february 2011csb presentation
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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)
Cool temperature correlates with longevity
Caloric Restriction
(Lane 1996)
Cool temperature correlates with longevity
Ames Dwarf
Ames Dwarf
(Hunter 1999)
Cool temperature correlates with longevity
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Gene Ontology Categories (Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)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(Curtis, Tavare’, Takano, Sanchez Larsen, unpublished)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
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
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
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
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
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
i) qRT-PCR
ii) Magic Red (IN PROGRESS)
Remaining:Quantitative analysisRepeat
asp-3F21F8.4
Control RNAi Control RNAi
How Effective is RNAi
(Sanchez, Larsen, unpublished)
asp-6
Control RNAi Control RNAi
How Effective is RNAicpr-1
(Sanchez, Larsen, unpublished)
cpr-7
Control
How Effective is RNAi
RNAi Control RNAi
(Sanchez, Larsen, unpublished)
K10C2.3
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
b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C
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
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
b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C
i) RNAi of temperature regulated
proteases
ii) Pharmacological lysosomal inhibitors
iii) RNAi of lysosomal components
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
b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C
i) RNAi of temperature regulated
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
RNAi of temperature regulated lysosomal proteases
p= 0.6739n=80Trials:3
p= 0.2731n=98Trials:2
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.7517n=100Trials:3
p= 0.8478n=111Trials:2
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p=0.15n=77Trials:4
p= 0.1419n=95Trials:2
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.3952n=103Trials:3
p= 0.0058n=84Trials:2
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
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
RNAi of temperature regulated lysosomal proteases
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
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
b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C
i) RNAi of temperature regulated
proteases
ii) Pharmacological lysosomal inhibitors
•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
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
b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C
ii) Pharmacological lysosomal inhibitors
*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*
Effect of lysosomal inhibitors on lifespan
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
Effect of lysosomal inhibitors on lifespan
n=81p= <.0001*
Alternative Explanations
• Affecting non-lysosomal processes
• Inhibiting bacterial growth
• Behavioral changesfood avoidancedietary restriction
Effect of lysosomal inhibitors on lifespan
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
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
b) Do lysosomal proteases contribute to healthy long-term survival at 15°C or 25°C
i) RNAi
ii) Pharmacological lysosomal inhibitors
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
RNAi of temperature regulated lysosomal proteases
p=0.6458n=65
p=0.4439n=79
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p=0.9367n=68
p=0.1064n=79
Prediction: Simple Model
Elevated proteases at 15C promote survivalReduced proteases at 25C promote aging
Pro-survival Pro-Aging
Prediction: Revised Model
Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote aging
Pro-survival Pro-Aging
Condition Specific Pro-survival
Prediction: Revised Model
Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival
Pro-survival Pro-Aging
Condition Specific Pro-survival
Prediction: Revised Model
Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival at 25C
Pro-survival Pro-Aging
Condition Specific Pro-survival
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
Determine if daf-2 has shared or independent mechanism of longevity with temperature
daf-2
Specific Aim 2
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
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
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
a) Is lysosome structure or number altered by elevated temperature?
i) Lysotracker staining
Specific Aim 2
a) Is lysosome structure or number altered by elevated temperature?
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
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
15˚C Day 1 25˚C Day 1
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
15˚C Day 1 25˚C Day 1
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
15˚C Day 1 25˚C Day 1
n=1Avg Area:1.02# of particles:369
n=5Avg Area:1.52# of particles:1213
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
25˚C Day 1 25˚C Day 7
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
25˚C Day 1 25˚C Day 7
n=5Avg Area:2.57# of particles:931.25
n=5Avg Area:1.52# of particles:1213
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
25˚C Day 7
n=5Avg Area:0.621# of particles:2136.7
15˚C Day 7
n=5Avg Area:2.57# of particles:931.25
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
Specific Aim 2
a) Is lysosome structure or number altered by elevated temperature?
i) Lysotracker stainingRemaining
Quantitative analysis Repeat experiments
(IN PROGRESS)
Specific Aim 2
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
a) Is lysosome structure or number altered by elevated temperature?
b) Is lysosome structure or number altered by protease reduction via RNAi?
15C Day 7 cpr-7 RNAi
n=5Avg Area:0.621# of particles:2136.7
15˚C Day 7
n=2Avg Area:2.19# of particles:971
Is lysosome structure or number altered by protease RNAi?Confocal Microscopy
Specific Aim 2
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
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?
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 15°C day 8Control 15°C day 8
cpr-7 25°C day 8Control 25°C day 8
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
(Sanchez, Larsen, unpublished)
92% 90/97 worms paralyzedvs17% 16/95 control
Specific Aim 2
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
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? 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
Drug Recommended Actual Paralyzed
PTZ 2.5-10mg/ml 20mg/ml 6/10
Drug Recommended Actual Paralyzed
Muscimol 3mM 3mM 0/10
Suggests temperature dependent dosage
Specific Aim 2
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
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? i) Neurotransmitter pharmacology
ii) Tissue-specific RNAi
(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?
ii) Tissue-specific RNAi
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
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
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
ii) Tissue-specific RNAi
(IN PROGRESS)
(READY TO START)
Specific Aim 1 Specific Aim 2 Specific Aim 3
Elucidate the function of lysosomal proteases regulated by temperature in healthy long-term survival at 15°C and 25°C
Determine if altered protease expression causes cellular and tissue changes that subsequently result in healthy long term survival
Determine if daf-2 has shared or independent mechanism of longevity with temperature
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)
Possible explanation for daf-2 abnormal temperature phenotype
• 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
Possible explanation for daf-2 abnormal temperature phenotype
• 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?
Prediction: Revised Model
Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival at 25C
Pro-survival Pro-Aging
Condition Specific Pro-survival
Specific Aim 3
A) microarray
B) qRT-PCR
C) RNAi of temperature regulated genes
Determine if daf-2 has shared or independent mechanism of longevity with temperature
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
Determine if daf-2 has shared or independent mechanism of longevity with temperature
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
Determine if daf-2 has shared or independent mechanism of longevity with temperature
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)
(Sanchez, Larsen, unpublished)
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
Specific Aim 1c RNAi of temperature regulated genes in daf-2 mutants
25˚C cpr-7 RNAi
Specific Aim 1c RNAi of temperature regulated genes in daf-2 mutants
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
Specific Aim 1c RNAi of temperature regulated genes in daf-2 mutants
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
Specific Aim 1c RNAi of temperature regulated genes in daf-2 mutants
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
Specific Aim 1c RNAi of temperature regulated genes in daf-2 mutants
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
Specific Aim 3c RNAi of temperature regulated genes in daf-2 mutants
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
Shared Mechanism of Longevity?
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
Shared Mechanism of Longevity?
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
Shared Mechanism of Longevity?
Promotes Aging
?
Revised Model
Occur concurrently
Pro-survival Pro-Aging
Condition Specific Pro-survival
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
n=5Avg Area:0.621# of particles:2136.7
15˚C Day 7
n=5Avg Area:2.57# of particles:931.25
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