This document summarizes research on mechanisms of longevity and aging in C. elegans. Key findings include: - 23-26% of longevity is genetic, with the remaining 75% due to environmental factors. - Genes involved in proteostasis and the unfolded protein response are differentially regulated by temperature and genotype. Inhibition of certain proteostasis genes increases lifespan at 25°C but decreases it at 15°C. - Neurodegeneration caused by RNAi of proteostasis genes starts in motor neurons with fewer synaptic connections. This suggests impaired neuronal maintenance or function at cooler temperatures. - In wild type worms, RNAi of UPR genes hsp-3 and hsp-4

1. Angela Sanchez
Mentor: Pamela Larsen
Mechanisms of Longevity
Gene by Environment
Interactions

2. How can we livea long healthylife?
23-26% oflongevity is genetic
Remaining 75% is environmental
Herskind 1996
+ =
How canIlivea long healthylife?
GENETICS

3. Environmentalinfluenceson aging andlongevity
Sunexposure
Smoking
Alcohol
BMI
Divorce
Guyuron 2009
Christensen 2009

4. We must understand each componentG, E, andGxE
What’stheprobabilitythatyouwilllivea longhealthylife?
What canwe dotomaximizeourlongevitypotential?
Life span is a complex trait
Phenotype = G + E+ (G xE)

5. Qa
Geneticand environmental manipulations that alter lifespan
Temperature daf-2
Identify mechanismsof aging andlongevity
by systematic analysis
Environmental Genetic

6. Qa
Furtherrationale for genotype selection
daf-2 25˚C
Wild type 25˚C
daf-2 15˚C
Wild type 15˚C
Homologous to human insulin/IGF receptor
Phenocopies aspects of cool temperature
Abnormalresponseto different cultivation temperatures
Larsen et al 1995
Gems et al 1998

7. Lifespan ofindependent daf-2alleles all missense
daf-2involved inacclimatization totemperature
unlike daf-2,temperature response is daf-16independent

8. TEMPERATURE REGULATION
daf-2REGULATON
WT WT15°C 25°C
daf-2 daf-215°C 25°C
vs
vs
vs vs
Microarraycomparisons: daf-2andtemperature

9. TEMPERATURE REGULATION
daf-2
Wild type + +15°C 25°C
15°C 25°C
vs
vs
vs vs
Microarraycomparisons: daf-2andtemperature
e1370
m41
m596
m577
m579
e1370
m41
m596
m577
m579
daf-2REGULATION

10. Qa
Number of Temperature Regulated Genes
(Curtis,Tavare’, Takano S. LarsenP.et al.)
700shared between all daf-2mutants
-4000
-3000
-2000
-1000
0
1000
2000
3000
WT m577 m41 e1370 m596 m579
NumberofDEGenes
WT WT15°C 25°C daf-2 daf-215°C 25°C
1000
2000
3000
4000
HIGHER at 25˚C
LOWER at 25˚C

11. Qa Qa
Number of daf-2regulatedgenes
(Curtis, Tavare’, Takano S. Larsen P. et al.)
-600
-400
-200
0
200
m577 m41 e1370 m596 m579
NumberofDEGenes
-6000
-4000
-2000
0
2000
m577 m41 e1370 m596 m579
WT 15°C
daf-2 15°C
WT 25°C
daf-2 25°C
m41 m577 e1370 m596 m579 m41 m577 e1370 m596 m579
HIGHER
LOWER
200
400
600
2000
4000
6000

12. Types of regulated genes
(Curtis, Tavare’, Takano S. Larsen P. et al.)
1. Protein synthesis
2. Lysosomalproteases
3. ER resident proteins

13. HYPOTHESIS
Proteostasis is a key mechanism
for healthylong-term survival
SIGNIFICANCE
Environmental changestrigger beneficial and detrimental responses.By
associating molecular signatureswith life expectancy we can predict
phenotypic outcome in different genotypes and environments.

14. SpecificAim 1
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) Determine theeffect ofRNAi ofselected genesat 15°Cand25°C onlife span

15. Qa
Islysosome structureor numberaltered by elevated temperature?
25˚C15˚C
0
1
2
3
15°C 25°C
AverageGreyValueper
worm(thousands)
DAY1
MR
LT
0
5000
10000
15000
20000
25000
30000
Day 1
#ofLysotrackerstainedfoci

16. Qa
Islysosome structureor numberaltered by elevated temperature?
0
2
4
6
8
10
12
14
15°C 25°C
AverageGreyValueperworm
(thousands)
25˚C15˚CDAY1
MR
LT
0
5000
10000
15000
20000
25000
30000
35000
Day 7
#ofLysotrackerstainedfoci

17. Qa
Islysosome structureor numberaltered by elevated temperature?
0
2
4
6
8
10
12
14
15°C 25°C
AverageGreyValueperworm
(thousands)
25˚C15˚CDAY1
MR
LT
0
0.5
1
1.5
2
2.5
3
3.5
Day 7
Averageareaoffoci(µm^2)

18. SpecificAim 1
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) Determine theeffect ofRNAi ofselected genesat 15°Cand25°C onlife span
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .

19. Effect of lysosomal inhibitors on lifespanTemperaturedependent phenotype of proteostasis inhibition
15˚C 25˚C
n=75
p= 0.002
n=64
p= <.0001*
0
20
40
60
80
100
0 5 10 15 20 25 30 35 40
%Survival
Adult Age (days)
Empty Vector
ifg-1 RNAi
0
20
40
60
80
100
0 5 10 15 20 25 30 35 40
%Survival
Adult Age (days)
Empty Vector
ifg-1 RNAi
(TakanoS.,Larsen P.etal.)

20. DECREASES LIFESPAN at 15˚C
10mM Chloroquine
15˚C 25˚C
DETRIMENTAL at 15˚C and BENEFICIAL at 25˚C
INCREASES LIFESPAN at 25˚C
n=75
p= 0.0028*
n=64
p= <.0001*
Temperaturedependent phenotype of proteostasis inhibition

21. SpecificAim 1
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) Determine theeffect ofRNAi ofselected genesat 15°Cand25°C onlife span
single protease RNAi at15°C and25°C(7 genes)
RNAiof V-ATPasesvha-15vha-1315°Cand25°C
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .

22. 0
20
40
60
80
100
0 5 10 15 20
PerecentNormal
Locomotion
Days of Adulthood
Empty Vector
cpr-7
0
20
40
60
80
100
0 5 10 15 20
PercentNormalVulva
Days of Adulthood
Empty Vector
egl-45
rpc-1
nol-5
F14B4.3
ifg-1
RNAi knockdown ofproteostasis genes resultsin defects at 15°C
(Takano S. , LarsenP.etal.) (Sanchez A.,Larsen P.etal.)

23. cpr-7 15°Cday2
cpr-7RNAi causes progressive paralysis
Control 15°Cday2
cpr-7 25°Cday2Control 25°Cday2

24. cpr-7 15°C day 8Control 15°C day 8
cpr-7 25°C day 8Control 25°C day 8
cpr-7 Progressive Paralysis: Day 8
cpr-7RNAi causes progressive paralysis

25. RNAi 15°C day 12
RNAi 25°C day 12Control 25°C day 12
Control 15°C day 12
Cpr-7 Progressive Paralysis: Day 12
cpr-7RNAi causes progressive paralysis

26. cpr-7 Progressive Paralysis
(15⁰C) Day 8
cpr-7RNAi causes progressive paralysis

27. SpecificAim 1
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) RNAiselected genes at 15°Cand25°C
d) Determine whetherparalysisis causedby neuronalor musculardysfunction
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .

28. Determining neuronalor musculardysfunction
•Tissuespecific RNAi
•Neuronsresistantto RNAi
•Leakage between tissues
•Neurotransmitterdrugs
•40% of animals died preventing longitudinal studies
•Neuromuscularexam without drugsor transgenics

29. •Feed adult worms RNAi for 8 days
•Transfer40 worms to individual plates
•Score on days 9, 10, 11, 12, 13, 15, and 16
•Blinded and unblinded examiner
•Exam consistsoftail tap, pause,head tap
•Establishes functionof
•Touchreception
•Musclefunction
•Neuronalfunction
•forward and backward motion circuits
•Switching ability
Determining neuronalor musculardysfunction

30. MUSCLE
C.eleganslocomotion circuits
Bodywall muscles
BACKWARDFORWARD
Type A motor neurons Type Bmotor neurons
Type D motor neurons
Bessereau Lab
Worm Atlas

31. But whatdoes it mean?
Determining neuronalor musculardysfunction

32. C. elegans locomotion circuit
Body wall muscles
BACKWARDFORWARD
Type A motor neurons Type B motor neurons
Type D motor neurons
Worm Atlas

33. Individual longitudinal data from neuromuscular exam
Control cpr-7 rpc-1

34. Vulva and locomotion circuits shareneurotransmittersand areadjacent
•Vulval musclesregulate egg laying
•VC4 and VC5 inhibit opening

35. Why is posterior half paralyzed first?
Number of motor neuronsare spread over
length of the animal in the ventral nervecord
Arethe number of synapses different overthe length of the animal?

36. Number of synapses in type A motor neurons
0
20
40
60
80
100
DA1 DA2 DA3 DA4 DA5 DA6 DA7 DA8 DA9
Post Synaptic
0
20
40
60
80
DA1 DA2 DA3 DA4 DA5 DA6 DA7 DA8 DA9
Presynaptic
0
20
40
60
80
100
VA1 VA2 VA3 VA4 VA5 VA6 VA7 VA8 VA9 VA10 VA11 VA12 VA13
Postsynaptic
0
20
40
60
80
100
VA1 VA2 VA3 VA4 VA5 VA6 VA7 VA8 VA9 VA10 VA11 VA12 VA13
Presynaptic

37. 0
20
40
60
80
100
DB1 DB2 DB3 DB4 DB5 DB6 DB7 DB8 DB9
Postsynaptic
0
20
40
60
80
100
DB1 DB2 DB3 DB4 DB5 DB6 DB7 DB8 DB9
Presynaptic
0
20
40
60
80
100
VB1 VB2 VB3 VB4 VB5 VB6 VB7 VB8 VB9 VB10 VB11 VB12
Postsynaptic
0
20
40
60
80
100
VB1 VB2 VB3 VB4 VB5 VB6 VB7 VB8 VB9 VB10 VB11 VB12
Presynaptic
Number of synapses in type B motor neurons

38. Proposed mechanismof neuraldysfunction
•Factorsnecessaryfor synaptic maintenance or neurotransmissionunder
cool conditions arenot reaching theirdestination
•Motor neuronswith fewer synapsesare sensitive

39. SpecificAim 1
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) RNAiselected genes at15°Cand25°C
d) Determine whetherparalysisis causedby neuronalor musculardysfunction
COMPLETE
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .

40. SpecificAim 1
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) RNAiselected genes at15°Cand25°C
d) Determine whetherparalysisis causedby neuronalor musculardysfunction
COMPLETE
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .

41. Data mining 15C and 25C molecular signatures
• Somegenes changesimilarly inallgenotypesbetween
thetwo temperatures
• Somegenes inwild typeandnotdaf-2
• Somegenes indaf-2andnotwild-type
• Associatingmolecularsignatures withlife expectancy
• SpecificAim2 and3 are different types ofsignatures

42. 15°C 25°C15°C 25°C
AGINGLONGEVITY
Environmentappropriate molecularresponses
Only a small fraction of the genes associate with lifespan
this way

43. Environmentappropriate responses
15°C 25°C15°C 25°C

44. 15°C 25°C15°C 25°C
Somechanges are necessaryfor long term healthy survival
Environmentappropriate responses

45. 15°C 25°C
25°C
AGING
15°C
LONGEVITY
65 genes
15°C 25°C
25°C
AGING
93 genes
Environmentappropriate responses

46. UnfoldedProtein Response
IRE-1PEK-1
BiP
ATF-5
Site 2
Protease
CHOP
PRO-AGING
BRANCHESof UPR

47. daf-2 has lowerlevels
Temperature andgenotype regulationof UPRgenes
MicroarrayExpressionLevels
S2P
WT daf-2
CHOP
WT daf-2
hsp-3
WT daf-2

48. Specific Aim 2
Determine whetherdifferentially expressed
genes that associates with aging causeaging at
25°C.
a) ValidatekeyDEgenes by qPCR
b) Determine theeffect ofRNAi ofputativewarmaginggenes at 15°Cand25°C
PROOFOFPRINCIPLETHATMOLECULAR SIGNATUREIS PREDICTITVEOF Adult
health, AGINGOR LONGEVITY

49. Twobip isoformsin C.elegans:hsp-3andhsp-4
IRE-1PEK-1
hsp-4hsp-3
Heatshock InducibleConstitutive/Non-Heatshock
BiP
•93% Identical 607/658 amino acids
•hsp-4isheatshock-inducible (>29°C)
•hsp-3constitutive

50. hsp-3and hsp-4RNAi increase lifespan at 25˚C in wild type

51. Inhibition ofUPR genes increases lifespanat 25˚C in wild type
%Survival
Adult Age (days)
Knockdownincreases lifespanunder warmconditions

52. RNAiof UPR genes does not alter lifespan at15˚C

53. Pro-agingbranchesof the Unfolded Protein Response
IRE-1PEK-1
Site 2
Protease
CHOPATF-5
BiPBEFORE
AFTER
?

54. IRE-1PEK-1
Site 2
Protease
CHOPATF-5
BiP
?
Pro-agingbranchesof the Unfolded Protein Response

55. SpecificAim 2
a) ValidatebyqPCR(partial complete need daf-2mutants)
b) Determine theeffect ofhsp-3andhsp-4at15°C and25°C
c) Determine whetherbranches of theunfolded protein response isrequired for Bipinhibition
longevity phenotype
Determine whetherdifferentially expressed
genes that associates with aging causeaging at
25°C.

56. 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

57. ire-1deletion reduceslifespan at 25˚C
DISCLAIMER: PRELIMINARYDATA ONUNBACKCROSSEDSTRAINS
*

58. hsp-3and hsp-4 RNAi rescuesire-1lifespan
**
* *
hsp-3and hsp-4 RNAi longevity might be
DOWNSTREAM ofire-1
(iftherearenoothermutations inthisstrain)

59. hsp-3and hsp-4 RNAi rescuesire-1to hsp-3/4RNAi levels
hsp-3and hsp-4RNAi longevity might be
DOWNSTREAM ofire-1
(iftherearenoothermutations inthisstrain)

60. pek-1and atf-6deletion increases lifespan at 25˚C
pek-1 and atf-6might be PRO-AGING

61. Whatistherelationshipbetweenpek-1andthetwobips
hsp-4RNAi does not increase pek-1lifespan
pek-1deletionincreaseslifespanandmightbehsp-3dependent

62. Whatistherelationshipbetweenpek-1andthetwobips

63. atf-6deletion increaseslifespan and might be hsp-3independent
Different responseto hsp-3RNAi
in atf-6and pek-1 deletion mutants
Whatistherelationshipbetweenatf-6andthetwobips

64. atf-6deletion increases lifespan might be hsp-3independent
Whatistherelationshipbetweenatf-6andthetwobips

65. hsp-3interacts withpek-1toregulatelongevity
atf-6and hsp-3inhibition have an additive affect on lifespan
pek-1mutant longevity partially dependenton hsp-3

66. hsp-4 RNAipromotes longevity by inhibition of atf-6

67. IRE-1PEK-1
hsp-4 hsp-3
PRO-AGING
PRO-AGING
hsp-4
Model of Pro-Aging Branches of the UPRVersion A
atf-6and pek-1inhibitionshould increaseire-1lifespan

68. hsp-3and hsp-4 RNAi rescuesire-1lifespan
atf-6and pek-1inhibitionshould increaseire-1lifespan

69. What is the relationship between atf-6and pek-1 to ire-1
0
20
40
60
80
100
0 5 10 15
ire-1(ok799) atf-6 RNAi
ire-1(ok799) control
0
20
40
60
80
100
0 5 10 15
ire-1(ok799) pek-1 RNAi
ire-1(ok799) control
atf-6andpek-1 RNAifail to rescueire-1deletionmutants

70. IRE-1PEK-1
hsp-4 hsp-3
PRO-AGING
hsp-4
Model of pro-aging branches of the UPRVersion B
PRO-AGING
BIPLONGEVITYMAYBE INDEPENDENTOF UPRUNDERPHYSIOLOGICAL
CONDITIONS

71. Features of a Bip longevity effector
1. ER resident or secretedprotein
2. Associates with a pro-aging signatureon microarray

72. Candidate for Biplongevityeffector: haf-6
1. ER resident protein
2. Peptide exporter
3. Analogous tohaf-1frommtUPR
4. Untested UPRfunction
5. Pro-aging signatureon microarray
Lower in daf-2 mutants at 15°C and 25°C

73. Does haf-6 deletion affect life span?
0
20
40
60
80
100
0 5 10 15 20 25
haf-6
WT
haf-6 deletion increaseslifespanat 25C

74. Candidate Bip longevity effector: Sec61 translocon
1. ER resident protein
2. Α, β,ᴽTransportsproteins intothe ER
3. Requireshsp-4 for cotranslational transport
4. Pro-aging signatureon microarrayalso dnj-27 = ERJ1
5. F55A4.2 is sil1 match from drosophila sil1
6. Grp170 homologue T24H7.2 has no change
TEMPERATURE REGULATION daf-2 15C vs WT 15C daf-2 25C vs WT 25C
Gene Name Clone ID Description WT m577 m41 m596 m579 e1370 m577 m41 m596 e1370 m579 m577 m41 M596 e1370 m579
trap-3 Y38F2AR.2
Translocon-associated TRAP,
gamma subunit
tram-1 C24F3.1
translocating chain associating
membrane transporter
191065
_s_at
dn
j-
27---
Y47H9
C.5 ///
Y73B6
BL.12
6118 // electron transport
// inferred from electronic
annotation --- ---
3.0
2
3.1
7
2.9
7
2.8
8
2.4
8
3.1
1
3.3
2
3.2
9
3.0
8
3.2
2 3.2
2.6
7
0.3810
5795
0.7787
4751
0.7729
5987
0.1178
7964
0.0005
1936
0.0217
5497
0.8282
166 1
0.8295
0984
0.0395
9742
0.8524
0529
0.9467
1984
0.4372
6907
0.7076
48
0.6923
6025
0.0005
533 1 0.01 0 0 0 0 1 2 # # # 1.23 1.08 1.08 1.27 1.64 0.74 1.12 0.97 0.91 0.69 1.07 0.98 0.84 0.93 0.92 0.63 slct dnj-27
Y47H9C.5 ///
Y73B6BL.12 191065_s_at
Molecular chaperone(DnaJ
superfamily)
6118 //
electron
transport //
inferred from
electronic
annotation dnj-27
Y47H9C.5 ///
Y73B6BL.12

75. Specific Aim 3:
Determine whether differentially expressedgenes that
associate with context dependent aging and longevity
cause aging and longevity in a context dependent manner

76. 15°C 25°C
15°C
AGING
25°C
LONGEVITY
62
15°C 25°C
25°C
AGING
15°C
LONGEVITY
Genesin Aim 1
Mirrorphenotypes shouldexist for both cool and warm

77. xbp-1 R74.3
X box binding
protein-1 2 2 2 2 2
sod-2 F10D11.1
Manganese
superoxide
dismutase 0 2 2 2 2 2
ttx-7
Required for
thermotaxis 0 2 2 2 2 2
flp-6 neuropeptide 0 2 2 2 2 2
Specificgenes in categories that associate with longevity in context dependent manner

78. Warm longevity/coolaging genes exist!
0
20
40
60
80
100
0 10 20 30 40 50 60
%Survival
Adult Age (days)
m596 asp-1
m596 Control
0
20
40
60
80
100
0 10 20 30 40 50 60%Survival
Adult Age (days)
m596 asp-1
m596 Control

79. Expectedconclusions in dissertation
• Identified mechanism forcontext dependent longevity and health
• Demonstrated appropriate physiological ER changesthat contribute to aging
• Refined the numberand direction ofpredictive categories for adult health aging and
longevity
• Shown causality for geneticby environmental interactions which are relevant to
adult life span and health

80. Presented a paper or poster at national meetings
Nathan Shock meeting Bandera 2009,2010
Genetics Society of America, Boston 2010
Gerontological Society of America,NewOrleans 2010
C. elegans International meeting, Los Angeles 2011
Contributed to writing a paper
temperature
Authoredhis/herown paper and review/book chapter
daf-2 and temperature
Genetics of Longevity
Other training
Barshop Student Day:Co-organizer2009
Participated annually
Barshop Student Oral Presentation: Annual
Barshop Student DayPoster Presentation: Annual
Neurobiology of Aging Course: AElectives complete
Meetings and authorship

81. Looking forward:post docs
Gordon Lithgow Buck Institute Novato, CA
MattKaeberlein University of Washington Seattle, WA
MaleneHansen Sanford-Burnham Institute San Diego, CA
Gary Ruvkun Mass General, Harvard University Boston, MA
Arturo Buylla UCSF San Francisco, CA
Graeme Davis UCSF San Francisco, CA
Ann Brunet Stanford University San Francisco, CA
Troy Littleton MIT Cambridge,MA
Alexey Terskikh Sanford-Burnham Institute San Diego, CA
Also interested in jobs outside academics!

82. SpecificAim 2 and 3: Done and To bedone
a) ValidatebyqPCR(partial havewt need daf-2andalsonulls)
b) Determine lifespans at 15°Cand25°C, three trials each
Backcrossmutants
atf-6
pek-1
ire-1
hsp-4 (5/10) 1 month
hsp-3 (8/10) 2 weeks
haf-6 (4/10) 6 weeks

83. SpecificAim 2 and 3 Lifespans tobe done
f-6
pek-1
ire-1
hsp-4
hsp-3
haf-6
hsp-4;haf-6
hsp-4;pek-1
hsp-4;atf-6
hsp-4;ire-1
atf-6;ire-1
pek-1;ire-1
hsp-3;ire-1
hsp-4;ire-1
hsp-3;pek-1
hsp-3;atf-6
hsp-3;ire-1
1. Constructdouble mutants 1 month?
2. Lifespans X3 trials
a)15C (2 months) or (4 months) daf-2
b) 25C (4 weeks) or (2 months) daf-2
Total : 6 months?
hsp-3;daf-2
hsp-4;daf-2

84. Acknowledgements
Committee MembersPast Larsen Lab Members
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

85. hsp-3 and hsp-4 RNAi in severe daf-2 mutants