This document summarizes research on the effects of temperature on longevity in C. elegans. Microarray analysis identified lysosomal protease genes that are differentially expressed at 15°C vs 25°C. Follow up experiments showed that lysosome structure and number are altered at elevated temperatures. RNAi of temperature-regulated proteases caused progressive paralysis specifically at 15°C, indicating a role in neuronal health. Further experiments traced this paralysis to dysfunction of motor neurons rather than muscles. The research suggests that proteostasis, particularly lysosomal function, is important for neuronal health under cool temperatures and influences longevity.

1. Angela Sanchez
Mentor: Pamela Larsen
Inhibitionof temperature-regulated
proteostasis genes causes an early onset
progressive dysfunction inmotorneurons

2. Cool temperature extends lifespan in C.elegans

3. Cool core temperature longevityis conserved
Drosophila
30˚c 18˚c
(Miquel 1976)
Mouse
(Conti 2006)
(Hunter 1999)
Ames Dwarf
(Lane 1996)
Caloric Restriction

4. Cool temperature is neuroandcardio protective
Enhance survival after stroke
(Kammersgaard LP J Am Heart Assoc 2002)
(Roth 2002)
Enhance survival after heart attack
(Varon J Chest 2008)
32°C 37°C33°C 37°C
(Campos 2012) (Chenoune 2010)

5. Universality of cooltemperature improving health and survival
Underlyingmechanismnot understood

6. Approach to understanding healthylong-term survival
Microarray
• AffymetrixC.elegansarray
• Genome wide snapshot
• UNBIASED
• PRIMARYbiological response to a
chronicchange

7. Primary cause occurs prior to evidence of aging

8. Experimentaldesign of samples for microarray
L1
L2
L3
L4
ImmatureAdult
Development
Total RNA Total RNA
MatureAdult
Visible Developmental Stage
15°C
15°C 25°C
Four biologicalsamples

9. Temperature regulatedgenes
19,762 Predicted Genes
16,896 Probes
383 Differentially Expressed
179
P<.01
280
Suggests partofmolecular responsemechanism Transcriptional

10. Number of Temperature Regulated Genes
Gene ontology categories
(Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)

11. Proteostasisgenes differentially expressedby temperature
Higher at 25˚C Lower at 25˚C
Proteostasis: 11 genes Proteostasis : 48 genes
Protein synthesis (0) Protein synthesis (26)
translation
ribosomal biogenesis
rRNA processing
tRNA processing
mRNA processing
rRNA/tRNA processing
rRNA transcription
rRNA/tRNA transcription
ribosome assembly
regulation of translation
proteolysis (4) proteolysis (18)
protein modification process (3) protein modification process (1)
protein folding protein folding
protein glycosylation
(Curtis,Tavare’, Takano, Sanchez Larsen, unpublished)

12. Differentiallyexpressed lysosomal proteases
cpr-1 Cathepsin B
cpr-4 Cathepsin B
W07B8.1 Cathepsin B
F21D5.2 Cathepsin B
cpr-7 Cathepsin B
K10C2.1 Cathepsin A
K10B2.2 Cathepsin A
asp-1 Cathepsin D
asp-3 Cathepsin D
asp-5 Cathepsin E
asp-6 Cathepsin D
F21F8.4 Cathepsin E
K10C2.3 Cathepsin C
C15C8.3 Cathepsin E
Y39B6A.24 Cathepsin D
Aspartyl Cysteine Serine
Classifiedbyactivesite
Onlysubsetaretemperatureregulated
19ingenome
24ingenome
10ingenome

13. Lysosomes
• Acidic organelle
• Containsproteases,glycosylases,lipases,nucleases
• Major degradationmachineryofthecell
• Roles in
• nutritionabsorption
• immunity,
• clearance ofmisfoldedproteins

14. Workingmodelofbiological responsetotemperature
Decreasedproteinsynthesisand
degradation
Increasedprotein
synthesisanddegradation

15. HYPOTHESIS
Lower expression level of temperature regulated
lysosmal proteases contributes to shorter lifespan at
elevated temperatures

16. SpecificAim 1
Determine whethergenesthat arelowered at 25°C
causelongevity or aging .
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) Determine theeffect ofRNAi ofselected genesat 15°Cand25°C onlife span

17. *
* *
*
*
*
*
*
* *
* *
*
Verify temperature regulation of lysosomal proteases
qRT-PCRverification
Normalized Ct levels
Temperature regulation confirmed
13/15
Range of expression levels
1000X

18. Further investigationwarranted
• Microarray result is VALID
• RT-PCRlarge differences

19. SpecificAim 1
Determine whethergenesthat arelowered at 25°C
causelongevity or aging .
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
c) Determine theeffect ofRNAi ofselected genesat 15°Cand25°C onlife span

20. SpecificAim 1
a) ValidatebyqPCR
b) Determine iforganelle structures are altered bytemperature
Magicred
Lysotracker red
Determine whethergenesthat arelowered at 25°C
causelongevity or aging .

21. In vivosubstrate
Linked to fluorophore
ACTIVITY
Cathepsin Substrate
Verify temperature regulation of lysosomal proteases
Magic red(MR)
LYSOSOME
Lysotracker red (LT)
Fluoresces inpH <5
Marks lysosomes
Number/Size

22. Image J Particle Analyzer
Calculates Area
Returnsnumberofparticles
Confocal Image Image J Drawing
Islysosome structureor numberaltered by elevated temperature?
ConfocalMicroscopy

23. 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

24. Qa
Islysosome structureor numberaltered by elevated temperature?
0
2
4
6
8
10
12
14
15°C 25°C
AverageGreyValueperworm
(thousands)
25˚C15˚CDAY7
MR
LT
0
5000
10000
15000
20000
25000
30000
35000
Day 7
#ofLysotrackerstainedfoci

25. Qa
Islysosome structureor numberaltered by elevated temperature?
0
2
4
6
8
10
12
14
15°C 25°C
AverageGreyValueperworm
(thousands)
25˚C15˚CDAY7
MR
LT
0
0.5
1
1.5
2
2.5
3
3.5
Day 7
Averageareaoffoci(µm^2)

26. 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
causelongevity or aging .
i) RNAi of temperatureregulated proteases
ii)Pharmacological lysosomalinhibitors
iii)RNAi of lysosomal components

27. •Cell permeable
•Protonated in acidic lysosome
•Protonated form becomes trapped
•IncreasespH
•Acidity requiredfor lysosomal enzyme activity
Lysosomal inhibitors
(Rejingoud 1976)
LYSOSOME
H+
pH

28. 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

29. Effect of lysosomal inhibitorson lifespan
alternativeexplanations
• Affecting non-lysosomal processes
• Inhibiting bacterial growth
• Behavioral changes
food avoidance
dietary restriction

30. Effect of lysosomal inhibitorson lifespan
AlternativeStrategies
• Vacuolar H+/ATPase
20 subunits in C.elegans
Pump H+ into lysosome
Resulting in acidification
LYSOSOME
H+
pH
H+
H+
Advantages
No food interference
increased specificity

31. Effect of lysosomal inhibitorson lifespan
AlternativeStrategies

32. 15˚C 25˚C
RNAiof temperature regulated lysosomal proteases
p=0.6458
n=65
p=0.4439
n=79

33. 15˚C 25˚C
RNAiof temperature regulated lysosomal proteases
p=0.9367
n=68
p=0.1064
n=79

34. 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)
Determine whethergenesthat arelowered at 25°C
in all genotypes causelongevity oraging .

35. Temperaturedependent 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.)

36. 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.)

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

38. 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

39. 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

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

41. 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 .

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

43. •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

44. But whatdoes it mean?
Determining neuronalor musculardysfunction

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

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

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

48. 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?

49. 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

50. 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

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

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

53. 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 .

54. 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.