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Transcript
Genes in flies: in search of biological
pathways and models of RLS
Subhabrata Sanyal
Emory University
Gene/Homolog
Mutations
Disease/
Therapy
Phenotypes
• An implicated gene
• Manipulate strongly conserved homolog
• Ability to assay expected phenotypes
• Link back to Human disease/therapy
Why flies?
Drosophila as a model organism
Behavior: systems
Organs: circuits
Cellular: neurons
Molecular: signaling
GFP
Techniques to manipulate genes: GAL4-UAS
GAL4
Gene of Interest
UAS elements
Dopaminergic neurons in the fly
TH-GAL4
UAS-GFP
UAS-RNAi(BTBD9)
• An implicated gene …
Bric-a-brac
Tramtrack
Broad Complex
Stefansson, H., Rye DB. et. al. N Engl J Med 2007
Winkelmann J., Schormair, B. et. al. Nat Genetics 2007
The Fly homolog of BTBD9 is CG1826
Homology Between Drosophila and Human BTBD9
48% identity
63% similarity
BTB domain
conserved
The Fly gene region:
Human
Drosophila
CG1826 is expressed in the nervous system
•Manipulate strongly conserved homolog
Transposon Excision to generate Deletions
P-element
5’-UTR
Coding region of GOI
Precise
3’-UTR
+ Transposase
Imprecise
A
B
C
D
Deletion mutants in CG1826
All deletion mutants are alive
• Ability to assay expected phenotypes
• Focus on sleep
Features of sleep
•
•
•
•
•
•
•
•
•
Humans
Drosophila


















Period of immobility
Increased arousal threshold
EEG correlates
Molecular correlates
Drug-dependent effects
Gender and individual differences
Circadian regulation
Homeostatic regulation
Fragmentation with aging
• Ability to assay expected phenotypes
• An implicated gene
• Manipulate strongly conserved homolog
• Ability to assay expected phenotypes
Results of BTBD9 knockdown in flies?
Knockout of BTBD9/CG1826 disrupts sleep
Total
sleep
time
Average
number of
sleep epochs
Average
length of
sleep epoch
Wild-type (n=112)
CG1826 ex13 (n=16)
BTBD9/CG1826 knockdown exclusively in
TH neurons is sufficient for sleep disruption
Average
number of
sleep epochs
Constant darkness
110
100
‡
90
♦
80
70
60
50
130
% of wild-type
Total
sleep
time
% of wild-type
12h:12h light:dark
Wild-type (n=112)
CG1826 RNAi (n=100)
TH control (n=32)
THxCG1826
RNAi (n=32)
120
110
100
90
80
Average
length of
sleep epoch
% of wild-type
120
110
100
90
80
70
60
50
*
§
*‡ p<0.05
p<0.01
§ p<0.005
♦ p<0.000001
General locomotion is normal
in BTBD9/CG1826 mutants
% of total flies
100
90
80
Wild-type
CG1826 RNAi
70
60
50
40
30
20
10
0
0/5
1/5
2/5
3/5
4/5
Number of successful climbs
5/5
• Link back to Human disease/therapy
a. Link to dietary iron (iron chelators/more iron)
b. Better link to dopamine (agonists/antagonists)
c. Genetic interactors (per, hth, Ataxin etc.)
d. Pharmacology (Mirapex, Requip)
e. Model for drug testing, pathway analysis.
Novel directions
• Gene-gene and gene-environment interactions
• BTBD9 function in neural development and plasticity
• Consequences of sleep loss on cognitive functions
Acknowledgements
• Amanda Freeman
(FIRST Fellowship)
• David Rye and lab. members, Emory
Emory URC and NARSAD