Thesis Poster 48x36_FinalFinal

1. Background/Introduc.on/ Results/
Methods/ Conclusions/
References/
Methods/
Results/
!!
!!!!!How!can!we!inves,gate!the!mechanisms!and!causes!of!neurological!diseases!
in!a!novel!way!that!may!lead!to!new!clues!to!their!cure?!In!epilepsy,!pa,ents!
suffer!from!recurring!seizures!due!to!hyperexcitability!and!synchronous!neural!
ac,vity!in!the!brain!(Fisher!et!al!2005).!Despite!our!general!understanding!of!
the!disease,!what!we!know!about!the!gene,c!and!molecular!causes!and!forces!
of!the!condi,on!are!not!well!understood.!The!insect!model&Drosophila&
melanogaster!stands!as!an!ideal!organism!for!studying!these!aspects!of!disease!
due!to!it’s!short!genera,on!type,!easy!level!of!care,!and!prevalence!of!several!
seizure!genotype!models!known!as!bangJsensi,ve!mutants,!which!produce!
certain!epilep,c!phenotypes!upon!receiving!a!mechanical!s,mulus!(a!bang!).!!
!!!!!In!this!study,!a!par,cular!bangJsensi,ve!mutant!known!as!slamdance!(sda)!is!
used!due!to!it’s!rela,vely!mild!(and!adjustable)!phenotype.!Previously,!this!
mutant!was!associated!to!a!muta,on!in!a!gene!for!in!an!aminopep,dase!
homolog!in!humans!(Zhang!et!al,!2002)!.!But!gene,c!studies!in!the!Deitcher!Lab!
have!shown!that!the!muta,on!is!actually!a!gene!known!as!CG14509,!which!has!
a!poorly!understand!protein!product!(Meghan!Horne,!unpublished!data).!
!!!!!Thus,!this!study!aims!to!complete!the!connec,on!of!CG14509!to!slamdance&
by!repor,ng!gene,c!rescues!of!the!original!sda&mutant!as!well!as!several!more!
severe!alleles!as!well!as!a!temperature!sensi,ve!GAL80!gene!using!GAL4J
mediated!expression!of!a!CG14509!transgene.!In!addi,on,!to!help!further!
characterize!the!ac,vity!of!the!CG14509,!we!also!used!this!system!to!guide!
expression!of!the!rescue!construct!in!different!neuronal!popula,ons!and!
determine!its!efficacy!at!blocking!or!reducing!seizure!severity,!and!express!GFP!
to!view!expression!paZerns.!
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!!
• Our!rescue!experiments!further!support!the!aZribu,on!of!slamdance!to!the!gene!CG14509!in!
Drosophila.!
• The!alleles!differ!in!seizure!severity,!thus!the!transgene!expression!can!rescue!differently!for!
each!one,!and!for!each!sort!of!expression!paZern.!
• CG14509!appears!to!have!a!developmental!impact!during!late!larva/early!pupa!stage!(rescues).!
• CG14509!appears!to!localize!in!the!op,c!lobes!and!along!neural!tracts!in!the!ventral!ganglion.!
(strong!level!of!expression)!
1. Armijo!JA,!Valdizán!EM,!De!Las!Cuevas!I,!Cuadrado!A.!Advances!in!the!physiopathology!of!epileptogenesis:!molecular!aspects.!Rev!
Neurol.!2002;20:409–429.![PubMed]!
2. Benzer!S!(1971),!From!the!gene!to!behavior.!JAMA!218(7):1015J22!!
3. Fisher!RS,!van!Emde!Boas!W,!Blume!W,!Elger!C,!Genton!P,!Lee!P,!Engel!J!Jr!(2005),!Epilep,c!seizures!and!epilepsy:!defini,ons!
proposed!by!the!Interna,onal!League!Against!Epilepsy!(ILAE)!and!the!Interna,onal!Bureau!for!Epilepsy!(IBE).!Epilepsia!46(4):
470J2. !!
4. Horne,!Meghan!(2014J2015),!Deitcher!Lab!unpublished!data.!
5. J.!O.!McNamara,!Y.!Z.!Huang,!and!A.!S.!Leonard,!Molecular!Signaling!Mechanisms!Underlying!Epileptogenesis.!Sci.!STKE!2006,!re12!
(2006)!
6. Pandey,!U.!B.,!&!Nichols,!C.!D.!(2011).!Human!Disease!Models!in!Drosophila&melanogaster!and!the!Role!of!the!Fly!in!Therapeu,c!
Drug!Discovery.!Pharmacological&Reviews,!63(2),!411–436.!doi:10.1124/pr.110.003293!
7. Sambandan,!D.,!Yamamoto,!A.,!Fanara,!J.J.,!Mackay,!T.F.C.,!Anholt,!R.R.H.!(2006).!Dynamic!gene,c!interac,ons!determine!odorJ
guided!behavior!in!Drosophila!melanogaster.!!Gene,cs!174(3):!1349—1363.!
8. Thurman,!D.!J.,!Beghi,!E.,!Begley,!C.!E.,!Berg,!A.!T.,!Buchhalter,!J.!R.,!Ding,!D.,!Hesdorffer,!D.!C.,!Hauser,!W.!A.,!Kazis,!L.,!Kobau,!R.,!
Kroner,!B.,!Labiner,!D.,!Liow,!K.,!Logroscino,!G.,!Medina,!M.!T.,!Newton,!C.!R.,!Parko,!K.,!Paschal,!A.,!Preux,!P.JM.,!!
9. Sander,!J.!W.,!Selassie,!A.,!Theodore,!W.,!Tomson,!T.,!Wiebe,!S.!and!for!the!ILAE!Commission!on!Epidemiology!(2011),!Standards!
for!epidemiologic!studies!and!surveillance!of!epilepsy.!Epilepsia,!52:!2–26.!doi:!10.1111/j.1528J1167.2011.03121.x!
10. Tunio,!Nahel!(2015),!Deitcher!Lab!unpublished!data.!!
11. Zhang,!H.,!Tan,!J.,!Reynolds,!E.,!Kuebler,!D.,!Faulhaber,!S.,!&!Tanouye,!M.!(2002).!The!Drosophila!slamdance!Gene:!A!Muta,on!in!
an!Aminopep,dase!Can!Cause!Seizure,!Paralysis,!and!Neuronal!Failure.!the!Gene,cs!Society!of!America,!1283J1299.!
!
Presented!by:!Glen!E!Francisco!
The!role!of!CG14509!on!seizure!suscep,bility!&!neuronal!morphology!in!Drosophila&melanogaster&
Lab!of!Dr.!David!Deitcher,!Cornell!University!
1. Fly!crosses!were!made!to!produce!offspring!of!desired!genotype,!including!mutant!background,!
desired!GAL4,!a!UAS,!and!GFP!or!GAL80!as!desired.!!
2. Flies!of!desired!genotype!were!vortexJtested!for!10!seconds,!10!flies!at!a!,me.!
3. Seizure!prevalence/rescue!was!determined!by!average!number!of!flies!that!seize.!
4. Seizure!severity!is!determined!by!average!halfJrecovery!,me:!number!of!seconds!required!for!
half!the!seizing!flies!to!fully!recover.!
5. To!test!development,!flies!were!raised!in!either!a!room!temperature!condi,on!(RT)!or!a!heated!
condi,on!(~29!degrees)(HT),!and!were!swapped!at!either!larval!or!pupa!stage!to!the!opposite!
condi,on,!and!vortexJtested!as!adults.!
6. To!visualize!expression,!GFP!expression!in!flies!was!driven!with!a!GAL4!under!control!of!
sequences!around!CG14509.!Brains!were!dissected!and!stained!using!immunohistochemistry,!
and!then!viewed!under!a!epiflourescence!microscope.!
Bloomington
Stock
Number
Description /
Function
25005 Deficiency in CG14509
18817 Transposon insertion in CG14509
5130 UAS construct to express GFP
458 Pan-neuronal GAL4 driver
48033 GABA -ergic neuronal GAL4 driver
46070 Expresses GAL4 under the control of
DNA sequences in or near CG14509 .
46416 Expresses GAL4 under the control of
DNA sequences in or near CG14509 .
7017 Ubiquitous expression of GAL80
ts

8760 Expresses GAL4 panneurally
!
Table/1./Descrip.on/of/various/Bloomington/Stocks/used/in/rescue/
crosses./Crosses!were!bred!to!produce!transgenic!flies!that!include!a!
mutant!background!of!homozygous!slamdance!or!a!combina,on!of!
slamdance!alleles.!They!also!included!a!GAL4!line!which!drove!
expression!of!a!wild!type!transgene!(UAS)!in!certain!neurons.!The!
GAL4!system!was!also!used!to!express!GFP!for!visualiza,on!and!GAL80!
in!a!developmental!study.!!
0!
20!
40!
60!
80!
100!
120!
C155! 48033! Control!
Pertentage/of/seizing/flies/
sda/sda!
sda/25005!
sda/18817!
0!
10!
20!
30!
40!
50!
60!
70!
80!
90!
100!
40670! 40670!Control! 46416! 46416!Control!
Percentage/of/flies/down/
sda/sda!
0!
10!
20!
30!
40!
50!
60!
70!
80!
90!
100!
Larva! Pupa! Adult!
Percentage/of/flies/down/
Room!Temperature!
Larva!
Pupa!
Adult!
Room!!
Heat!
0!
10!
20!
30!
40!
50!
60!
70!
80!
90!
100!
Larva! Pupa! Adult!
Percentage/of/flies/down/
Heat!Treatment!
Larva!
Pupa!
Adult!
Heat!
Room!
Figure/1./Results/of/gene.c/rescue/using/different/GAL4Ldriven/expression/of/
the/UAS/transgene/CG14509./(Lev)!Results!show!rescuing!ability!of!both!a!panJ
neuronal!and!GABAJergic!driver!against!different!mutant!backgrounds,!with!the!
heteroallelics!being!more!severe!in!phenotype.!Controls!did!not!include!a!GAL4!
gene.!(Right)!Results!also!show!the!ability!of!two!GAL4!lines!controlled!by!DNA!
sequences!in!or!near!CG14509.!Controls!here!contain!no!UAS.!
Figure/2./Results/of/a/developmental/study/using/expression/of/the/CG14509/
transgene/by/8760,/a/different/panLneuronal/driver,/which/is/addi.onally/controlled/
by/GAL80
ts
,/a/temperatureLsensi.ve/inhibitor/of/GAL4/that/becomes/inac.ve/in/
heated/condi.ons./Included/are/bar/graphs/illustra.ng/.me/spent/in/heated/or/room/
temperatures,/rela.ve/to/.me/from/birth/to/eclosing.//
Figure/3./Drosophila/brain/expressing/GFP/in/the/paYern/of/the/GAL4/driver/
40670./
Medulla!
Lamina!
Re,na!
Lower!
Thoracic!
Ganglia!
Axon!Tracts!