infusion of egf blocks locomotor activity in hamsters · sleep in worms! egf and egf-r! target...
TRANSCRIPT
Infusion of EGF blocks locomotor activity in hamsters
Running wheel assay
Period of infusion; therefore it’s reversible
TGFα, an EGF like factor, disrupts sleep-wake behavior
Mouse EGF Receptor mutant is defective in light response
Together, these experiments suggest EGF is necessary and sufficient for activity/sleep cycle
Sleep in worms
EGF and EGF-R
Target neuron - ALA
Neuropeptides and receptors
CHROMOSOME_IIcctaagcctaagcctaaaatagtgactctggcagttctctaaaataagtgactctggcagttcaccaaaaattgtgactctgaccgttcaccaaaaatagtgactctgaccgttcaccaaaaatagtgactctgaccgttcacaaaaaatagtgactctgaccgttcaccaaatatagtgactctgaccgttcaccaaaaattgtgacaatgaccgttcaccaaaaattgtgactctgaccgtcactatttttattgaactgccagagtcactatttttagtgaacttccagagtcacaatttttagtgaactgccagagtcactatttttagtgaactgccagagtcacttattttggtgcactggggtgggtcacgcccccagttctcagttatgggtactctgatccactcgggacccactttatcgtgttccccgtgcctcatttaccctagagcttcctcctttacctctcctctcgctatctctaacattccaatggaaactcctatttgaattaccgccaccgatgtgcccgacgcgacttactgttagcccttgttttgcacaaatctgttggcttccatatttaaaagttaattaatgacccaatgttctttttttctctaaatctccacaagatgttctgttttccctactggacactatcgttcactgcgtctcaccaattcacattgtctctactttaccttttttgtcatagtacacgttcgccaacggtgtcgacggccaaatgctttgggcagcgtttgctttttttataattagttttattttattaaaacaatagctctaaagtttacaagtcatttgttataggctaaatgagttatgtctaataagtaatttgaactagatacttccgtgtaagtgacaatgtatcggaaaagtcctcaaagtgcgatgtagaagttcacatgtactttgtttggcatgttagtaaaagagccagtatgctgattcattttatattctatatactcatgtaatatgcccatgtaaggtttaattccaaaaatatgagcgtgttctattttataatattttactaaaatacctttcagttaattgcactcaaatttgttgttcttcattctctcgttatgatttaatcttattgcgtcaaggtcattattttaggtccattagttatcgatctgaaacatgttgttgtatttttctattcttgtgagctcaggacacctcatacaactccagagaaaatgtgtctcattattcttgtcttttttcaagatctaatcaattttctacattaacgacgtttttgtcgttctgcttctttttttcgttcgtttgtctcgtccatcagctgtccactcatttctctcccactcactaggcagtgctttgtttggttccgattggcagctggctgcagggcctgcatctcttctatgtctctcatttacttgcattcttttcttcgttaatttttgttatgatatttaaacgggaagaagagtttgtggttcttctttttataatcactaaaacttttggataagtaacaattttctgataaaatattttcacggcgaagaaaaaagaaaaagaagagtagtttttgcacgttttcatataattattttcgttgatcaaatgttcttctggagttttctaataaatttcttatcgactttttttcagaaatttttctcaacttgtcatgtcaatggtaagaaatgtatcaaatcagagcgaaaaattggaagtaagttctttataatttcatttatatactataagttttctcgatcacaggagaaacaaaaacaacagacaacacaaaaaacaataaaacaatattgctctagtaatcaatagtgttgtaaagagggaagaaaattgttatctgtgtagcagtcaacgttgattgagatgttgtgtttgactatagagttgaaaataataacttcaaacttgcaagtcatgacttatcaaacactgccggaacttattctggatcaaaggaaagttgtccaactgtagagtcatgtttttcaaaagaaaacacaatttttaagtataaatattttgaaaaagtatgttttagaagtatgtcaaattaaaaaaaaaatccttggttaaaaaatgatttttttggatatatgtgtatttttaactaaaaatatatactttacatatatattttggcgcagttatttgatctataaatcaaactttttgatagacatttttttatatttacaacaactagggttgttatgaaaacgcctattattctacaaactaaattattttaatcatacattccccactatctaaaaactaatgcaattttcagattttgtcatgtaaatgggtaggatgtctcaaatcaacagaagtgttcaaaacggttgaaaagttattagatcatgttacggctgatcatattccagaagttattgtaaacgatgacgggtcggaggaagtcgtttgtcagtgggattgctgcgaaatgggtgccagtcgtggaaatcttcaaaaaaaggtatttttaatttaatgtgcattttataatataaattcttcagaaagagtggatggagaatcacttcaaaacacgtcatgttcgcaaagcaaaaatattcaaatgcttaattgaggattgccctgtggtaaagtcaagtagtcaggaaattgaaacccatctcagaataagtcatccaataaatccgaaaaaaggtattcacaatttgcatgatattgttataatctaattttcagagagactgaaagagtttaaaagttctaccgaccacatcgaacctactcaagctaatagagtatggacaattgtgaacggagaggttcaatggaagactccaccgcggtaagtgtgtttctttaaaaattacttccttttttcaattgtttgaaattaacaagaaacctgttggagcgtatttctgaacttttaaatcgaaaatatcatttgcaaaaaaacttgaaaattgagaaacttttttaaaagtggagtagcgtctgcgggtttttttgccctaaatgacagaatacatacccaatataccgaatataaccgtaataaaattatgcgatttttatttttatttttcatgaatgttaggggcaaaaaacccacatgcgctactccgccttaagaagaatcagctgtgagcactatccactatacattggaaatttacaaataaaatagagattaagtaatataatttttaagggttaaaaaaaagactgtgatatactatgatgatgggccgaggtatgtatttccaacgggatgtgcgagatgcaactatgatagtgacgaatcagaactggaatcagatgagttttggtcagccacagagatgtcagataatgaagagtacgttgttttgcaaattgattaaaagtggagtagcgtcagttaaaaactctaacatgtcttaggtttttcaaaagtttggtcaaagttttggcaaactgccaacttcttgaaaacttcgttaaaaaaattcttgaaatgatttgaaaatttgtattatgttattctcttatttctgcactattctatatggcgctactatacttttaattgatttcttgaaagcagttcaataataattaattttagagtatatgtgaacttccgtggaatgaactgtatctcaacaggaaagtcggccagtatggtcccgagcaaacgaagaaattggccaaaaagagtgaagaaaaggctatcgacacaaagaaacaatcagaaaactattcgaccaccagagctgaataaaaataatatagagataaaagatatgaagtaagtcgaaattgacaaacagtggtttttgtttaagtttattgcgaaatattcaaaattagacatgttaaaattttgcgagataatctaaagattaggtatacagattttttcatgtaaagttacattcatcaaaatttttgtgttcaccaaattagacaaaaaatgttagttacacagtatatttattttttatatcaataaaacctttttcagctcaaataaccttgaagaacgcaacagagaagaatgcattcagcctgtttctgttgaaaagaacatcctgcattttgaaaaattcaaatcaaatcaaatttgcattgttcgggaaaacaataaatttagagaaggaacgagaagacgcagaaagaattctggtgaatcggaagacttgaaaattcatgaaaactttactgaaaaacgaagacccattcgatcatgcaaacaaaatataagtttctatgaaatggacggggatatagaagaatttgaagtgtttttcgatactcccacaaaaagcaaaaaagtacttctggatatctacagtgcgaagaaaatgccaaaaattgaggttgaagattcattagttaataagtttcattcaaaacgtccatcaagagcatgtcgagttcttggaagtatggaagaagtaccatttgatgtggaaataggatattgattttataacgtgtaattgagttttggccaaaaaggtatggaaaggtggctgtttagttatatatttttctattatttatttgaaacatgcaaaattgaagtgaacaataagtgatgttcatggaaatttaaactgttttatgatacttttttgagaaattgaaaaatctgttcattttagaaacaatgtccacatggttctaagagctaaaatttttattttcatccatttagagtactttctcttttagagtacggccccagagcgatgttagaaacctgagatcggtcaacacagaccgttaattttgggaagttgagaaattcgctagtttctgacacgaatttcagctaataccaaataatgtgcaattgcattttgcatgtcagcattcagcattcatacaaaaatttcaaagagccaacttttcatacgtttatggtcaacactgtatgtgttacattgaacttttttaaattgt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6
C. elegans SLEEP: molecules & circuits
graphic by Chris Grove [WormBase-‐Caltech]
Elly Chow Julie Cho Cheryl Van Buskirk Erich Schwarz Daniel A. Lee, David Prober
302 neurons
Paul Sternberg, Cal tech
Neuropeptide signaling network promotes EGF-dependent sleep
Sleep dampens sensory response by
multilevel modulation of a sensory-motor circuit
1
2
SEM-5 Grb2 LET-341 SOS
LET-60 RAS
[IP3]
Vulval development
PLCγ [PIP2]
Ovulation
ITR-1 IP3 Receptor
SLI-1
Cbl LFE-2 IP3K
LET-23 EGF-R
LIN-3 EGF
[DAG]
Y~P Y~P
Wild-type adult worms are almost constantly moving
Cheryl Van Buskirk Van Buskirk & Sternberg Nat Neuro 2007
EGF expression severely inhibits locomotion
(no movement unless perturbed)!Cheryl Van Buskirk
WT lethargus hs:EGF adult
unperturbed perturbed
Octanol latency
hs:EGF induces sleep-like behavior outside of lethargus
cessaQon of feeding and locomoQon
reduced sensory responsiveness
quick reversibility
hatch!
embryogenesis L1 ! L2 ! L3 ! L4 ! Adult!
molt molt molt molt
Cheryl Van Buskirk
second
s unQ
l respo
nse
EGF promotes quiescence during molting via ALA neuron
Time (min)
Velocity (m
m/sec)
0 50 100 150 200 0 50 100 150 200
0.20
0.15
0.10
0.05
0
-‐.05
0.20
0.15
0.10
0.05
0
-‐.05
N2 let-‐23(sy12)
plc-‐3(tm1340) ALA-‐
Wild-‐type, intact
Wild-‐type ALA-‐ablated
EGF-‐receptor mutant
PLC-‐γ mutant
Van Buskirk & Sternberg Nat Neuro 2007
EGF promotes quiescence during molting via ALA neuron
Time (min)
Velocity (m
m/sec)
0 50 100 150 200 0 50 100 150 200
0.20
0.15
0.10
0.05
0
-‐.05
0.20
0.15
0.10
0.05
0
-‐.05
N2 let-‐23(sy12)
plc-‐3(tm1340) ALA-‐
Wild-‐type, intact
Wild-‐type ALA-‐ablated
EGF-‐receptor mutant
PLC-‐γ mutant
Van Buskirk & Sternberg Nat Neuro 2007
SEM-5 Grb2 LET-341 SOS
LET-60 RAS
[IP3]
Vulval development
PLCγ [PIP2]
Ovulation
ITR-1 IP3 Receptor
SLI-1
Cbl LFE-2 IP3K
LET-23 EGF-R
LIN-3 EGF
[DAG]
Sleep
Synaptic Release UNC-13, etc.
EGF signaling mediates stress-induced sleep
Time (min)
Velocity (m
m/sec)
0 50 100 150 200 0 50 100 150 200
0.20 0.15
0.10
0.05
0
-‐.05
WT let-‐23(rf)
lethargus behavior EGF/EGFR
STRESS
stress-‐induced sleep Van Buskirk Lab, Cal State Northridge
The single ALA neuron is necessary for
EGF-dependent sleep
~ALA-‐ablated animals are insomniac ~Expression of EGFR only in ALA is is sufficient for EGF-‐induced sleep ~ALA synapses not necessary for funcQon
Cheryl Van Buskirk
Micro-dissection & Transcriptional profiling of single ALA neurons
L4
L4 Lethargus 4-‐5hr
Single cell RT-‐PCR cDNA amplificaQon 9 cells (2 pools)
Igor Antoshechkin, Caltech Jacobs Genome Center
Elly Chow Erich Schwarz, Miriam Goodman, Mihoko Kato Schwarz, Kato & Sternberg [PNAS 2012]
Erich Schwarz
ALA enriched genes: 10 neuropeptide genes
FRMFamide-Like Peptide
Ex
pre
ssio
n L
eve
l
Enrichment
8110 protein coding genes detected in ALA
ALA-‐enriched neuropepQdes (>12-‐fold over whole larvae)
Validation of expression in ALA neuron using enhancers predicted from “ALA motif”
FLP-24
FLP-7
FLP-13
VPSAGDMMVRF!!
SPMQRSSMVRF!TPMQRSSMVRF!SPMERSAMVRF!SPMDRSKMVRF!
!
AMDSPLIRF!AADGAPLIRF!APEASPFIRF!ASPSAPLIRF!SPSAVPLIRF!ASSAPLIRF!SAAAPLIRF!
VPSAGDMMVRFG!
VPSAGDMMVRFamide!
KRVPSAGDMMVRFGKR!
FLP-13
NLP-8
FLP-24
FLP-7
NPR-7
NPR-22
sleep EGF
ALA neuron
neuropeptides receptors
FRPR-3
?
STRESS
EGFR
Elly Chow
sleep neuropeptides receptors
sleep neuropeptides receptors
Elly Chow
35°C, 30 min recover RT, 10 min
score
4 neuropeptides necessary for EGF response
Qui
esce
nce
Time (min)
WT
sleep neuropeptides receptors
sleep neuropeptides receptors
sleep neuropeptides receptors
Elly Chow
Qui
esce
nce
Time (min)
Multiple receptors are necessary
sleep neuropeptides receptors
sleep neuropeptides receptors
sleep neuropeptides receptors
sleep neuropeptides receptors
Elly Chow
FLP peptides increase response latency
sleep neuropeptides receptors
sleep neuropeptides receptors
sleep neuropeptides receptors
sleep neuropeptides receptors
sleep neuropeptides receptors
Elly Chow
receptor mutant block peptide overexpression
FLP-13
NLP-8
FLP-24
FLP-7
NPR-7
NPR-22
sleep EGF
ALA neuron
neuropeptides receptors
FRPR-3
?
STRESS
EGFR
Elly Chow