research talk

1. Reduction of proteostasis gene expression negatively
impacts adult healthspan or increases life span,
depending on the environment
Angela Sanchez
Cellular & Structural Biology

2. Healthy Living
Fauja Singh ,97 Philip Rabinowitz, 104 Mavis Lindgren, 90
What interventions can we use?

3. Cool Temperatures Favors Healthy Long-Term Survival
Long Lifespan Short Lifespan
Cool Temperature Warm Temperature
(Lamb MJ, 1968) (Kibler HH, 1961)(Liu RK, 1973)

4. Low Temperature Enhances Survival
• Enhance survival after heart attack
(Varon J Chest 2008)
• Enhance survival after stroke
(Kammersgaard LP J Am Heart Assoc 2002)
• Long-lived Individuals naturally lower CBT
(Shock, NW 1984)
MECHANISM FOR THIS IS UNKNOWN

5. How does temperature affect survival
in C. elegans ?

6. What accounts for this difference?
• Molecular mechanism of acclimatization
• Which cellular processes are altered?
?
How can we find the cause of this?

7. Temperature Dependent Survival in C. elegans
PercentSurvival
Days
25°C
15°C
25°C
15°C
Lifespan is doubled at 15°C

8. Microarray Analysis
• Collaboration with S. Tavare’ (USC)
• Different from affymetrix software
• Calculated Background
• Not estimated

9. Microarray Overview
• Post-development
• Look early before there is a difference
• Molecular mechanism of acclimatization
• Which cellular processes are altered?

10. Microarray Overview
L1
L2
L3
L4
Immature
Development
Total RNA Total RNA
Mature
Visible Developmental
Stage
15°C
15°C 25°C
Four Biological Samples

11. Temperature Regulated Genes
19,762 Predicted Genes
16,896 Probes
386 Differentially Expressed
178
P<.01
208
Suggests part of acclimatization mechanism Transcriptional

12. Temperature Regulated Genes
386 total altered genes
Gene Ontology

13. Cellular Response To Temperature
REPRESSION of protein synthesis genesChanges in the activity of the endoplasmic reticulumREPRESSION of lysosomal proteasesDisproportionate impact on membrane associated proteins
REPRESSED
REPRESSED
ALTERED

14. Cellular Response To Temperature
REPRESSED
REPRESSED
ALTERED

15. Hypothesis
Temperature regulated proteases contribute to
increased healthy survival observed at 15°C

16. 15°C Elevated Proteases mRNA
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
Classified by active site
31 in genome
39 in genome
75 in genome

17. Is the array result true?
• Validation by QPCR
• Designed primers
• Tested
• Assay three biological samples
• Normalized to cyc-1 for control

18. RT PCR of 15C elevated Proteases
0
1
2
3
4
5
6
7
8
9
10
FoldIncrease

19. Specific Aims
1. Elucidate the biological function of proteases
differentially expressed between 15°C and 25°C
2. Determine the effect of altered protease
expression on the proteome

20. Specific Aim One
1. Elucidate the function of differentially expressed proteases
a) Determine if reduction by RNAi shows a phenotype at 15°C or
25°C.
b) Determine if protease-eGFP transgenics shows a phenotype at
15°C or 25°C
c) Determine spatial and temporal expression of lifespan altering
proteases

21. 15°C Elevated Proteases mRNA
cpr-1
cpr-4
W07B8.1
F21D5.2
cpr-7
K10C2.1
K10B2.2
asp-1
asp-3
asp-5
asp-6
F21F8.4
K10C2.3
C15C8.3
Y39B6A.24
Aspartyl Cysteine Serine
(RNAi Library Ahringer et al. 2003)

22. cpr-7 15°C day 2
cpr-7 Progressive Paralysis:Young
Control 15°C day 2
cpr-7 25°C day 2Control 25°C day 2

23. 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

24. cpr-7 Progressive Paralysis
(15⁰C) Day 8

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

26. RNAi knockdown of cpr-7 results in paralysis at 15°C

27. Conclusions
• Reduction of cpr-7 results in DECREASED
health at 15C
• Results in 80-90% affected
• Reduction of cpr-7 may be BENEFICIAL at 25C
PARADOX

28. Does cpr-7 RNAi impact lifespan?

29. cpr-7 RNAi effect on lifespan
0
20
40
60
80
100
120
0 5 10 15 20 25
Control
cpr-7 RNAi
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35
15°C
Control
cpr-7 RNAi
25°C
DECREASES INCREASES
OPPOSITE EFFECTS AT DIFFERENT TEMPERATURES

30. Conclusions
• cpr-7 RNAi DECREASES lifespan at 15°C
• cpr-7 RNAi causes progressive paralysis at 15°C
• cpr-7 RNAi slightly INCREASES lifespan at 25°C
• Opposite effects at different temperatures

31. daf-2 mutation
temperature15°C
25°C
15°C
0
20
40
60
80
100
0 10 20 30 40 50 60
N2
daf-2(m41)
%Survival
Adult age (days)
It is a shared mechanism
Translation gene eif3A temperature regulated similarly
in WT and 5 daf-2 alleles
Daf-2 independent.
Cpr-7 is both temperature and daf-2
regulated it is different in the daf-2 allleles
It is a distinct mechanism

32. Daf-2 mutations
• are over 40 independently isolated alleles
•How to choose which ones
• Go by severity of phenotypes, particularly adult
•Choose some strong
•Some mild

33. Low Temperature and Mutation of the daf-2
Gene are Similar for Many Traits
Temperature affects
– development (rate and dauer formation)
– metabolism
– reproduction
– life span
DAF-2 pathway affects
– development (dauer formation and rate)
– metabolism (oxygen consumption)
– reproduction
– life span

34. Shared mechanisms?
How to sort this out
Use different alleles

35. DAF-2 receptor
DAF-16
DAF-2 Signaling and mutations
m41
G380E
m577
C1042Y
Class I
m579
R434C
m596
G544S
e1370
P1465S
Class II
(strong) (mild)
Longevity

36. Worm ins Genes
• 39 insulin-like loci identified in the genome
• ins-1 and ins-18 act as antagonists (Pierce et al., 2001)
• daf-28 is an agonist. Overexpression of ins-4 & -6 can
suppress the daf-28 mutation, so they are predicted to
function similarly. (Li et al., 2003)
• ins-7 acts as agonist (Murphy et al., 2003)
Is the regulation specific for these ins genes?

37. Summary: Receptor Mutations Result in
Multiple Changes in Insulin-like Signaling
Wild type (39 “insulin like” genes)
DAF-2 missense mutants- different amounts of agonist and
antagonist ligand mRNAs. More receptor mRNA.
INS binding affinity changed?
Signals transduced?
Altered downstream preferences?

38. daf-2 Mutants Altered Temperature Response
daf-2 involved in acclimatization to temperatureunlike daf-2, temperature response is daf-16 independentLive longer, maintain temperature responseAltered temperature response
(Reference et al.)
REMOVE DAF-16 lifespan line

39. Daf-2 mutants respond to temperature
differently

40. Can daf-2 mutants sense temperature?
• Thermotaxis (Ttx) assay
• AIY sensory neurons (FIND OUT NEURONS)
• Migrate preferentially toward growth temperature
• Will associate growth temperature with food
• Learned behavior

41. ttx-1 and ttx-3
Or cloned and where expressed
Compare them too each other in table parallel
Cryophyllic or athermotactic this is
what is important about them

42. daf-2 ttx-assay
m41
e1370
m579
121416182022242628
Wildtype
No difference
(Sanchez, Takano, Larsen)

43. Conclusion
• Adults carrying a daf-2 mutation do Not
sensing temperature differently from wild
type or from each other
• Mechanism for Age
– In daf-2 mutant adults
– In adults at cool or warm temperature
– Is unlikely to originate from neuronal signals due
to sensation of temperature in the circuit that
controls taxis to a particular temperature

44. daf-2 mutation
temperature15°C
25°C
15°C
0
20
40
60
80
100
0 10 20 30 40 50 60
N2
daf-2(m41)
%Survival
Adult age (days)
Shared or distinct mechanism?

45. Number of Genes with Altered Expression in
Response Temperature
179
461
703 645
828
1618
208
441
779 810
1048
1792
0
500
1000
1500
2000
2500
3000
3500
4000
N2 m577 m41 m596 m579 e1370
Numberofgene
UP
DOWN
(Curtis, Tavare’, Takano S. Larsen P. et al.)

46. daf-2 Mutants Altered Temperature Response
Flip this to be like next one

47. 200
220
240
260
280
300
320
340
10 15 20 25 30 35 40 45 50
Age at Last Quartile (days)
N2
daf-16
daf-16; daf-2
fer-15
daf-2(m577)
daf-2(m41)
fer-15; daf-2
daf-2(m596)
daf-2(m579)
r=0.34, P=0.368
MetabolicRate
(pmolO2individual-1hour–1)
SharedWarmDifferentiallyExpressed
Genes

48. category number example
Secreted 24 Purple acid phosphatase, lysozyme,
metalloproteinase,
Lysosomal 15 Sugar degradation, protease inhibitors
Fat 25 Lipid transport, desaturase lipid binding protein
Endoplasmic Reticulum 38 Endocytosis, lipid biosynthesis and degradation
C-type lectin 10
Udp-glucosyltransferase 3
Mitochondria 18 Thioredoxin, glutathione-s-transferase
Transport 22
Ion transport 7
Sugar transport 3
Lipid transport 4
Amino acid transport 3
Amino Acid Recycling 8 Histidine catabolism, Methionine salvage, pyrimidine
degradation
Increased at 25C Daf-2 different from wildtype

49. Category Number Examples
Neuronal
28 Jnk-1, mak-2, synaptic vesicle, jkk-1
ER/golgi 15 Endocytosis, glucosyl transferase
Integral membrane transport 19
cell swelling 2 osr-1 aqp-4
ion transport 9 Calcium and sodium transporters
lipid transport 3 lipid endocytosis
other 5 amino acid, lactate, transporter
lysosomal 3 atg-1 , cysteine protease,
muscle 3 Tropomoduline, myosin phosphatase
structural 5 act-4, act-3
Hedgehog related proteins 9 grl-5 grl-7 ptr-5 hyperdermis
Transcription Factors 8 fkh-7, jun-1, nhr-255, peb-1
About 160 differentially expressed in the strong alleles
Increased at 25C Daf-2 STRONG ALLELES only

50. Daf-2 mutants accentuate wildtype response
• Increased fatty acid desaturases
• Increased sugar binding proteins
• Increased glucosyltransferases
• Increased inhibition of translation
• Skewing of lysosomal function away from
protein degradation and towards sugar
degradation

52. Daf-2 mutants have altered membranes
Ray’s fat data

53. Do daf-2 alleles respond differently to
temperature regulated RNAi?

54. N2
m41
m577
m596
m579
e1370
0
20
40
60
80
100
120
0 10 20 30 40
0
20
40
60
80
100
120
0 20 40 60
0
20
40
60
80
100
120
0 20 40 60
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 20 40 60

55. 0
20
40
60
80
100
120
0 10 20 30
0
20
40
60
80
100
120
0 10 20 30 40 50
N2
m41
m577
cpr-7 rnai
0
20
40
60
80
100
120
0 10 20 30 40 50
m596
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
m579
e1370
0
20
40
60
80
100
120
0 10 20 30 40 50
eif3A RNAi 25C

56. N2
m41
m577
m596
m579
e1370
0
20
40
60
80
100
120
0 10 20 30 40
0
20
40
60
80
100
120
0 20 40 60
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50 60
0
20
40
60
80
100
120
0 20 40 60

57. N2
m41
m577
m596
m579
e1370
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 20 40 60
0
20
40
60
80
100
120
0 10 20 30 40 50 60
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 20 40 60

58. N2
m41
m577
m596
m579
e1370
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 20 40 60
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 20 40 60
0
20
40
60
80
100
120
0 20 40 60

59. 0
20
40
60
80
100
120
0 10 20 30
0
20
40
60
80
100
120
0 10 20 30 40 50
eif3A RNAi 25C
N2
m41
0
20
40
60
80
100
120
0 10 20 30 40 50
m577
0
20
40
60
80
100
120
0 10 20 30 40 50
m596
0
20
40
60
80
100
120
0 10 20 30 40 50
m579
0
20
40
60
80
100
120
0 10 20 30 40 50
e1370

60. 0
20
40
60
80
100
120
0 5 10 15 20 25
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40
N2
m41
m577
eiF4?
0
20
40
60
80
100
120
0 10 20 30 40 50
m596
0
20
40
60
80
100
120
0 10 20 30 40 50
m579
0
20
40
60
80
100
120
0 10 20 30 40 50
e1370
eif3A RNAi 25C

61. 0
20
40
60
80
100
120
0 5 10 15 20 25
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
0
20
40
60
80
100
120
0 10 20 30 40 50
N2
m41
m577
m596
0
20
40
60
80
100
120
0 10 20 30 40 50
m579
e1370
0
20
40
60
80
100
120
0 10 20 30 40 50
eif3A RNAi 25C

62. Allows to definite what context of
different phenotypes
tie to molecular/cellular mechanism

63. daf-2
++++++++++++++++++++
15C 25C
+++WT benefits +/-
daf-2 (refined)
++++++++++++++++++++

64. What is the Relationship between
Metabolic Rate and Longevity?

65. • larvae were raised at 15˚C, each of the 9 strains has a
different adult life span expectancy
• young adults were placed at 15˚ or 22.5˚
• on day 4 adults were suspended in medium in the
absence of E. coli in air-tight glass vials
• measurements were made after 7 hours with a
polarographic oxygen electrode
Oxygen Consumption Protocol

66. 200
220
240
260
280
300
320
340
10 15 20 25 30 35 40 45 50
Metabolic Rate and Life Span at 22.5ºC
Age at Last Quartile (days)
N2
daf-16
daf-16; daf-2
fer-15
daf-2(m577)
daf-2(m41)
fer-15; daf-2
daf-2(m596)
daf-2(m579)
r=0.34, P=0.368
MetabolicRate
(pmolO2individual-1hour–1)

67. 200
220
240
260
280
300
320
340
10 20 30 40 50
r=0.22, P=0.577
N2
daf-16(m26)
daf-16; daf-2(m41)
fer-15(b26)daf-2(m577)
daf-2(m41)fer-15; daf-2(m41)
daf-2(m596)
daf-2(m579)
Metabolic Rate and Life Span at 15ºC
Age at Last Quartile (days)
MetabolicRate
(pmolO2individual-1hour-1)

68. Metabolic Rate and Longevity
• there is not a correlation between oxygen consumption and
longevity in C. elegans
• reduction in metabolic rate alone is not the physiological
mechanism for increase in adult longevity in the daf-2 mutants
• other metabolic events such as insulin-like sensitivity and lipid
levels may contribute to the anti-aging mechanisms of the daf-2
mutations

69. 200
220
240
260
280
300
320
340
360
15 22.5
Temperature (ºC)
pmolO2individual-1hour-1
N2
fer-15
daf-16
daf-2(m41)
daf-2(m577)
daf-2(m579)
daf-2(m596)
Metabolic Rate Response to Temperature
non-Age:
Age:

70. Shared mechanisms
daf-2 mutation temperature
15°C
25°C
15°C
0
20
40
60
80
100
0 10 20 30 40 50 60
N2
daf-2(m41)
%Survival

71. Life of a protein

72. • Protein Synthesis
• Lysosomal Proteases
• Glucosyltransferases
Temperature Regulated Genes
Repressed at 25C Elevated at 25C
• C-type lectins
• Lysosomal Proteases
• Fatty Acid Desaturases
• Glucosyltransferases