Last research talk

1. Angela Sanchez
Mentor:Dr. Pamela Larsen
Inhibitionof temperature-regulated proteostasis
genes causes an early onset progressive dysfunction in
motorneurons

2. Cool temperature extends lifespan in C.elegans

3. Cool core temperature longevityis conserved
Drosophila
30˚c 18˚c
(Miquel 1976)
Mouse
(Conti 2006)
(Hunter 1999)
Ames Dwarf
(Lane 1996)
Caloric Restriction

4. Cool temperature is neuroandcardio protective
Enhance survival after stroke
(KammersgaardLPJAmHeartAssoc 2002)
(Roth 2002)
Enhance survival after heart attack
(Varon JChest2008)
32°C 37°C33°C 37°C
(Campos 2012) (Chenoune 2010)

5. Universality of cooltemperature improving health and survival
Underlyingmechanismnot understood

6. Approach to understanding healthylong-term survival
Microarray
• AffymetrixC.elegansarray
• Genome wide snapshot
• UNBIASED
• PRIMARYbiological response to a
chronicchange

7. Primary cause occurs prior to evidence of aging

8. Experimentaldesign of samples for microarray
L1
L2
L3
L4
ImmatureAdult
Development
Total RNA Total RNA
MatureAdult
Visible Developmental Stage
15°C
15°C 25°C
Four synchronizedbiologicalsamples

9. Temperature regulatedgenes
19,762 Predicted Genes
16,896 Probes
383 Differentially Expressed
179
P<.01
280
Suggests partofmolecular responsemechanism Transcriptional

10. Number of Temperature Regulated Genes
Gene ontology categories
(Curtis, Tavare’, Takano, Sanchez, Larsen, unpublished)

11. Proteostasisgenes differentially expressedby 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(Curtis,Tavare’, Takano, Sanchez Larsen, unpublished)

12. SpecificAim 1: Abbreviated for short talk format
Determine whethergenesthat aretemperature
regulated causelongevity or aging .
a) ValidatebyqPCR
b) Determine theeffect ofRNAi ofselected genesat 15°Cand25°C onlife span

13. Differentiallyexpressed lysosomal protease genes
Gene name Clone ID Description
asp-1 Y39B6A.20 Aspartyl protease
asp-3 H22K11.1 Aspartyl protease
asp-5 F21F8.3 Aspartyl protease
asp-6 F21F8.7 Aspartyl protease
F21F8.4 Aspartyl protease
K10C2.3 Aspartyl protease
C15C8.3 Aspartyl protease
Y39B6A.24 Aspartyl protease
cpr-1 C52E4.1 Cysteine proteinase Cathepsin L
cpr-4 F44C4.3 Cysteine proteinase Cathepsin L
F57F5.1 Cysteine proteinase Cathepsin L
W07B8.1 Cysteine proteinase Cathepsin L
F21D5.2 Cysteine proteinase Cathepsin L
K10C2.1 Serine carboxypeptidases (lysosomal cathepsin A)
K10B2.2 Serine carboxypeptidases (lysosomal cathepsin A)
Only specificgenes are temperature regulated
8regulatedof
19ingenome
5regulatedof
24ingenome
2regulatedof
10ingenome
Classified by active site

14. Differentiallyexpressed protein synthesis genes
Gene name Clone ID Description
eIF-1A ZK856.11 translational initiation
iff-2 F54C9.1 translational initiation
eif-3.I Y74C10AR.1 translational initiation
eIF-2C F20D12.1 translational initiation
eIF-4G M110.4 translational initiation
ife-3 B0348.6 translational initiation
eif-3.G F22B5.2 protein biosynthesis
rpl-24.2 C03D6.8 protein biosynthesis
F53F4.11 protein biosynthesis
eIF-2C C16C10.3 protein biosynthesis
eif-3.E B0511.10 protein biosynthesis
gld-1 T23G11.3 regulation of translation
M28.5 ribosome biogenesis and assembly
K12H4.3 ribosome biogenesis and assembly
Y54E10A.10 ribosome biogenesis and assembly
K07C5.4 ribosome biogenesis and assembly
W01B11.3 ribosome biogenesis and assembly
C43E11.9 ribosome biogenesis and assembly
fib-1 T01C3.7 rRNA processing
ZK430.7 rRNA processing
T07A9.8 rRNA processing
Y53C12B.1 rRNA processing
Only 37of the 589protein synthesis-related
genes on
C.elegans Affymetrixmicroarrray were DE
by temperature

15. *
* *
*
*
*
*
*
* *
* *
*
Verify temperature regulation of lysosomal proteases
qRT-PCRverification
Normalized Ct levels
Temperatureregulation confirmed for 13/15
Range of expression levels: 1000 fold

16. 10mM Chloroquine
15˚C 25˚C
n=75
p= 0.=.0162*
n=75
p= 0.4774
Temperaturedependent phenotype of proteostasis inhibition

17. Temperaturedependent phenotype of proteostasis inhibition
15˚C 25˚C
n=63
p= 0.0011 *n=64
p= 0.1161
(TakanoS.,Larsen P.etal.)
0
20
40
60
80
100
0 5 10 15 20 25 30 35 40
%Survival
Adult Age (days)
Empty Vector
egl-45 RNAi
0
20
40
60
80
100
0 5 10 15 20 25 30 35 40
%Survival
Adult Age (days)
Empty Vector
egl-45 RNAi

18. RNAi knockdown occurs at both 15˚C and 25˚C
10mM Chloroquine
cpr-7
0
1
2
Control
15°C
cpr-7
RNAi 15°C
Control
25°C
cpr-7
RNAi 25°C
RelativemRNAlevels
0
0.5
1
Control
15°C
egl-45
RNAi 15°C
Control
25°C
egl-45
RNAi 25°C
RelativemRNAlevels
egl-45

19. cpr-7 15°Cday8Control 15°Cday8
cpr-7 Progressive Paralysis: Day 8
cpr-7RNAi causes progressive paralysis

20. RNAi 15°Cday12Control 15°Cday12
cpr-7RNAi causes progressive paralysis

21. Inhibition oftranslation not universally beneficial
Surprisethat inhibition oftranslation can causeparalysis in middle-aged adults
Translationincreaseslife span
Theseresultsdemonstrate that this is not a universal outcome
Similar to positive/negative/neutral responsestodietary restriction depending on
genotype
Really need to find out what went wrongvery negativeside effect

22. SpecificAim 3
a) Quantifyincidence ofdefects at 15°C
Determine the causeofmidlife paralysis

23. 0
20
40
60
80
100
0 5 10 15 20
PerecentNormal
Locomotion
Days of Adulthood
Empty Vector
cpr-7
0
20
40
60
80
100
0 5 10 15 20
PercentNormalVulva
Days of Adulthood
Empty Vector
egl-45
rpc-1
nol-5
F14B4.3
ifg-1
RNAi knockdown ofproteostasis genes resultsin defects at 15°C
(Takano S. , LarsenP.etal.) (Sanchez A.,Larsen P.etal.)
Maximum life span 35 days
Therefore2/3 ofadult life spent paralyzed

24. SpecificAim 3
a) Quantifyincidence ofdefects at 15°C
b) Distinguishbetween musclularor andneuronal origin ofthe paralysis
Determine the causeofmidlife paralysis

25. Possible approachesto determine neuronalor musculardysfunction
underlyingparalysis
•Tissuespecific RNAi
•Neuronsresistantto RNAi
•Leakage between tissues
•Neurotransmitterdrugs
•40% of animals died preventing longitudinal studies
•Neuromuscularexam without drugsor transgenics

26. MUSCLE
C.elegans locomotion circuits
Bodywall muscles
BACKWARDFORWARD
Type A motor neurons Type Bmotor neurons
Type D motor neurons
Bessereau Lab
Worm Atlas

27. •Feed adult worms RNAi for 8 days
•Transfer40 worms to individual plates
•Score on days 9, 10, 11, 12, 13, 15, and 16
•Blinded and unblinded examiner
•Exam consistsoftail tap, pause,head tap, and scorevulva morphology
•Establishes functionof
Touchreception
Musclefunction
Neuronalfunction
forward and backward motion circuits
switching ability
Experimentaldesign

28. But whatdoes it mean?
Exampleof early defect at 15°C
individual# 18 treated withrpc-1 RNAi

29. How I will present the data
9 10 11 12 13 15 16
Day ofadulthood

30. Individuallongitudinaldata from neuromuscularexam
Control
Control #17 This individual would mistakenly
be scored as effected in a cross-sectional
study rather than never normal
Control #25 This individual was moving
slowly on day 13 and paralyzed by day 16
Control #9 and 11 These individuals slow
down prior to having a backwards defect

31. Individual longitudinal data fromneuromuscularexam: Control
Control
Control #17/18 These individuals develop a
protruding vulva after the reproductive
period is over

32. Defects in backwards movement
precedes paralysis in 70% of cases
SHOWS UP EARLIER AND IN GREATER
FREQUENCY THAN CONTROLS
Individual longitudinal data fromneuromuscularexam: cpr-7
Progressive, similar to neurodegenerative
diseases in humans

33. Individual longitudinal data fromneuromuscularexam: rpc-1
Defects in backwards movement
precedes paralysis in 54% of cases

34. Establishes functionof
•Touchreception- Normal
•Musclefunction-Normal
•Neuronalfunction
•locomotion motion circuits-Abnormal
Interpretation ofthe origin of the dysfunctionleading to paralysis
Body wall muscles
BACKWARDFORWARD
Type A motor neurons Type B motor neurons
Type D motor neurons

35. Vulval protrusionsareconcurrentwith locomotion defects
Control cpr-7 rpc-1

36. Vulva and locomotion circuits shareneurotransmittersand areadjacent
•Vulval musclesare essential foregg laying
•VC4 and VC5 cholinergic motor neuronsinhibit opening

37. Why is posterior half paralyzed first?
Number of motor neuronsare spread over
length of the animal in the ventral nervecord
Arethe number of synapses different overthe length of the animal?

38. Number of synapses in type A motor neurons
0
20
40
60
80
100
DA1 DA2 DA3 DA4 DA5 DA6 DA7 DA8 DA9
Post Synaptic
0
20
40
60
80
DA1 DA2 DA3 DA4 DA5 DA6 DA7 DA8 DA9
Presynaptic
0
20
40
60
80
100
VA1 VA2 VA3 VA4 VA5 VA6 VA7 VA8 VA9 VA10 VA11 VA12 VA13
Postsynaptic
0
20
40
60
80
100
VA1 VA2 VA3 VA4 VA5 VA6 VA7 VA8 VA9 VA10 VA11 VA12 VA13
Presynaptic
Ventral
muscle
innervation
Dorsal
muscle
innervation

39. 0
20
40
60
80
100
DB1 DB2 DB3 DB4 DB5 DB6 DB7 DB8 DB9
Postsynaptic
0
20
40
60
80
100
DB1 DB2 DB3 DB4 DB5 DB6 DB7 DB8 DB9
Presynaptic
0
20
40
60
80
100
VB1 VB2 VB3 VB4 VB5 VB6 VB7 VB8 VB9 VB10 VB11 VB12
Postsynaptic
0
20
40
60
80
100
VB1 VB2 VB3 VB4 VB5 VB6 VB7 VB8 VB9 VB10 VB11 VB12
Presynaptic
Number of synapses in type B motor neurons
Ventral
muscle
innervation
Dorsal
muscle
innervation

40. Deducedmechanismof neuraldysfunction
•Factorsnecessaryfor synaptic maintenance or neurotransmissionunder
cool conditions arenot reaching theirdestination
•Motor neuronswith fewer synapsesare sensitive
•Dysfunction startsnearthe tail and progressestoward the head
•Protrusionfromthe vulva is similarly neuronalin origin and occurs
concurrentwith locomotion dysfunction

41. SIGNIFICANCE
Environmental changestrigger beneficial and detrimental responses.By
associating molecular signatureswith life expectancywe can predict
phenotypic outcome in different genotypes and environments.

42. Acknowledgements
Committee MembersPast Larsen Lab Members
CollaboratorsFunding
Dr.Shelly Buffenstein
Dr.Ellen Kraig
Dr.Merry Lindsey
Dr.Jim Lechleiter
Dr.Syuichi Takano
Wendy Lee
Dr.Hal Boylston
Dr.Pamela Larsen
NIA Training Grant
Glenn Medical Foundation
Ellison Medical Foundation
Dr.Simon Tavaré
Dr.Christina Curtis