february 2011csb presentation
TRANSCRIPT
![Page 1: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/1.jpg)
Proteostasis: A Key Node in Longevity
CSB SeminarFebruary 24th 2011Angela Sanchez
![Page 2: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/2.jpg)
Aging is a universal process
![Page 3: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/3.jpg)
• 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?
![Page 4: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/4.jpg)
Cool Temperature Doubles Lifespan in C. elegans
![Page 5: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/5.jpg)
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.
![Page 6: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/6.jpg)
Cool temperature extends lifespan
Drosophila
30˚c
18˚c
VERTEBRATES? (Miquel 1976)
![Page 7: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/7.jpg)
Cool temperature extends lifespan
Mouse
INCREASED 20% (Conti 2006)
![Page 8: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/8.jpg)
Cool temperature correlates with longevity
Naked Mole Rat
(McNab 1966)
![Page 9: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/9.jpg)
Cool temperature correlates with longevity
Caloric Restriction
(Lane 1996)
![Page 10: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/10.jpg)
Cool temperature correlates with longevity
Ames Dwarf
Ames Dwarf
(Hunter 1999)
![Page 11: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/11.jpg)
Cool temperature correlates with longevity
Long-lived human
(Roth 2002)
![Page 12: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/12.jpg)
Why does cool temperature improve health and survival?
Underlying mechanism not understood
![Page 13: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/13.jpg)
Understanding healthy long term-survivalMicroarray
• Affymetrix C. elegans array
• Genome wide snapshot
• UNBIASED
• PRIMARY biological response to a chronic change
![Page 14: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/14.jpg)
Looking early for an initiating cause
![Page 15: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/15.jpg)
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
![Page 16: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/16.jpg)
Temperature Regulated Genes
19,762 Predicted Genes
16,896 Probes
383 Differentially Expressed
179
P<.01
280
Suggests part of molecular response mechanism Transcriptional
![Page 17: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/17.jpg)
Number of Temperature Regulated Genes
Gene Ontology Categories (Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)
![Page 18: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/18.jpg)
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)
![Page 19: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/19.jpg)
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
![Page 20: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/20.jpg)
Working model of biological response to chronic temperature conditions
Decreased protein synthesis and degradation
Increased protein synthesis and degradation
![Page 21: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/21.jpg)
HYPOTHESIS
Proteostasis is a key mechanismfor healthy long-term survival in animals
with cool core body temperature
![Page 22: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/22.jpg)
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
![Page 23: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/23.jpg)
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
![Page 24: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/24.jpg)
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
![Page 25: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/25.jpg)
** *
**
**
** *
* *
*
Verify temperature regulation of lysosomal proteasesqRT-PCR verification (1a-i)
Normalized Ct levels
Temperature regulation confirmed13/15
Range of expression levels1000X
![Page 26: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/26.jpg)
Further investigation warranted
• Microarray result is VALID
• RT-PCR large differences
![Page 27: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/27.jpg)
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
![Page 28: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/28.jpg)
• In vivo substrate linked to fluorophore
• Measure ACTIVITY
Cathepsin Substrate
Verify temperature regulation of lysosomal proteasesMagic Red (1a-ii)
![Page 29: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/29.jpg)
15°C25°CEffect of temperature on Cathepsin Activity
Cathepsin Activity is HIGHER at 15°C
(Sanchez, Larsen, unpublished)
![Page 30: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/30.jpg)
Further investigation warranted
• Microarray result is VALID
• RT-PCR large differences
• Cathepsin activity is elevated at 15⁰C
![Page 31: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/31.jpg)
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
![Page 32: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/32.jpg)
asp-3F21F8.4
Control RNAi Control RNAi
How Effective is RNAi
(Sanchez, Larsen, unpublished)
![Page 33: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/33.jpg)
asp-6
Control RNAi Control RNAi
How Effective is RNAicpr-1
(Sanchez, Larsen, unpublished)
![Page 34: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/34.jpg)
cpr-7
Control
How Effective is RNAi
RNAi Control RNAi
(Sanchez, Larsen, unpublished)
K10C2.3
![Page 35: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/35.jpg)
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
![Page 36: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/36.jpg)
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
![Page 37: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/37.jpg)
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
![Page 38: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/38.jpg)
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
![Page 39: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/39.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.9255n=105Trials:3
p= 0.1148n=87Trials:2
![Page 40: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/40.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.6739n=80Trials:3
p= 0.2731n=98Trials:2
![Page 41: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/41.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.7517n=100Trials:3
p= 0.8478n=111Trials:2
![Page 42: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/42.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p=0.15n=77Trials:4
p= 0.1419n=95Trials:2
![Page 43: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/43.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.3952n=103Trials:3
p= 0.0058n=84Trials:2
![Page 44: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/44.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p= 0.7940n=100Trials:2
p= 0.3681n=94Trials:2
![Page 45: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/45.jpg)
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
![Page 46: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/46.jpg)
exon-1
asp-1-1 RNAi clone
212bpRT-PCR
479bp
asp-1 RNAi clone design
567bpasp-1-2 RNAi clone
• Two independent clones
• Overlapping
![Page 47: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/47.jpg)
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
![Page 48: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/48.jpg)
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
![Page 49: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/49.jpg)
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
![Page 50: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/50.jpg)
•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
![Page 51: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/51.jpg)
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
![Page 52: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/52.jpg)
• Lysosensor yellow/blue• Excitation is pH dependent• More blue = more basic• More yellow = more acidic
Verifying deacidification of lysosomes
BLUE YELLOW
ACIDICBASIC
![Page 53: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/53.jpg)
Control
20mM Chloroquine
15C Lysosensor Yellow/BlueCFP YFP CFP/YFP
![Page 54: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/54.jpg)
Quantitation of 15C Lysosensor Yellow/Blue
• Measured Grey ValueCFPYFP
• Calculate ratio ofYFP/CFP
No detectable differenceLarge variability
![Page 55: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/55.jpg)
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*
![Page 56: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/56.jpg)
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
![Page 57: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/57.jpg)
Effect of lysosomal inhibitors on lifespan
n=81p= <.0001*
Alternative Explanations
• Affecting non-lysosomal processes
• Inhibiting bacterial growth
• Behavioral changesfood avoidancedietary restriction
![Page 58: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/58.jpg)
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
![Page 59: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/59.jpg)
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
![Page 60: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/60.jpg)
Effect of lysosomal inhibitors on lifespanAlternative Strategies
![Page 61: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/61.jpg)
Control
vha-15 RNAi
15C Lysosensor Yellow/BlueCFP YFP CFP/YFP
![Page 62: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/62.jpg)
Quantitation of 15C Lysosensor Yellow/Blue
No detectable differenceLarge variability
![Page 63: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/63.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p=0.6458n=65
p=0.4439n=79
![Page 64: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/64.jpg)
15˚C 25˚C
RNAi of temperature regulated lysosomal proteases
p=0.9367n=68
p=0.1064n=79
![Page 65: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/65.jpg)
Prediction: Simple Model
Elevated proteases at 15C promote survivalReduced proteases at 25C promote aging
Pro-survival Pro-Aging
![Page 66: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/66.jpg)
Prediction: Revised Model
Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote aging
Pro-survival Pro-Aging
Condition Specific Pro-survival
![Page 67: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/67.jpg)
Prediction: Revised Model
Elevated proteases at 15C promote survival at 15CReduced proteases at 25C promote survival
Pro-survival Pro-Aging
Condition Specific Pro-survival
![Page 68: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/68.jpg)
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
![Page 69: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/69.jpg)
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
![Page 70: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/70.jpg)
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?
![Page 71: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/71.jpg)
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
![Page 72: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/72.jpg)
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
![Page 73: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/73.jpg)
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?
![Page 74: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/74.jpg)
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
![Page 75: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/75.jpg)
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
![Page 76: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/76.jpg)
15˚C Day 1 25˚C Day 1
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
![Page 77: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/77.jpg)
15˚C Day 1 25˚C Day 1
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
![Page 78: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/78.jpg)
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
![Page 79: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/79.jpg)
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
![Page 80: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/80.jpg)
25˚C Day 1 25˚C Day 7
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
![Page 81: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/81.jpg)
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
![Page 82: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/82.jpg)
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
![Page 83: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/83.jpg)
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
![Page 84: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/84.jpg)
Is lysosome structure or number altered by elevated temperature?Confocal Microscopy
![Page 85: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/85.jpg)
Specific Aim 2
a) Is lysosome structure or number altered by elevated temperature?
i) Lysotracker stainingRemaining
Quantitative analysis Repeat experiments
(IN PROGRESS)
![Page 86: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/86.jpg)
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?
![Page 87: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/87.jpg)
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
![Page 88: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/88.jpg)
![Page 89: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/89.jpg)
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?
![Page 90: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/90.jpg)
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
![Page 91: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/91.jpg)
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
![Page 92: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/92.jpg)
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
![Page 93: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/93.jpg)
cpr-7 Progressive Paralysis
15⁰C Day 8
![Page 94: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/94.jpg)
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
![Page 95: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/95.jpg)
Quantification of cpr-7 RNAi induced paralysis at 15°C
(Sanchez, Larsen, unpublished)
92% 90/97 worms paralyzedvs17% 16/95 control
![Page 96: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/96.jpg)
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
![Page 97: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/97.jpg)
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
![Page 98: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/98.jpg)
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)
![Page 99: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/99.jpg)
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
![Page 100: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/100.jpg)
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
![Page 101: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/101.jpg)
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
![Page 102: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/102.jpg)
![Page 103: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/103.jpg)
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)
![Page 104: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/104.jpg)
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
![Page 105: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/105.jpg)
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.
![Page 106: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/106.jpg)
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
![Page 107: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/107.jpg)
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)
![Page 108: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/108.jpg)
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)
![Page 109: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/109.jpg)
Temperature
Manipulations That Double Lifespan in C. elegans
GeneticEnvironment
daf-2 gene
Wild Typedaf-2
![Page 110: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/110.jpg)
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
![Page 111: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/111.jpg)
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
![Page 112: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/112.jpg)
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)
![Page 113: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/113.jpg)
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)
![Page 114: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/114.jpg)
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?
![Page 115: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/115.jpg)
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
![Page 116: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/116.jpg)
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?
![Page 117: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/117.jpg)
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
![Page 118: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/118.jpg)
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
![Page 119: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/119.jpg)
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
![Page 120: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/120.jpg)
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
![Page 121: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/121.jpg)
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
![Page 122: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/122.jpg)
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
![Page 123: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/123.jpg)
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
![Page 124: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/124.jpg)
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)
![Page 125: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/125.jpg)
qRT-PCR Lysosomal Proteases: daf-2(m596)
ELEVATED mRNA levels of proteases in daf-2 (m596)
asp-6 cpr-7 cpr-4 asp-1
![Page 126: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/126.jpg)
qRT-PCR Lysosomal Proteases: daf-2(m41);daf-16
Some daf-2 regulated proteases are daf-16 dependent
K10C2.3 cpr-1 F21F8.4
![Page 127: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/127.jpg)
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
![Page 128: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/128.jpg)
daf- 2 qRT-PCR Lysosomal ProteasesRemaining Experiments
![Page 129: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/129.jpg)
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)
![Page 130: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/130.jpg)
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)
![Page 131: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/131.jpg)
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
![Page 132: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/132.jpg)
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
![Page 133: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/133.jpg)
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
![Page 134: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/134.jpg)
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
![Page 135: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/135.jpg)
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
![Page 136: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/136.jpg)
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
![Page 137: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/137.jpg)
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
![Page 138: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/138.jpg)
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
![Page 139: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/139.jpg)
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?
![Page 140: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/140.jpg)
Temperature
Shared Mechanism of Longevity?
GeneticEnvironment
daf-2 gene
Wild Typedaf-2
![Page 141: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/141.jpg)
DAF-2Temperature
Lysosomal Proteases
Protein Synthesis
Proteostasis genes are regulated by
BOTH daf-2 and temperature
REGULATION IS SHARED
Shared Mechanism of Longevity?
![Page 142: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/142.jpg)
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
?
![Page 143: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/143.jpg)
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
?
![Page 144: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/144.jpg)
Revised Model
Occur concurrently
Pro-survival Pro-Aging
Condition Specific Pro-survival
![Page 145: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/145.jpg)
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∑
![Page 146: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/146.jpg)
![Page 147: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/147.jpg)
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
![Page 148: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/148.jpg)
Pharmacological lysosomal inhibitors
(Seglan 1976)(Klempner 1983)(Jones 1984)
Chloroquine NH4ClpH
• Decreases pH
• Vacuolization
![Page 149: February 2011CSB presentation](https://reader035.vdocuments.net/reader035/viewer/2022062902/58ef918d1a28ab9e288b4613/html5/thumbnails/149.jpg)
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