsp-3
PRO-AGING PRO-AGING
hsp-4
pek-1 deletion increases lifespan and is hsp-3 dependent
hsp-4 RNAi promotes longevity by pek-1 inhibition
pek-1 deletion increases lifespan and is hsp-3 dependent
hsp-4 RNAi promotes longevity by pek-1 inhibition
atf-6 deletion increases lifespan and is hsp-3 independent
Different response to hsp-3 RNAi in atf-6 and pek-1 deletion mutants
atf-6 deletion increases lifespan and is hsp-3 independent
hsp-4 RNAi promotes longevity by inhibition of atf-6
hsp-3 interacts with pek-1 to regulate longevity
atf-6 and hsp-3 inhibition have an additive affect on lifespanpek-1 mutant longevity partially dependent on hsp-3
hsp-4 RNAi promotes longevity by inhibition of atf-6
Links between Unfolded Protein Response and Insulin
ATPADP
SubstrateBindingDomain
NucleotideBindingDomain
LIDCLEFT
Pro-Aging Branches of the
Unfolded Protein Response
daf-2: Candidate for cool temperature mediated longevity
• Homologous to human insulin/IGF receptor• Found to have increased lifespan
Wild typedaf-2(m41)
BiP
What is the role of UPR genes in longevity?
IRE-1PEK-1
Translation
eIF2
ATF-5
ATF-6
Site 2Protease
CHOP
hsp-3/4 hsp-3/4 hsp-3/4
RNAi of wild type animals
N2 15 m41 15 m577 15 m596 15 m579 15 e1370 150
1
2
3
4
5
6
atf-5 15Catf-5 25C
***
****
Insignificant response to temperature in mild alleles% Δ WT Temp % Δ m41 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 -9.23 -20.14 ** -11.16 -23.28 * * -25.65 ** -29.19 **
Temperature Regulation of atf-5 in Wild Type and daf-2M
icro
arra
y E
xpre
ssio
n Le
vels
WT m41 m577 m596 m579 e1370
atf-5 (15˚C)atf-5 (25˚C)
daf-2 allele
N2 15 m41 15 m577 15 m596 15 m579 15 e1370 150
1
2
site-2 protease 15Csite-2 protease 25C
****
** **
**
**++
++ + ++
++
Longest lived have lower levels
% Δ WT Temp % Δ m41 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 S2PM
icro
arra
y E
xpre
ssio
n Le
vels
S2P (15˚C)S2P (25˚C)
daf-2 allele
WT m41 m577 m596 m579 e1370
N2 15 m41 15 m577 15 m596 15 m579 15 e1370 150
1
2
3
4
CHOP 15CCHOP 25C
**** **
** **++
++ ++
Lower levels correlate with longevity
% Δ WT Temp % Δ m41 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 **
Mic
roar
ray
Exp
ress
ion
Leve
ls
WT m41 m577 m596 m579 e1370
CHOP (15˚C)CHOP (25˚C)
daf-2 allele
Temperature and Genotype Regulation of CHOP
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