february 2011csb presentation

Proteostasis: A Key Node in Longevity

CSB SeminarFebruary 24th 2011Angela Sanchez

Aging is a universal process

• Need to compare populations with different life spans

• Use C. elegans

– Short-lived

– Cultivate large numbers

– Amenable to genetic manipulation

– Validated model system for humans

How can we uncover mechanism?

Cool Temperature Doubles Lifespan in C. elegans

Is cool-mediated longevity conserved?

• Biological response to temperature is ancient• Temperature fluctuated before oxygen

existed• We hypothesize that molecular responses to

non-stress inducing temperatures are conserved.

Cool temperature extends lifespan

Drosophila

30˚c

18˚c

VERTEBRATES? (Miquel 1976)

Cool temperature extends lifespan

Mouse

INCREASED 20% (Conti 2006)

Cool temperature correlates with longevity

Naked Mole Rat

(McNab 1966)


Caloric Restriction

(Lane 1996)


Ames Dwarf

Ames Dwarf

(Hunter 1999)


Long-lived human

(Roth 2002)

Why does cool temperature improve health and survival?

Underlying mechanism not understood

Understanding healthy long term-survivalMicroarray

• Affymetrix C. elegans array

• Genome wide snapshot

• UNBIASED

• PRIMARY biological response to a chronic change

Looking early for an initiating cause

Preparation of samples for microarray

L1

L2

L3

L4

Immature Adult

Development

Total RNA Total RNA

Mature Adult

Visible Developmental Stage

15°C

15°C 25°C

Four Biological Samples

Temperature Regulated Genes

19,762 Predicted Genes

16,896 Probes

383 Differentially Expressed

179

P<.01

280

Suggests part of molecular response mechanism Transcriptional

Number of Temperature Regulated Genes

Gene Ontology Categories (Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)

Proteostasis Genes Differentially Expressed by 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(Curtis, Tavare’, Takano, Sanchez Larsen, unpublished)

Differentially Expressed Proteases

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 siteOnly subset are temperature regulated

19 in genome

24 in genome

10 in genome

Working model of biological response to chronic temperature conditions

Decreased protein synthesis and degradation

Increased protein synthesis and degradation

HYPOTHESIS

Proteostasis is a key mechanismfor healthy long-term survival in animals

with cool core body temperature

Specific Aim 1 Specific Aim 2 Specific Aim 3

Determine whether differentially expressed proteases functions in life-limiting or survival-enhancing processes. 15°C and 25°C

Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival

daf-2

Determine if daf-2 has shared or independent mechanism of longevity with temperature

Specific Aim 1

Elucidate the function of lysosomal proteases regulated by temperature in healthy long-term survival at 15°C and 25°C

a) Verify temperature regulation of lysosomal proteases mRNA

b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C

c) Determine if daf-2 has shared or independent mechanism of longevity with temperature

Specific Aim 1


a) Verify temperature regulation of lysosomal proteases

i) qRT-PCR

ii) Magic Red

** *

**

**

** *

* *

*

Verify temperature regulation of lysosomal proteasesqRT-PCR verification (1a-i)

Normalized Ct levels

Temperature regulation confirmed13/15

Range of expression levels1000X

Further investigation warranted

• Microarray result is VALID

• RT-PCR large differences

Specific Aim 1



i) qRT-PCR

ii) Magic Red

• In vivo substrate linked to fluorophore

• Measure ACTIVITY

Cathepsin Substrate

Verify temperature regulation of lysosomal proteasesMagic Red (1a-ii)

15°C25°CEffect of temperature on Cathepsin Activity

Cathepsin Activity is HIGHER at 15°C

(Sanchez, Larsen, unpublished)

Further investigation warranted

• Microarray result is VALID

• RT-PCR large differences

• Cathepsin activity is elevated at 15⁰C

Specific Aim 1



i) qRT-PCR

ii) Magic Red (IN PROGRESS)

Remaining:Quantitative analysisRepeat

asp-3F21F8.4

Control RNAi Control RNAi

How Effective is RNAi


asp-6

Control RNAi Control RNAi

How Effective is RNAicpr-1


cpr-7

Control

How Effective is RNAi

RNAi Control RNAi


K10C2.3

Specific Aim 1




Prediction: Simple Model

Everything at 15C is pro-survivalMimic cool to live long

Everything that 25C is promotes agingMimic warm to promote aging

Pro-survival Pro-Aging

Specific Aim 1




i) RNAi of temperature regulated

proteases

ii) Pharmacological lysosomal inhibitors

iii) RNAi of lysosomal components

Specific Aim 1





proteases

15˚C 25˚C

RNAi of temperature regulated lysosomal proteases

p= 0.9255n=105Trials:3

p= 0.1148n=87Trials:2

15˚C 25˚C


p= 0.6739n=80Trials:3

p= 0.2731n=98Trials:2

15˚C 25˚C


p= 0.7517n=100Trials:3

p= 0.8478n=111Trials:2

15˚C 25˚C


p=0.15n=77Trials:4

p= 0.1419n=95Trials:2

15˚C 25˚C


p= 0.3952n=103Trials:3

p= 0.0058n=84Trials:2

15˚C 25˚C


p= 0.7940n=100Trials:2

p= 0.3681n=94Trials:2

RNAi of a temperature regulated gene in wild type: cpr-7

p<.001n=75 Trials:3

15˚C 25˚C

n=75Trials:3

p=.0162

exon-1

asp-1-1 RNAi clone

212bpRT-PCR

479bp

asp-1 RNAi clone design

567bpasp-1-2 RNAi clone

• Two independent clones

• Overlapping

15˚C 25˚C


Similar results with RNAiasp-6 asp-5 K10C2.3

asp-3 asp-3 F21F8.4

p=0.15n=77Trials:2

p=0.97n=82Trials:2

Model Testing

• mRNA change is real • Is this a correlation or functionally relevant• Assay in vivo protease activity• manipulate lysosomal protease activity and

assay is functionally important for longevity– Pharmacological inhibitor– RNAi

Specific Aim 1





proteases


•Cell permeable•Protonated in acidic lysosome•Protonated form becomes trapped•Increases pH•Acidity required for lysosomal enzyme activity

Lysosomal inhibitors

(Rejingoud 1976)

LYSOSOME

H+

pH

Specific Aim 1





*Dosage response curves*Verify deacidification of

lysosomes

• Lysosensor yellow/blue• Excitation is pH dependent• More blue = more basic• More yellow = more acidic

Verifying deacidification of lysosomes

BLUE YELLOW

ACIDICBASIC

Control

20mM Chloroquine

15C Lysosensor Yellow/BlueCFP YFP CFP/YFP

Quantitation of 15C Lysosensor Yellow/Blue

• Measured Grey ValueCFPYFP

• Calculate ratio ofYFP/CFP

No detectable differenceLarge variability

Effect of lysosomal inhibitors on lifespan

DECREASES LIFESPAN at 15˚C

10mM Chloroquine15˚C 25˚C

Lysosomal Inhibition DETRIMENTAL at 15˚C and BENEFICIAL at 25˚C

INCREASES LIFESPAN at 25˚C

n=75p= 0.0028*

n=64p= <.0001*


DECREASES LIFESPAN at 15˚C

25mM NH4Cl15˚C 25˚C

Lysosomal Inhibition DETRIMENTAL at 15˚C and BENEFICIAL at 25˚C

INCREASES LIFESPAN at 25˚C

n=81p <.0001*

n=80p= 0.3578


n=81p= <.0001*

Alternative Explanations

• Affecting non-lysosomal processes

• Inhibiting bacterial growth

• Behavioral changesfood avoidancedietary restriction


n=81p= <.0001*

Alternative Strategies

• Vacuolar H+/ATPase13 subunits in c elegansPump H+ into lysosomeResulting in acidification

LYSOSOMEH+

pH

H+

H+

AdvantagesNo food interferenceincreased specificity

Specific Aim 1




i) RNAi


Effect of lysosomal inhibitors on lifespanAlternative Strategies

Control

vha-15 RNAi

15C Lysosensor Yellow/BlueCFP YFP CFP/YFP

Quantitation of 15C Lysosensor Yellow/Blue

No detectable differenceLarge variability

15˚C 25˚C


p=0.6458n=65

p=0.4439n=79

15˚C 25˚C


p=0.9367n=68

p=0.1064n=79

Prediction: Simple Model

Elevated proteases at 15C promote survivalReduced proteases at 25C promote aging


Prediction: Revised Model

Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote aging


Condition Specific Pro-survival


Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival




Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival at 25C




Determine whether differentially expressed proteases functions in life-limiting or survival-enhancing processes. 15°C and 25°C



daf-2

Specific Aim 2


a) Is lysosome structure or number altered by elevated temperature?

b) Is lysosome structure or number altered by protease reduction via RNAi?

c) Are tissues differentially impacted by temperature or protease reduction?

Specific Aim 2



i) Lysotracker staining

Specific Aim 2


i) Lysotracker staining

Accumulates in acidic organelles

Fluorescent red15C 25C Control RNAi

v v

Expect accumulation of lysotracker

DAY 2 DAY 7 DAY 13Control (15⁰C)

F57F5.1 (15⁰C)

Control (25⁰C)

Temperature accelerates accumulation of lysotrackercpr-7 RNAi accelerates accumulation of lysotracker

Specific Aim 2a lysosomes altered by elevated temperature?

Is lysosome structure or number altered by elevated temperature?Confocal Microscopy

• 60X Magnification with 2X zoom

• 8um/pixel

• 1um/slice Z-stacks

• Anterior intestinal region

• Have whole worm data

Image J Particle AnalyzerCalculates Area

Returns number of particles

Confocal Image Image J Drawing


15˚C Day 1 25˚C Day 1


15˚C Day 1 25˚C Day 1

n=1Avg Area:1.02# of particles:369



25˚C Day 1 25˚C Day 7


25˚C Day 1 25˚C Day 7

n=5Avg Area:2.57# of particles:931.25



25˚C Day 7


15˚C Day 7


Specific Aim 2


i) Lysotracker stainingRemaining

Quantitative analysis Repeat experiments

(IN PROGRESS)

Specific Aim 2




15C Day 7 cpr-7 RNAi


15˚C Day 7


Is lysosome structure or number altered by protease RNAi?Confocal Microscopy

Specific Aim 2




c) Are tissues differentially impacted by temperature or protease reduction?

RNAi of a temperature regulated gene in wild type: cpr-7

p<.001n=75

15˚C 25˚C

n=75p=.0162

Healthspan alert!>90% of animals are paralyzed as middle aged adults at

15C when treated with RNAi

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 Progressive Paralysis: Middle Aged

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

Quantification of cpr-7 RNAi induced paralysis at 15°C


92% 90/97 worms paralyzedvs17% 16/95 control

Specific Aim 2




c) Are tissues differentially impacted by temperature or protease reduction? i) Neurotransmitter pharmacology

ii) Tissue-specific RNAi

Pharmacological Neurotransmitters Electrical Stimulation

PREDICTIONTEST

No muscle contractionNo muscle contraction

Specific Aim 2c Paralysis in cpr-7 RNAi :muscles or neurons?Neurotransmitter Expected Results

MUSCLE

Specific Aim 2

c) Is the paralysis observed in cpr-7 RNAi treated animals at 15C due to decreased function of muscles or neurons?

i) Neurotransmitter

pharmacology

•302 Neurons

•74 motor neurons

• GABA-nergic (INHIBITORY)

•Cholinergic (EXCITATORY)

Specific Aim 2

b) Is the paralysis observed in cpr-7 RNAi treated animals at 15C due to decreased function of muscles or neurons?

i) Neurotransmitter

pharmacology

HYPER CONTRACTION

• (-)GABA-nergic (INHIBITORY)

• (+) Cholinergic (EXCITATORY)

FLACCID

• (+) GABA-nergic (INHIBITORY)

• (-) Cholinergic (EXCITATORY)

Types of Paralysis

PREDICTION

DrugAldicarb (cholinesterase inhibitor)

Specific Aim 2b Paralysis in cpr-7 RNAi :muscles or neurons?Neurotransmitter Expected Results

NEURONAL

Levamisole (cholinergic agonist)

PTZ (GABA antagonist)

Muscmimol (GABA agonist)

Hyper-Contract Flaccid(-)GABA (+) GABA(+) Cho (-) Cho

No EffectNo Effect No EffectNo Effect No EffectNo Effect Contract

Relax

No Effect No Effect No EffectContractRelax No Effect No Effect No Effect

Specific Aim 2c Paralysis in cpr-7 RNAi :muscles or neurons?Preliminary Data

Drug Recommended Actual Paralyzed

Aldicarb 500uM 1mM 0/10


PTZ 2.5-10mg/ml 20mg/ml 6/10


Muscimol 3mM 3mM 0/10

Suggests temperature dependent dosage

Specific Aim 2




c) Are tissues differentially impacted by temperature or protease reduction? i) Neurotransmitter pharmacology


(IN PROGRESS)

Specific Aim 2

c) Is the paralysis observed in cpr-7 RNAi treated animals at 15C due to decreased function of muscles or neurons?


rde-1(ne219)required for DICER complextissue-specific rescueRNAi occurs only in targeted

tissue

Specific Aim 2b Paralysis in cpr-7 RNAi :muscles or neurons?ii Tissue specific RNAi

*Neuronalunc-25p:rde-1 ;rde-1(ne219)

RNAi rescue in GABA-nergic neurons (McIntire 1993)

cha-1p:rde-1;rde-1(ne219) RNAi rescue in cholinergic neurons.

*Muscle specific rde-1(ne219);neIs9[myo-3::HA::RDE-1 + pRF4(rol-6)]

RNAi rescue only in body wall muscle.

*rde-1(ne219) kzIs20[pDM#715(hlh-1p::rde-1) + pTG95(sur-5p::nls::GFP)]

RNAi rescue only in body wall muscle.

Specific Aim 2b Paralysis in cpr-7 RNAi :muscles or neurons?Tissue specific RNAi: Expected Results

Neuronal-specific

Muscle-specific

Neuronal Muscle

Paralysis

No Effect

No Effect

Paralysis

RNAi Both

Paralysis

Paralysis

Other

No Effect

No Effect

Specific Aim 2


a) Is lysosome structure or number altered by elevated temperature or protease reduction via RNAi?

b) Is the paralysis observed in cpr-7 RNAi treated animals at 15C due to decreased function of muscles or neurons?

i) Neurotransmitter pharmacology


(IN PROGRESS)

(READY TO START)





daf-2

(IN PROGRESS)

Temperature

Manipulations That Double Lifespan in C. elegans

GeneticEnvironment

daf-2 gene

Wild Typedaf-2

ins genes are temperature regulated

HIGHER AT 15Cdaf-28ins-28ins-29ins-35ins-25ins-20ins-34ins-5

ins-23ins-4

ins-22ins-30

LOWER AT 15Cins-19ins-11 ins-16ins-27ins-31

What is daf-2?• Putative receptor kinase• Homologous to human insulin/IGF receptor/insulin-

related receptor• Recessive hypomorphs

m41G380E

m579R434C

m596G544S

m577C1042Y

e1370P1465S

N- -CLigand binding Tyrosine kinase

Gene x Environment Interactiondaf-2 cool vs warm

daf-2 mutants have a non-wild type response to temperatureand are long lived

WTm41

e1370m596

m5790

20

40

60

80

15˚C22.5˚C

daf-2 allele

Maximal Adult

Life Span(DAYS)

Possible explanation for daf-2 abnormal temperature phenotype

• daf-2 mutations not as mutant at 15˚C

WTm41

e1370m596

m5790

20

40

60

80

15˚C22.5˚C

daf-2 allele

Maximal Adult

Life Span(DAYS)


• daf-2 mutations do live longer than WT at 15˚C• Are daf-2 mutants less responsive to temperature?

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


• daf-2 mutations do live longer than WT at 15C• daf-2 mutants are hyper responsive to temperature• Expression of daf-2 gene altered in mutants?


Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival at 25C



Specific Aim 3

A) microarray

B) qRT-PCR

C) RNAi of temperature regulated genes


daf-2

daf-2: Shared or independent mechanisms with temperature Microarray Analysis

•Temperature regulation lost in some daf-2 mutants

•Elevated levels by daf-2 at 25˚C for some

daf-2: Shared or independent mechanisms with temperature Microarray Analysis

•daf-2 mutation induces temperature regulation

•Elevated levels by daf-2 at 25˚C for some

Temperature Regulation p values Gene Description N2 daf-2(m41) daf-2(m577) daf-2(m596) daf-2(m579) asp-4 Aspartyl protease 1 0.008807 0.027883 0.009639 0.026397

T14F9.3 Beta-N-acetylhexosaminidase 0.626127 4.20E-08 1.40E-05 2.24E-06 8.53E-07 pho-14 Lysosomal phosphatases 1 0.001881 0.008924 0.008726 0.011879

Y37D8A.2 phospholipase-B 0.34562 0.026767 0.000792 4.62E-05 1.52E-06 acl-12 Lysophosphatidic acyltransferase 0.980377 0.000231 2.60E-02 0.006811 1.98E-05

T19B10.3 Beta-galactosidase 0.357722 0.012278 7.49E-03 6.42E-05 0.000119 K11G9.5 Sialin Solute transport 0.716823 0.049115 0.007736 0.000675 0.000449

Temperature Regulation p values Gene Description N2 daf-2(m41) daf-2(m577) daf-2(m596) daf-2(m579)

F28A12.4 Aspartyl protease 0.837792 0.274683 0.499305 0.011814 0.005419 cpz-1 Cysteine proteinase Cathepsin L 0.682801 0.151837 0.307674 0.001988 0.008102

H22K11.2 Beta-galactosidase 0.978491 0.958974 0.512632 0.00008 0.013544 ZK563.6 Lysosomal phosphatases 0.894114 0.838573 9.72E-01 0.010806 0.00852

lys-3 N-acetylmuraminidase/lysozyme 1 0.65643 0.84658 0.044525 0.009436

Additional lysosomal proteins regulated by temperature in daf-2 mutants

Temperature regulated in all daf-2 not WT

Temperature regulated in strong daf-2

daf-2: Shared or independent mechanisms with temperature

•Loss of temperature regulation in some proteases

•Comparable levels by microarray

•Additional temperature regulated in daf-2

•Suggests ENHANCED lysosomal function in daf-2

Microarray Analysis

Specific Aim 3


a Determine if regulation of temperature regulated proteases is altered by daf-2 mutation

i) microarray

ii) qRT-PCR

daf-2

qRT-PCR Lysosomal Proteases: daf-2(m41)

cpr-1 F21F8.4 F21D5.2 K10C2.3 W07B8.1

ELEVATED mRNA levels of proteases in daf-2 (m41)

qRT-PCR Lysosomal Proteases: daf-2(m596)

ELEVATED mRNA levels of proteases in daf-2 (m596)

asp-6 cpr-7 cpr-4 asp-1

qRT-PCR Lysosomal Proteases: daf-2(m41);daf-16

Some daf-2 regulated proteases are daf-16 dependent

K10C2.3 cpr-1 F21F8.4

daf-2: Shared or independent mechanisms with temperature

•Loss of temperature regulation in some proteases

•Increased mRNA levels of some lysosomal proteases

•Some are daf-16 dependent

•Suggests ENHANCED lysosomal function in daf-2 that is

partially daf-16 dependent

qRT-PCR Preliminary results

daf- 2 qRT-PCR Lysosomal ProteasesRemaining Experiments

Specific Aim 3

A) microarray

B) qRT-PCR

C) RNAi of temperature regulated genes


daf-2

(IN PROGRESS)

0 5 10 15 20 250

102030405060708090

100

0 5 10 15 20 250

20

40

60

80

100

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

0 5 10 15 20 250

102030405060708090

100

0 5 10 15 20 250

102030405060708090

100

2 4 6 8 10 12 14 16 18 20 220

20

40

60

80

100m596

2 4 6 8 10 12 14 16 18 20 220

20

40

60

80

100

wild type

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)


wild type

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)p<.001

p= 0.84p=.3364

p=0.1809 p= 0.0006

p<.0001

Specific Aim 1c RNAi of temperature regulated genes in daf-2 mutants

15˚C cpr-7 RNAi

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)p=.0162

p= 0.5658p= 0.7916

p= .0007 p= 0.5424

p= 0.1239wild type


25˚C cpr-7 RNAi


25˚C asp-1 RNAi

p= 0.3800n= 87

p= 0.0025*n= 76

RNAi of temperature regulated genes in daf-2 mutants15˚C asp-1 RNAi

p= 0.0009*n= 80

p= <.0001*n= 77

p= <.0001*n= 59

p= 0.0013*n= 56

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)p<.0001

p<.0001p<.0001

p<.0001 p<.0001

p<.0001

wild type


25˚C eiF3A RNAi

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)p<.0001

p= 0.6831 p= .0014

p= 0.4785 p= <.0001

p= 0.0012wild type


25˚C eiF4G RNAi

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)p= 0.0799

p= .4760p= .0034

p= 0.2573

p= 0.0694

p= 0.838

wild type


15˚C eiF3A RNAi

daf-2(m41)

daf-2(m577)

daf-2(m596)

daf-2(m579)

daf-2(e1370)p= 0.2576

p= 0.9445p= 0.938

p= 0.619 p= 0.3825

p= 0.0003wild type


15˚C eiF4G RNAi

DAF-2

Temperature 25C (WARM)

Lysosomal Proteases

Protein Synthesis

Promotes Survival at 25C

DAF-2

Temperature 15C (COOL)

Lysosomal Proteases

Protein Synthesis

Promotes Survival at 15C

Signaling is Active

Signaling is Reduced?

Temperature

Shared Mechanism of Longevity?

GeneticEnvironment

daf-2 gene

Wild Typedaf-2

DAF-2Temperature

Lysosomal Proteases

Protein Synthesis

Proteostasis genes are regulated by

BOTH daf-2 and temperature

REGULATION IS SHARED


DAF-2Temperature

Lysosomal Proteases

Protein Synthesis

Temperature Promotes survival by manipulation of this node

Does not appear to be a shared mechanism between daf-2 and temperature


Promotes Survival

?

DAF-2Temperature

Lysosomal Proteases

Protein Synthesis

In the event of daf-2 mutation

Proteostasis node becomes disregulated

Can no longer respond appropriately to an environmental change

Presents a liability for daf-2 mutants


Promotes Aging

?

Revised Model

Occur concurrently



AcknowledgementsCommittee Members Larsen Lab

CollaboratorsFunding

Dr. Shelly Buffenstein

Dr. Ellen Kraig

Dr. Merry Lindsey

Dr. Olivia Smith

Dr. Syuichi Takano

Wendy Lee

Dr. Hal Boylston

Dr. Pamela Larsen

NIA Training GrantGlenn Medical FoundationEllison Medical Foundation

Dr. Simon Tavaré

Dr. Christina Curtis

ff =∫ d x≠ ⅔∑

Pharmacological lysosomal inhibitors

• 0, 10, 25, 50, 75 and 100 mM NH4Cl at 15°C and 25°C (Broeks 1995)

• 0,1,5,10,20 mM chloroquine at 15°C and 25°C (Hujova 2005)

• NOT used for lifespans

dose response curves

Pharmacological lysosomal inhibitors

(Seglan 1976)(Klempner 1983)(Jones 1984)

Chloroquine NH4ClpH

• Decreases pH

• Vacuolization

25˚C Day 7


15˚C Day 7


february 2011csb presentation

Science