Barshop December 2011

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)


Ames Dwarf

(Hunter 1999)


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(Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)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?


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


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


Long-lived human

(Roth 2002)

Reduced Bip Levels in Offspring of Long-lived Individuals

Reduced Bip in other longevity promoting interventions?


Caloric Restriction

(Lane 1996)

Reduced Bip levels in Dietary Restricted Mice


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






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







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







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

Science

Barshop December 2011