Reduction of proteostasis gene expression negatively impacts adult healthspan or increases life span,

depending on the environment

Angela SanchezCellular & Structural Biology

Healthy LivingFauja Singh ,97 Philip Rabinowitz, 104 Mavis Lindgren, 90

What interventions can we use?

Cool Temperatures Favors Healthy Long-Term Survival

Long Lifespan Short Lifespan

Cool Temperature Warm Temperature

(Lamb MJ, 1968) (Kibler HH, 1961)(Liu RK, 1973)

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

How does temperature affect survival in C. elegans ?

What accounts for this difference?

• Molecular mechanism of acclimatization

• Which cellular processes are altered?

?

How can we find the cause of this?

Temperature Dependent Survival in C. elegansPe

rcen

t Su

rviv

al

Days

25°C

15°C

25°C

15°C

Lifespan is doubled at 15°C

Microarray Analysis

• Collaboration with S. Tavare’ (USC)

• Different from affymetrix software

• Calculated Background

• Not estimated

Microarray Overview

• Post-development

• Look early before there is a difference

• Molecular mechanism of acclimatization

• Which cellular processes are altered?

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

Temperature Regulated Genes

19,762 Predicted Genes

16,896 Probes

386 Differentially Expressed

178

P<.01

208

Suggests part of acclimatization mechanism Transcriptional

Temperature Regulated Genes

386 total altered genes

Gene Ontology

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

Cellular Response To Temperature

REPRESSED

REPRESSED

ALTERED

Hypothesis

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

15°C Elevated Proteases mRNA

cpr-1 Cathepsin Bcpr-4 Cathepsin BW07B8.1 Cathepsin BF21D5.2 Cathepsin Bcpr-7 Cathepsin B

K10C2.1 Cathepsin AK10B2.2 Cathepsin A

asp-1 Cathepsin Dasp-3 Cathepsin Dasp-5 Cathepsin Easp-6 Cathepsin DF21F8.4 Cathepsin EK10C2.3 Cathepsin CC15C8.3 Cathepsin EY39B6A.24 Cathepsin D

Aspartyl Cysteine Serine

Classified by active site

31 in genome

39 in genome

75 in genome

Is the array result true?

• Validation by QPCR • Designed primers• Tested • Assay three biological samples • Normalized to cyc-1 for control

RT PCR of 15C elevated Proteases

asp-1

asp-3

asp-4

asp-6

F21F8

.4

K10C2.3

C15C8.3

Y39B6A.24

CPR-1CPR-4

W07B8.1

F21D5.2

F57F5

.1

K10B2.2

K10B2.10

1

2

3

4

5

6

7

8

9

Fold

Incr

ease

asp-5

Specific Aims1. 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

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

15°C Elevated Proteases mRNA

cpr-1cpr-4W07B8.1F21D5.2cpr-7

K10C2.1K10B2.2

asp-1asp-3asp-5asp-6F21F8.4K10C2.3C15C8.3Y39B6A.24

Aspartyl Cysteine Serine

(RNAi Library Ahringer et al. 2003)

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

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-7 Progressive Paralysis

(15⁰C) Day 8

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

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

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

Does cpr-7 RNAi impact lifespan?

cpr-7 RNAi effect on lifespan

0 5 10 15 20 250

20

40

60

80

100

120

Controlcpr-7 RNAi

0 5 10 15 20 25 30 350

20

40

60

80

100

120

15°C

Controlcpr-7 RNAi

25°C

DECREASES INCREASES

OPPOSITE EFFECTS AT DIFFERENT TEMPERATURES

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

daf-2 mutationtemperature15°C

25°C15°C

0

20

40

60

80

100

0 10 20 30 40 50 60

N2

daf-2(m41)

% S

urvi

val

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

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

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

Shared mechanisms?

How to sort this out

Use different alleles

DAF-2 receptor

DAF-16

DAF-2 Signaling and mutations

m41G380E

m577C1042Y

Class I

m579R434C

m596G544S

e1370P1465S

Class II(strong) (mild)

Longevity

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?

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?

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

Daf-2 mutants respond to temperature differently

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

ttx-1 and ttx-3

Or cloned and where expressedCompare them too each other in table parallel

Cryophyllic or athermotactic this is what is important about them

daf-2 ttx-assay

m41

e1370

m579

121416182022242628Wildtype

No difference (Sanchez, Takano, Larsen)

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

daf-2 mutationtemperature15°C

25°C15°C

0

20

40

60

80

100

0 10 20 30 40 50 60

N2

daf-2(m41)

% S

urvi

val

Adult age (days)

Shared or distinct mechanism?

Number of Genes with Altered Expression in Response Temperature

N2 m577 m41 m596 m579 e13700

500

1000

1500

2000

2500

3000

3500

4000

179461 703 645 828

1618

208

441

779 8101048

1792

UP DOWN

Num

ber

of g

ene

(Curtis, Tavare’, Takano S. Larsen P. et al.)

daf-2 Mutants Altered Temperature Response

Flip this to be like next one

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=0.368Met

abol

ic R

ate

(pm

ol O

2 ind

ivid

ual-1

hou

r –1)

Shar

ed W

arm

Diff

eren

tially

Exp

ress

edGe

nes

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

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

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

Daf-2 mutants have altered membranes

Ray’s fat data

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

N2

m41

m577

m596

m579

e1370

0 5 10 15 20 25 30 35 400

20

40

60

80

100

120

0 10 20 30 40 50 600

20

40

60

80

100

1200 10 20 30 40 50 60

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

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80

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120

0 5 10 15 20 25 30 35 40 450

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40

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120

0 10 20 30 40 50 600

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40

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120

0 5 10 15 20 250

20

40

60

80

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120

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

N2

m41

m577

cpr-7 rnai

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

m596

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120 m579

e1370

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

eif3A RNAi 25C

N2

m41

m577

m596

m579

e1370

0 5 10 15 20 25 30 35 400

20

40

60

80

100

120

0 10 20 30 40 50 600

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

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40

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120

0 5 10 15 20 25 30 35 40 450

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40

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100

120

0 10 20 30 40 50 600

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40

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80

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120

0 10 20 30 40 50 600

20

40

60

80

100

120

N2

m41

m577

m596

m579

e1370

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

0 10 20 30 40 50 600

20

40

60

80

100

120

0 10 20 30 40 50 600

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40

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120

0 5 10 15 20 25 30 35 40 450

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120

0 5 10 15 20 25 30 35 40 450

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40

60

80

100

120

0 10 20 30 40 50 600

20

40

60

80

100

120

N2

m41

m577

m596

m579

e1370

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

0 10 20 30 40 50 600

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

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80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

0 10 20 30 40 50 600

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40

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120

0 10 20 30 40 50 600

20

40

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120

0 5 10 15 20 25 300

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

eif3A RNAi 25CN2

m41

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120m577

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120m596

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120 m579

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

e1370

0 5 10 15 20 250

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

0 5 10 15 20 25 30 35 400

20

40

60

80

100

120

N2

m41

m577

eiF4?

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120 m596

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120 m579

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

e1370

eif3A RNAi 25C

0 5 10 15 20 250

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120N2

m41

m577

m596

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120m579

e1370

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

eif3A RNAi 25C

Allows to definite what context of different phenotypes

tie to molecular/cellular mechanism

daf-2

++++++++++++++++++++

15C 25C

+++ WT benefits +/-

daf-2 (refined)

++++++++++++++++++++

What is the Relationship between Metabolic Rate and Longevity?

• 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

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=0.368Met

abol

ic R

ate

(pm

ol O

2 ind

ivid

ual-1

hou

r –1)

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)

Met

abol

ic R

ate

(pm

ol O

2 ind

ivid

ual-1

hou

r -1)

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

200

220

240

260

280

300

320

340

360

15 22.5

Temperature (ºC)

pmol

O2 i

ndiv

idua

l-1 h

our -1

N2fer-15daf-16

daf-2(m41)daf-2(m577)daf-2(m579)daf-2(m596)

Metabolic Rate Response to Temperature

non-Age:

Age:

Shared mechanisms

daf-2 mutation temperature15°C

25°C15°C

0

20

40

60

80

100

0 10 20 30 40 50 60

N2

daf-2(m41)

% S

urvi

val

Adult age (days)

Life of a protein

• Protein Synthesis• Lysosomal Proteases• Glucosyltransferases

Temperature Regulated GenesRepressed at 25C Elevated at 25C

• C-type lectins• Lysosomal Proteases• Fatty Acid Desaturases• Glucosyltransferases