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The role of karyopherin-alpha in the pathogenesis of TDP-43 proteinopathy Armstrong, Bryson Walter 2013

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!!!The!Role!of!Karyopherin0alpha!in!the!Pathogenesis!of!TDP043!Proteinopathy!!by##Bryson#Walter#Armstrong########A#THESIS#SUBMITTED#IN#PARTIAL#FULFILLMENT#OF#THE#REQUIREMENTS#FOR#THE#DEGREE#OF##MASTER#OF#SCIENCE##in##The#Faculty#of#Graduate#and#Postdoctoral#Studies##(Neuroscience)##THE#UNIVERSITY#OF#BRITISH#COLUMBIA##(Vancouver)##September#2013#####?Bryson#Walter#Armstrong,#2013## ii#Abstract!#Aberrant#cellular#processing#and#targeting#of#TDPT43#has#been#implicated#in#a#wide#variety#of#neurological#diseases#such#as# frontotemporal# lobar#degeneration#with# ubiquitinated# inclusions# (FTLDTU)# and# amyotrophic# lateral# sclerosis# (ALS).#These#diseases#are#characterized#by#the#sequestration#of#TDPT43#into#the#cytoplasm#of# afflicted# neurons,# leading# to# the# formation# of# ubiquitinated,# cytoplasmic#inclusions# and#an# increased# susceptibility# to# cellular# insults.#While# the#underlying#causes# of# TDPT43# proteinopathy# are# unknown,# we# are# investigating# the# role# of# a#protein#family#known#as#the#karyopherins#in#the#nuclear#targeting#of#TDPT43.#Using#coTimmunoprecipitation# in# SHTSY5Y# cells# we# determined# that# a# major# binding#partner# of# TDPT43# is# karyopherinTalpha# 2# (KPNA2).# Next,# utilizing# a# highTdensity#peptide#array#comprised#of#overlapping#peptide#sequences#derived#from#TDPT43#we#identified#six#regions#where#KPNA2#may#directly#interact#with#TDPT43.#From#these#regions#we# developed# six# small,# cellTpenetrating# peptides# designed# to# specifically#inhibit# the# interaction# between# KPNA2# and# TDPT43.# Through# the# use# of# these#synthetic#peptides,#we#were#able#to#interfere#with#the#binding#of#KPNA2#to#TDPT43#in#vitro.#We#found#that#the#disruption#of#this#specific#proteinTprotein#interaction#was#not# sufficient# to# induce# TDPT43# cytoplasmic# sequestration,# as# determined# by# coTimmunoprecipitation#and#subcellular#fractionation#assays.##As# our# research# focused# primarily# on# healthy# SHTSY5Y# cells,# future# studies#will# focus#on# investigating# the# effects# of# peptideTmediated#TDPT43#nuclear# import#impairment# paired# alongside# oxidative# insult.# We# will# also# investigate# whether#compensatory# mechanisms# within# SHTSY5Y# cells# are# responsible# for# the# nuclear#localization#of#TDPT43#in#the#absence#of#KPNA2Tmediated#nuclear#import.## iii#Preface!This# thesis# is# based# on#work# conducted# by# Dr.#Max# Cynader,# Dr.#William# Jia,# and#Bryson#Armstrong#in#the#Cynader#laboratory#of#the#UBC#Brain#Research#Centre.#Dr.#Max# Cynader,# Dr.# William# Jia# and# I# identified# and# designed# the# project.# I# was#responsible# for#conducting#all#experiments#and#performing#all#data#analysis.# I#was#tasked#with#the#writing#of#this#thesis,#and#Dr.#Max#Cynader#and#Dr.#William#Jia#both#were#involved#in#the#editing#process.#!!!!!!!!!!!!!!# iv#Table!of!Contents!Abstract.......................................................................................................................................ii!Preface........................................................................................................................................iii!Table!of!Contents.....................................................................................................................iv!List!of!Figures...........................................................................................................................vi!Acknowledgements...............................................................................................................vii!Dedication...............................................................................................................................viii!1! Introduction..................................................................................................................1!1.1#Structure#and#Function#of#TDPT43.........................................................................................1#1.2#TDPT43#and#Disease.....................................................................................................................3#1.3#TDPT43#and#ALS.............................................................................................................................5#1.4#TDPT43#and#FTLD..........................................................................................................................7#1.5#Karyopherins................................................................................................................................12#1.6#Classical#Nuclear#Import#Cycle.............................................................................................15#1.7#KPNA#and#TDPT43......................................................................................................................16#1.8#Specific#Aims.................................................................................................................................18#2! Peptide0Mediated!Inhibition!of!TDP043!Nuclear!Targeting.......................19!2.1#Introduction..................................................................................................................................19#2.2#Materials#and#Methods.............................................................................................................21## 2.2.1#Cell#Culture........................................................................................................................21## 2.2.2#HighTDensity#Peptide#Array#Synthesis..................................................................21## 2.2.3#ProteinTProtein#Interaction#Assay..........................................................................22## 2.2.4#Peptide#Array#Regeneration......................................................................................23## 2.2.5#Peptide#Synthesis...........................................................................................................24## 2.2.6#Peptide#Treatments.......................................................................................................24## 2.2.7#Protein#Extraction#for#Western#Blotting..............................................................24## 2.2.8#CoTImmunoprecipitation#...........................................................................................25## 2.2.9#Western#Blot#...................................................................................................................26## 2.2.10#Subcellular#Fractionation........................................................................................27## 2.2.11#Immunocytochemistry.............................................................................................28## v##2.3#Results.............................................................................................................................................29## 2.3.1#Human#Neuroblastoma#SHTSY5Y#Cells#Express#KPNA2#and#KPNA4.......29## 2.3.2#KaryopherinT?2#Interacts#with#TDPT43#in#SHTSY5Y#Cells............................29## 2.3.3#Recombinant#KaryopherinT?2#Binds#with#Six#Distinct#Regions#of#TDPT43#on#HighTDensity#Peptide#Array.......................................................30#2.3.4#The#Interaction#Between#Endogenous#KaryopherinT?2#and##TDPT43#Can#Be#Disrupted#by#Interference#Peptides#In#Vitro...................31#2.3.5#Inhibition#of#the#Interaction#Between#KPNA2#and#TDPT43#is#Not#Sufficient#to#Induce#Cytoplasmic#Mislocalization#of#TDPT43......32#2.4#Discussion......................................................................................................................................32#3! Conclusions!and!Future!Directions.....................................................................43!Bibliography............................................................................................................................45!# ### ################ vi##List!of!Figures##Figure#1# Schematic#representation#of#classical#nuclear#import#cycle....................37#Figure#2# KaryopherinT?2#interacts#with#TDPT43#in#SHTSY5Y#cells.........................38#Figure#3# TDPT43Tderived#highTdensity#peptide#array..................................................39#Figure#4# Peptides#interfere#with#interaction#between#KPNA2#and#TDPT43.......40#Figure#5# Subcellular#localization#of#TDPT43#following#24Thour#in#vitro#peptide#treatment#and#nuclear#fractionation............................................41#Figure#6# Immunocytochemical#analysis#of#the#subcellular#distribution#of#TDPT43#following#24#hour#in#vitro#peptide#treatment.....................42## ################ vii#!Acknowledgements!#I#would#like#to#express#my#sincere#gratitude#to#Dr.#Max#Cynader#and#Dr.#William#Jia#for#supplying#me#with#the#knowledge#and#wisdom#I#sorely#needed#to#see#this#project#through.#I#would#also#like#to#thank#my#committee#members,#Dr.#Yu#Tian#Wang#and#Dr.#Neil#Cashman,#for#all#of#their#excellent#feedback#in#regards#to#my#experiments.#A#special#thanks#goes#to#Rui#Liu,#Carlo#Smith,#and#Shelly#Fan#for#always#being#there#for#me#when#I#needed#help#troubleshooting.#Most#of#all#I#would#like#to#thank#my#mother#for#continuing#to#support#me,#despite#everything#I#have#put#her#through.#I#couldn?t#have#done#it#without#you.############## viii#####   Dedicated to my Mother and Grandmother #######!! 1!1:#Introduction#1.1 #Structure#and#Function#of#TDP643#Transactive!Response!DNA3Binding!Protein!43!(TDP343)! is!a!highly!conserved,!ubiquitously! expressed! nuclear! protein! encoded! by! the! TARDBP! gene! located! on!human!chromosome!1P36.2!(Banks,!Kuta!et!al.!2008).!TDP343!was!first!described!by!Ou!et!al.!(1995)!as!a!transcriptional!regulator!which!represses!both!basal!and!TAT3induced! expression! of! the! human! immunodeficiency! virus! type! 1! (HIV31)! gene.!Subsequent! studies! revealed! that! TDP343! plays! a! critical! role! in! the! alternative!splicing! of! a! variety! of! mammalian! pre3mRNAs,! such! as! those! coding! for! CFTR!(Buratti,!D?rk et al. 2001; Buratti and Baralle 2001),!SKAR!(Fiesel,!Weber!et!al.!2012),!and!ApoA3II!(Mercado,!Ayala!et!al.!2005)!proteins.!!More!recent!investigations!have!hinted!at!an!increasingly!complex!role!for!TDP343.!In!mice,!Polymenidou!et!al.!(2011)!showed!that!TDP343!is!involved!in!regulating!the! largest! set! of! cassette! exons! reported! to! date.! They! determined! that! the!reduction!of!TDP343!in!the!adult!mouse!nervous!system!was!sufficient!to!alter!the!expression! levels! of! 601! mRNAs,! as! well! as! the! splicing! patterns! of! 965! mRNAs!(Polymenidou,!Lagier3Tourenne!et!al.!2011).!In!humans,!TDP343!has!been!shown!to!influence!the!generation!of!a!number!of!alternative!mRNA!isoforms,!many!of!which!encode! for! proteins! that! have! been! implicated! in! neuronal! development! and!neurological!diseases! (Tollervey,!Curk!et!al.!2011).! Interestingly,!TDP343!has!been!! 2!found!to!act!in!an!autoregulatory!manner,!controlling!its!own!expression!by!binding!to!TARDBP!mRNA!(Tollervey,!Curk!et!al.!2011).!TDP343,!which!consists!of!414!amino!acids,!structurally!appears!to!belong!to!a!group! of! proteins! known! as! 2! RBD3Gly! RNA!Binding! Proteins! (Wang,!Wang! et! al.!2004).!This!group!of!proteins,!which!also!includes!heterogeneous!ribonucleoprotein!particles! (hnRNPs)! and! other! factors! involved! in! RNA! splicing! and! transport,! is!characterized!by!the!presence!of! two!RNA!recognition!motifs!(RRMs)!along!with!a!carboxy3terminal!glycine3rich!domain! (Wang,!Wang!et! al.!2004).!TDP343!has!been!shown!to!directly!interact!with!transcripts!via!these!RNA!recognition!motifs!(Buratti!and!Baralle!2001).!In!addition!to!these!motifs,!TDP343!contains!two!closely!spaced,!specific! clusters! of! basic! residues! near! the! N3terminal! (Winton,! Igaz! et! al.! 2008).!This!region!is!hypothesized!to!act!as!a!bipartite!nuclear!localization!signal.!!Through! the!use!of! site!directed!mutagenesis,!Winton!et! al.! (2008)! found! that!the!bipartite!signal!sequence!found!on!TDP343!is!necessary!for!the!nuclear!import!of!the! protein.! While! TDP343! is! localized! primarily! within! the! nuclei! of! healthy!neuronal! and! non3neuronal! cells,! it! must! be! continuously! shuttled! between! the!nucleus!and!the!cytoplasm!in!order!for! it! to!perform!its!cellular!functions.!Nuclear!TDP343!is!believed!to!be!targeted!to!the!cytoplasm!via!a!leucine3rich!nuclear!export!signal!located!near!the!middle!of!the!protein.!Site!directed!mutagenesis!studies!have!provided!support!for!this!hypothesis!(Winton,!Igaz!et!al.!2008).!!!! 3!1.2 #TDP643#and#Disease#Aberrant! cellular! processing! of! TDP343! has! been! implicated! in! a! number! of!debilitating!neurodegenerative!diseases.!In!2006,!TDP343!was!discovered!to!be!the!primary!protein!component!of! the!pathological! inclusions!observed!in!the!affected!neurons! of! patients! suffering! from! either! Amyotrophic! Lateral! Sclerosis! (ALS)! or!Frontotemporal! lobar! degeneration! with! ubiquitinated! inclusions! (FTLD3U)!(Neumann,! Sampathu! et! al.! 2006).! In! both! diseases,! TDP343,! which! is! primarily!localized! to! the! nuclei! of! healthy! neurons,! was! found! to! redistribute! to! the!cytoplasm! where! it! aggregates! into! insoluble,! ubiquitin3positive! inclusions.! The!pathologic! TDP343! found! in! these! inclusions! has! been! shown! to! be! ubiquitinated,!hyperphosphorylated!and!cleaved! into!C3terminal! fragments!(Neumann,!Sampathu!et!al.!2006).!!While!pathological!TDP343!is!a!primary!feature!of!both!ALS!and!FTLD3U,!there!are!a!number!of!other!neurological!diseases!that!exhibit!signs!of!abnormal!TDP343!processing.! Alzheimer?s! disease,! hippocampal! sclerosis,! corticobasal! degeneration!and!the!Parkinsonism3dementia!complex!of!Guam!have!all!been!observed!to!present!with! secondary! TDP343! pathology! (Geser,! Martinez3lage! et! al.! 2009).! In! these!diseases,!TDP343!pathology!is!generally!present!to!a!lesser!degree!than!is!observed!in!primary!TDP343!proteinopathies!such!as!ALS!and!FTLD3U!(Geser,!Martinez3lage!et! al.! 2009).! Additionally,! while! TDP343! pathology! exhibits! a! rather! diffuse!distribution!in!ALS!and!FTLD3U!patients,!the!pathology!observed!in!secondary!TDP3! 4!43!proteinopathies!is!generally!localized!within!more!discrete!brain!regions!(Geser,!Martinez3lage!et!al.!2009).!In!the!primary!TDP343!proteinopathies!ALS!and!FTLD3U,!afflicted!cells!exhibit!a!substantial!loss!of!nuclear!TDP343!along!with!an!increase!in!pathologic!TDP343!and!TDP343! fragments!within! the! cytoplasm! (Neumann,! Sampathu! et! al.! 2006).!While!TDP343! cytoplasmic! sequestration! and! aggregation! has! been! shown! to! occur! in!these!neurodegenerative!diseases,!it!is!unclear!what!role!pathological!TDP343!plays!in!cellular!toxicity.!!There!is!evidence!to!suggest!that!TDP343!proteinopathies!result! in!a!toxic!gain!of!function!(GOF)!in!afflicted!cells.!A!study!utilizing!cultured!rodent!neurons!found!that! the! degree! of! cytoplasmic! TDP343! expression! correlated! with! neurotoxicity!(Halliday,!Bigio!et!al.!2012).! In!rodent!models,!overexpression!of!WT!and!mutated!TDP343!has!been!shown!to!be!neurotoxic,!and!this!toxicity!was!observed!in!a!dose3dependent! fashion! (Halliday,! Bigio! et! al.! 2012).! Additionally,! disease! progression!has!been! found! to! correlate!with! levels!of!TDP343!C3terminal! fragments! (CTFs)! in!some!rodent!models!(Halliday,!Bigio!et!al.!2012).!Furthermore,!a!number!of!studies!have! shown! that!mutations! in! the!TARDBP!gene!may! lead! to! cellular! toxicity! in! a!gain! of! function! manner,! including! elevated! stress! granule! formation,! enhanced!toxic! TDP343! CTF! generation! and! increased! production! of! prion3like,! protease3resistant!fragments!(Halliday,!Bigio!et!al.!2012).!While! there! is! evidence! that! TDP343! proteinopathy! results! in! a! toxic! GOF! in!affected!cells,!there!is!also!reason!to!believe!that!pathogenesis!may!arise!due!to!the!! 5!loss! of! functional! TDP343! (LOF).! As! TDP343! is! implicit! in! many! critical! cellular!processes,! it! follows! that! a! disruption! in! its! normal! functions! may! have! drastic!consequences.! The! sequestration! of! TDP343! into! cytoplasmic! aggregates! may!prevent! sufficient! levels! of! TDP343! from! translocating! into! the! nucleus,! where! it!normally! regulates! a! variety! of! mRNA! processing! (Halliday,! Bigio! et! al.! 2012).!Although! LOF! and! GOF! mechanisms! underlying! the! toxicity! of! TDP343! are! often!presented!as!competing!hypotheses,!there!is!no!evidence!that!these!mechanisms!are!mutually! exclusive,! and! both! are! likely! to! play! a! role! in! the! etiology! of! TDP343!proteinopathies.!!1.3 #TDP643#and#ALS#Amyotrophic!Lateral!Sclerosis!(ALS)!is!a!debilitating!neurodegenerative!disease!that! is! characterized! by! progressive! paralysis,! muscular! atrophy! and! death!(Siddique!and!Ajroud3Driss!2011).!Recent! studies!have!estimated! that! the! lifetime!risk!of!acquiring!spontaneous!ALS!before!the!age!of!70!is!between!1!in!1000!to!1!in!400,! with! males! being! slightly! more! at! risk! than! females! (Wijesekera! and! Leigh!2009).!The!average!life!expectancy!of!a!patient!suffering!from!ALS!is!233!years!after!diagnosis,! with! respiratory! failure! being! the! leading! cause! of! mortality! (Pratt,!Getzoff!et!al.!2012).!!While! the! majority! of! ALS! cases! are! sporadic! in! origin! (sALS),! it! has! been!reported! that! approximately! 10%! are! of! the! familial! variety! (fALS)! (Siddique! and!Ajroud3Driss! 2011).! Familial! ALS! is! inherited! in! either! an! autosomal! dominant! or!autosomal!recessive!fashion!(Siddique!and!Ajroud3Driss!2011).!A!number!of!genetic!! 6!mutations! have! been! found! to! be! associated!with! fALS.!Mutations! in!SOD1,!which!encodes! for! the! enzyme! Superoxide! dismutase! (Rosen,! Siddique! et! al.! 1993),! and!FUS,! a! gene! encoding! for! RNA3binding! protein! Fused! in! Sarcoma! (FUS)! (Vance,!Rogelj!et!al.!2009),!have!both!been!observed!in!patients!presenting!with!fALS.!!A! major! pathological! characteristic! of! ALS! is! the! presence! of! ubiquitinated!cytoplasmic! inclusions! in! afflicted! cells;! most! commonly,! spinal! motor! neurons!(Siddique! and! Ajroud3Driss! 2011;! Deng,! Zhai! et! al.! 2010).! Recently,! it! was!discovered!that!the!major!protein!component!of!inclusions!seen!in!non3SOD1!ALS!is!TDP343,!which!is!found!to!colocalize!with!FUS!and!the!ubiquitin3binding!protein!p62!in! the! cytoplasm!of! diseased! cells! (Sreedharan,! Blair! et! al.! 2008;!Deng,! Zhai! et! al.!2010).!Mutations!in!the!TARDBP!gene!have!been!observed!in!cases!of!both!sALS!and!fALS,! and! studies! suggest! that!TARDBP!mutations!may! be! present! in! up! to! 4%!of!fALS!cases!(Sreedharan,!Blair!et!al.!2008;!Deng,!Zhai!et!al.!2010).!While! ALS! and! FTLD! have! historically! been! regarded! as! separate,! distinct!pathologies,!it!is!becoming!clear!that!these!diseases!may!share!similar!etiologies.!A!2003!review!on!motor!neuron!disease!(MND)!reported!that!as!many!as!20340%!of!patients! suffering! from! ALS! exhibit! cognitive! impairments! such! as! those! seen! in!FTLD,! and! approximately! 5%! of! MND! patients! are! diagnosed! as! having! motor!neuron! disease! with! dementia! (MND3D),! or! FTLD3MND! (Leigh,! Abrahams! et! al.!2003).! Interestingly,! cognitive! impairments! associated! with! FTLD3MND! are! most!commonly! observed! in! patients! with! marked! bulbar! symptoms,! although! these!! 7!impairments! have! been! reported! in! patients! exhibiting! non3bulbar! ALS! as! well!(Leigh,!Abrahams!et!al.!2003).!Currently,! there! are! not!many! options! available! for! the! treatment! of!ALS.! The!majority! of! post3diagnosis! support! focuses! on! palliative! care! measures,! such! as!providing!non3invasive!ventilation!and!supplying!the!patient!with!specially!modified!foodstuffs! designed! for! easy! ingestion! (Pratt,! Getzoff! et! al.! 2012).!While! palliative!care,!in!some!cases,!has!been!shown!to!improve!the!quality!of!life!and!the!prognosis!of! the! patient,! there! is! a! severe! lack! of! pharmacological! options! targeting! the!progression! of! ALS! (Pratt,! Getzoff! et! al.! 2012).! A! greater! understanding! of! the!pathophysiology!of!this!disease!is!necessary!for!the!development!of!more!effective!therapeutics.!#1.4 #TDP643#and#FTLD#Frontotemporal! lobar! degeneration! (FTLD)! is! one! of! the! leading! causes! of!dementia,! accounting! for! approximately! 5%310%!of! recorded! cases! (Rademakers,!Baker!et!al.!2007).!With!an!age!of!onset!of!as!early!as!30!years!of!age!(Rabinovici!and!Miller!2010),!it!represents!approximately!10%320%!of!all!dementia!cases!occurring!before! the! age! of! 65! years! (Rademakers,! Baker! et! al.! 2007).! Interestingly,! there!appears! to! be! a! strong! familial! component! associated! with! the! risk! of! acquiring!FTLD:!it!has!been!reported!that!nearly!half!of!all!FTLD!patients!have!a!family!history!of!a!resemblant!dementia!(Rademakers,!Baker!et!al.!2007).!!! 8!FTLD!consists!of!three!distinct!clinical!variants,!which!are!defined!by!their!initial!and! predominant! symptoms! (Rabinovici! and! Miller! 2010).! Behavioural3variant!frontotemporal! dementia! (bvFTD)! is! characterized! by! significant! changes! of!personality! and! behaviour! in! patients,! as!well! as! the! progressive! development! of!apathy! and! disinhibition! (Rabinovici! and! Miller! 2010).! While! the! behavioural!changes!of!these!patients!are!substantial,!this!variant!of!FTLD!has!less!of!an!impact!on! overall! cognition! (Rabinovici! and! Miller! 2010).! Notably,! while! ALS! has! been!found!to!co3occur!with!any!of!the!FTLD!variants,!it!is!most!commonly!coupled!with!bvFTD!(Rabinovici!and!Miller!2010).!Semantic!dementia!(SD)!is!another!variant!of!FTLD.!Patients!suffering!from!SD!initially! present! with! fluent,! anomic! aphasia,! which,! over! time,! may! progress! to!multimodal! agnosia! (Rabinovici! and! Miller! 2010).! Semantic! dementia,! in! some!cases,!is!also!characterized!by!marked!behavioural!changes!that!overlap!with!those!seen!in!bvFTD!patients!(Rabinovici!and!Miller!2010).!Patients!exhibiting!SD!tend!to!perform!poorly!during!tests!on!semantic!memory,!such!as!confrontation!naming!and!category! fluency! tests,! whereas! their! spatial! abilities,! executive! functions! and!episodic!memory!remain!intact!(Rabinovici!and!Miller!2010).!The! third! variant! of! FTLD! is! known! as! progressive! nonfluent! aphasia! (PNFA),!which!is!characterized!by!the!gradual!deterioration!of!motor!speech!and!expression!of! language! (Rabinovici! and! Miller! 2010).! PNFA! is! frequently! associated! with!corticobasal!degeneration!(CBD)!and!progressive!supranuclear!palsy!(PSP);!patients!! 9!with!PNFA!tend!to!exhibit!Parkinsonism!and!limb!apraxia,!as!well!as!supranuclear!gaze!palsies!(Rabinovici!and!Miller!2010).!!A! major! component! of! FTLD! pathology! is! the! presence! of! inclusion! bodies!within!cells!located!in!the!frontal!and!temporal!lobes!of!afflicted!patients.!There!are!two! distinct! types! of! inclusion! bodies! observed,! which! are! defined! by! their!immunoreactivities:! tau3positive! inclusions! and! tau3negative,! ubiquitin3positive!inclusions! (Rabinovici! and! Miller! 2010).! The! majority! of! FTLD! cases! can! be!subclassified! as! either! tau3positive! FTLD! (FTLD3Tau)! or! ubiquitin3positive! FTLD!(FTLD3U)!based!on!these! inclusions,!with!FTLD3U!presenting!as!the!most!common!clinical!form!of!FTLD!(Dickson,!Kouri!et!al.!2011).!Tau3positive!FTLD!is!characterized!by!the!presence!of!tau3positive!inclusions!in!the! cytoplasm! of! neurons! and! glia.! Examples! of! FTLD3related! tauopathies! include!Pick?s!disease,!CBD,!PSP!and!argyrophilic!grain!disease!(Rabinovici!and!Miller!2010).!The! majority! of! familial! FTLD3Tau! patients,! as! well! as! some! sporadic! FTLD3Tau!patients,! have! been! found! to! harbor! mutations! in! the! microtubule3associated!protein! tau! (MAPT)! gene,!which!encodes! for! the! tau!protein! (Dickson,!Kouri! et! al.!2011).!#While!FTLD3Tau!represents!a!large!proportion!of!cases!of!FTLD,!there!are!many!patients! suffering! from! FTLD! that! do! not! exhibit! tau! immunoreactivity.! Once!immunohistochemical! techniques! became! more! refined,! it! was! discovered! that! a!majority! of! tau3negative! FTLD! cases! exhibit! staining! for! ubiquitin3positive!! 10!inclusions;! this! subcategory! of! FTLD! was! termed! FTLD3U! (Rabinovici! and! Miller!2010).!!In!2006,!Neumann!et! al.! discovered! that,! for!most! cases! of! FTLD3U,! the!major!protein! component! of! the! observed! ubiquitinated! inclusions! is!hyperphosphorylated,! truncated! TDP343.! Unlike! in! ALS,! where! ubiquitin3positive!inclusions! are! found! primarily! in! spinal! motor! neurons,! the! neuronal! inclusions!observed! in! FTLD3U! are! characteristically! observed! in! both! frontal! and! temporal!cortices,!as!well!as!in!the!hippocampus!(Baker,!Mackenzie!et!al.!2006).!As! frontotemporal! dementia! exhibits! a! strong! familial! inheritance!pattern,! the!pathogenesis!of!this!disease!is!believed!to!contain!a!genetic!component.!In!2006,!a!study!by!Baker!et!al.!revealed!an!association!between!FTLD3U!and!mutations!in!GRN,!a!gene!encoding!for!the!protein!progranulin!(PGRN).!Further!epidemiologic!studies!have!revealed!that!mutations!in!GRN!may!account!for!5%311%!of!all!cases!of!FTD,!and!up!to!11%326%!of!cases!of!familial!FTD!(Mackenzie!2007).!Progranulin,! also! known! as! proepithelin,! is! a! heavily! glycosylated,! 5933amino!acid! protein! involved! in! a! wide! variety! of! physiological! processes,! such! as!embryonic!development,!inflammation!and!wound!healing!(Ahmed,!Mackenzie!et!al.!2007).!PGRN!has!an!estimated!molecular!weight!of!68.5!kDa;!due!to!glycosylation,!however,! its!weight! is! observed! to! be! closer! to! 90! kDa! (Ahmed,!Mackenzie! et! al.!2007).! Progranulin! consists! of! seven! granulin3like! domains! and! is! cleaved! by!extracellular! proteases,! resulting! in! the! generation! of! small! peptides! known! as!granulins! (GRNs).! These! GRNs! have! been! shown! to! mediate! numerous! biological!! 11!functions! such! as! cell! cycle! progression,! tumorigenesis! and! inflammation! (He! and!Bateman!2003).!While!the!function!of!PGRN!in!the!periphery!has!been!the!subject!of!numerous!studies,!there!is!little!known!about!the!role!of!PGRN!in!the!central!nervous!system.!In!a! recent! study,!Tang!et!al.! (2011)!discovered! that!progranulin!binds!directly! to!TNF3receptors!(TNFR)!in!both!mouse!and!human!cells.!They!found!that!PGRN!acts!to! reduce! inflammatory! signaling! by! blocking! the! interaction! between! TNF3?! and!TNFRs,!leading!to!increased!cell!viability!during!periods!of!inflammation!(Tang,!Lu!et!al.!2011).!!PGRN!mutations! linked! to! FTLD3U! have! been! shown! to! lead! to! the! nonsense3mediated!decay!(NMD)!of!mutant!transcripts,!resulting!in!PGRN!haploinsufficiency!(Baker,! Mackenzie! et! al.! 2006).! It! follows! that! PGRN! haploinsufficiency,! and! the!resulting!disinhibition!of!neuronal!TNF3?!signaling,!may!lead!to!decreased!neuronal!survivability!over! time.!By!utilizing!RNAi! technology,!Guo!et!al.! (2010)!discovered!that! PGRN! haploinsufficiency,! induced! in! mouse! primary! cortical! neurons,! was!sufficient! to! induce! cytoplasmic! mislocalization! of! TDP343,! increase! cellular!vulnerability!to!H2O2!and!NDMA!insults!and!enhance!caspase33!activation.!!While! it! is! clear! that! cytoplasmic! mislocalization! of! TDP343! and! the!pathogenesis! of! clinical! FTLD3U! are! strongly! linked,! the! mechanisms! behind! the!cytoplasmic!sequestration!of!TDP343!are!still!unknown.!Recent!studies!suggest!that!an! impairment! of! the! nuclear! transport! system,! responsible! for! returning!cytoplasmic!TDP343!to!the!nucleus,!may!be!involved!in!the!development!of!TDP343!! 12!proteinopathy! (Nishimura,! Zupunski! et! al.! 2010).! The! proteins! implicated! in! the!nuclear! transport! of! TDP343! are! known! as! karyopherins! (KPNs)! (Nishimura,!Zupunski!et!al.!2010).!1.5 #Karyopherins#The! transportation! of! a! protein! from! the! cytoplasm! to! the! nucleus! is! a! highly!selective,!regulated!process.!There!are!a!number!of!different!pathways! involved!in!the!nucleocytoplasmic!transport!of!proteins!within!a!cell,!the!most!well!understood!being! classical! nuclear! localization.! Classical! nuclear! localization! utilizes!monopartite! and! bipartite! classical! nuclear! localization! signals! (cNLS)! and! is!mediated!by!a!superfamily!of!proteins!known!as!karyopherins!(Conti,!Muller!et!al.!2006).!The!label!karyopherin!encompasses!a!family!of!conserved!proteins!comprised!of!both!importins!and!exportins,!proteins!involved!in!shuttling!target!proteins!to!and!from!the!nucleus,!respectively!(Goldfarb,!Corbett!et!al.!2004).!There!are!a!number!of!proteins! involved! in! classical! nucleocytoplasmic! shuttling,! primarily! the!karyopherin3?s!(KPNA)!and!karyopherin3?s!(KPNB)!(Goldfarb,!Corbett!et!al.!2004).!The! karyopherin3?! family! consists! of! three! distinct! subfamilies:! Importin! ?P!(Imp?P),! Importin! ?Q! (Imp?Q),! and! Importin! ?S! (Imp?S)! (Yasuhara,! Oka! et! al.!2009).!Each!KPNA!is!encoded!by!a!distinct!gene!in!mammals!(Tejomurtula,!Lee!et!al.!2009).!To!date,!there!have!been!seven!KPNA3encoding!genes!discovered!in!humans,!and!six!found!in!mice!(Tejomurtula,!Lee!et!al.!2009).!The!Imp?P!subfamily!consists!! 13!of!karyopherin3?2!(KPNA2)!and!the!recently!discovered!karyopherin3?7!(KPNA7),!the! Imp?Q! subfamily! consists! of! karyopherin3?3! (KPNA3),! karyopherin3?4!(KPNA4)! and! karyopherin3?1! (KPNA1),! and! the! subfamily! Imp?S! consists! of!karyopherin3?5!(KPNA5)!and!karyopherin3?6!(KPNA6)!(Yasuhara,!Oka!et!al.!2009;!Tejomurtula,! Lee! et! al.! 2009).!While! the!majority! of! human! karyopherins! exist! as!mouse!orthologues,!the!presence!of!a!human!KPNA5!orthologue!has!not!been!found!to!be!expressed!in!mice!(Tejomurtula,!Lee!et!al.!2009).!Although!each!karyopherin3?! is!distinct,! there!exists!a!great!deal!of!homology!between!members!of! this!protein! family.!Karyopherin3?! isoforms!within! the!same!subfamily! exhibit! roughly! 85%! sequence! homology,! while! KPNA! proteins! within!different!subfamilies!exhibit!approximately!50%!sequence!homology!(Tejomurtula,!Lee!et!al.!2009).!All!known!KPNA!proteins!contain!the!same!structural!motifs.!The!two!major!motifs!seen!are!the!importin3?!binding!(IBB)!domain,!located!near!the!N3terminal,! and! multiple! repeated! armadillo! (ARM)! motifs! (Tejomurtula,! Lee! et! al.!2009).! The! ARM!motifs! have! been! shown! to! form! an! anionic! NLS3binding! pocket!(Tejomurtula,!Lee!et!al.!2009;!Yasuhara,! Shibazaki!et! al.!2007).!Notably,!when!not!bound! to!KPNB,! the! IBB! domain! of! KPNA! is! bound! to! its! own!ARM!domain! in! an!autoinhibitory!fashion!(Chook!and!Blobel!2001).!!Karyopherin! expression! is! highly! regulated,! and! the! expression! levels! of!individual!KPNA! isoforms!have! been! shown! to! vary! between!both! the! tissue! type!and! the! stage! of! development! of! the! organism! (Kohler,! Ansieau! et! al.! 1997;!Yasuhara,!Shibazaki!et!al.!2007).!Karyopherin3?!proteins!are!ubiquitously!expressed!! 14!and,! while! each! KPNA! recognizes! both! classical! bipartite! and! monopartite! NLSs,!each! exhibits! distinct! affinities! and! transport! efficiencies! for! specific! proteins!(Kohler,! Speck! et! al.! 1999).! The! variety! of! KPNA! proteins! involved! in! nuclear!transport! allows! for! redundancy!within! the! system.! A! number! of! studies! suggest!that! some! proteins! may! be! targeted! to! the! nucleus! by! more! than! one! KPNA!(Nishimura,!Zupunski!et!al.!2010;!Ma!and!Cao!2006).!Karyopherin3?1!(KPNB1)! is!a!member!of! the!karyopherin3?! family,!a! family!of!proteins!involved!in!both!classical!and!non3classical!nuclear!localization!(Chook!and!Blobel! 2001).! KPNB1! has! been! shown! to! directly! interact! with! nucleoporins,! a!family!of!proteins!that!make!up!nuclear!pore!complexes!(NPC)!(Kobe!1999).!NPCs!are!large,!multimeric!protein!channels!that!allow!the!passive!movement!of!ions!and!small! molecules! across! the! nuclear! envelope! while! restricting! the! exchange! of!macromolecules,!such!as!proteins!over!20!kDa!(Kobe!1999).!Macromolecules!must!contain!the!appropriate!signals,!such!as!a!cNLS,! in!order!to!cross!through!the!NPC!into!the!nucleus!(Kobe!1999).!While! some!KPNBs!have!been!shown! to!bind!directly! to!non3classical!NLSs,! in!the! case! of! classical! nuclear! localization! KPNB1! binds! to! the! IBB! domain! of! the!target!KPNA!within! the! cytoplasm.!This! binding! results! in! the! disinhibition! of! the!KPNA!ARM!domain,!allowing!the!KPNA!to!bind!the!cNLS!of!the!target!protein!(Kobe!1999).!!!! 15!1.6 #Classical#Nuclear#Import#Cycle#The!nuclear!import!of!a!protein!begins!in!the!cytoplasm!of!the!cell!and!utilizes!the! RanGTP3RanGDP! nucleocytoplasmic! gradient! to! drive! the! process! (Kutay,!Bischoff! et! al.! 1997).! First,! KPNB1! binds! to! the! IBB! region! of! KPNA! within! the!cytoplasm,!disinhibiting! the!KPNA!ARM!domain!and! increasing! its!binding!affinity!for! cNLSs! (Kobe! 1999).! After! KPNA! binds! the! cNLS! of! the! protein! destined! for!nuclear! import,! the! resulting! trimeric! complex! is! shuttled! to! the!nuclear! envelope!where!KPNB1!mediates! the! translocation!of! the!protein!complex! through! the!NPC!(Lange,!Mills! et! al.! 2007).! Once! inside! the! nucleus,! KPNB1! binds! to! RanGTP.! This!binding!induces!a!conformational!change,!causing!KPNB1!to!release!the!IBB!domain!of!KPNA! (Lee,!Matsuura!et! al.! 2005).! !The! IBB!domain,!now! free! to!bind! the!ARM!domain,! initiates! the! release!of! the!cargo!protein! from!KPNA!(Lee,!Matsuura!et!al.!2005).!!In! order! for! a! cell! to! continue! to! shuttle! proteins! into! the! nucleus,! the!karyopherins! involved! must! first! be! recycled! back! to! the! cytoplasm.! Kutay! et! al.!(1997)! found! that,! after! the! dissociation! of! the! trimeric! nuclear! import! complex,!nuclear! KPNA! rapidly! associates! with! a! protein! dimer! consisting! of! cellular!apoptosis! susceptibility! protein! (CAS)! and! RanGTP.! The! binding! of! KPNA! and!RanGTP!to!CAS!was!observed!to!be!highly!cooperative,!and!CAS!was!found!to!have!a!higher! affinity! for! NLS3free! KPNA! as! opposed! to! NLS3occupied! KPNA! (Kutay,!Bischoff!et!al.!1997)!!!! 16!Cellular!apoptosis!susceptibility!protein!is!a!nuclear3export!factor!that!interacts!with!the!nuclear!pore!complexes,!allowing!it!to!cross!the!nuclear!membrane!without!the!need!for!other!soluble!factors!(Kutay,!Bischoff!et!al.!1997).!Once!the!KPNA3CAS3RanGTP! trimer! leaves! the! nucleus! via! the! NPC,! cytoplasmic! proteins! Ran3specific!binding!protein!1!(RanBP1)!and!Ran!GTPase3activating!protein!(RanGAP1)!catalyze!the!hydrolysis!of!Ran3GTP!to!Ran3GDP!(Kutay,!Bischoff!et!al.!1997).!As!CAS!has!very!low!affinity!for!Ran3GDP,!the!cytosolic!hydrolysis!of!Ran3GTP!causes!the!dissociation!of! the!trimeric!complex! into!the! individual!protein!components!(Kutay,!Bischoff!et!al.!1997).!The!newly!released,!cytoplasmic!KPNA!is!free!to!bind!KPNB1!and!the!NLS!of!cargo!proteins,!reinitiating!the!classical!nuclear!import!cycle.!1.7 #KPNA#and#TDP643##In! 2010,! Nishimura! et! al.! utilized! a! SMARTpool! human! small! interfering! RNA!library,!targeted!against!82!known!nuclear!transport!proteins,!in!order!to!determine!which!proteins!were! involved! in!shuttling!TDP343!to! the!nucleus!of!SH3SY5Y!cells.!They! found! that! knocking! down! either! CAS! or! KPNB1! in! SH3SY5Y! cells! led! to! the!cytoplasmic! mislocalization! and! aggregation! of! TDP343.! Similar! findings! were!reported! utilizing! mouse! neuroblastoma! N2a! cells! and! mouse! primary! cortical!neurons!(Nishimura,!Zupunski!et!al.!2010).!!Nishimura! et! al.! (2010)!went! on! to! investigate! the! effects! of! CAS! and! KPNB1!knockdowns! on! the! subcellular! distribution! of! Karyopherin3?s.! They! discovered!that!the!knockdown!of!CAS!results!in!the!accumulation!of!KPNAs!within!the!nucleus!! 17!while,!conversely,!the!knockdown!of!KPNB1!leads!to!the!cytoplasmic!sequestration!of!KPNAs!(Nishimura,!Zupunski!et!al.!2010).!!In! order! to! determine! which! KPNA! isoforms! are! involved! in! the! subcellular!transport!of!TDP343,!Nishimura!et!al.!(2010)!performed!a!glutathione!S3transferase3pull3down!on!SH3SY5Y!cell!lysate,!utilizing!GST3karyopherin3?!fusion!proteins.!They!found! that! endogenous! TDP343! was! able! to! bind! to! the! five! KPNA! isoforms!examined:!KPNA1,!KPNA2,!KPNA3,!KPNA4,!and!KPNA6.!This!suggested!that!multiple!KPNA! isoforms!may!be! involved! in! the!nuclear! import!of!TDP343.!Nishimura!et!al.!went!on!to!discover!that!GST3KPNA!fusion!proteins!do!not!bind!to!TDP343?NLS,!a!modified! form! of! TDP343! lacking! an! NLS;! this! demonstrated! that! the! association!between!KPNA!and!TDP343!requires!a!cNLS!(Nishimura,!Zupunski!et!al.!2010).!Nishimura! et! al.! (2010)! went! on! to! analyze! the! expression! levels! of! nuclear!transport! factors! in! both! the! affected! cortical! regions! of! FTLD3U! patients! and! the!spinal! cords! of! ALS! patients.! Interestingly,! they! found! that! there! was! a! dramatic!reduction! of! CAS! and! KPNA2! expression! in! the!medial! temporal! lobes! of! FTLD3U!patients.!In!contrast,!they!found!no!significant!difference!in!the!expression!of!KPNB,!KPNA1!or!KPNA6!when!compared!to!healthy!controls.!Upon!analysis!of!spinal!cord!lysates! derived! from! ALS! patients,! the! team! found! that! there! was! an! increase! in!KPNA2!expression,! a!decrease! in!KPNA6!expression,! and!no!observable! change! in!KPNB!and!KPNA1!expression.!CAS!expression!was!also!unchanged.!Through!the!use!of! immunohistochemistry,! they! found! that! cytoplasmic! KPNA2! staining! was!! 18!significantly!higher! in! the!spinal! cords!of!ALS!patients! (Nishimura,!Zupunski!et!al.!2010).!!1.8 #Specific#Aims#While! the! findings! by! Nishimura! et! al.! (2010)! suggest! that! disruption! of! the!nucleocytoplasmic!transport!system!may!be!involved!in!the!pathogenesis!of!TDP343!proteinopathies,!any!cause!and!effect!relationship!remains!unclear.!As!pathological!TDP343! is! observed! in! a! number! of! devastating! neurological! diseases,! an!understanding! of! the! mechanisms! underlying! its! nuclear! import! is! of! great!importance.!Aim! 1:! Identify! the! endogenous! KPNA! isoforms! that! interact! with! TDP343! in! SH3SY5Y!cells!!Aim!2:!Utilizing!a!high3density!peptide!array,!determine!the!regions!of!TDP343!that!interact!with!KPNA!isoforms!expressed!in!SH3SY5Y!cells!and,!from!these!sequences,!develop!small!interfering!peptides!that!are!able!to!specifically!block!the!interaction!between!relevant!KPNA!isoforms!and!TDP343!Aim!3:!Utilizing!our!putative!interference!peptides,!determine!whether!blocking!the!interaction! between! TDP343! and! KPNA! is! sufficient! to! cause! cytoplasmic!mislocalization!and!aggregation!of!TDP343!in!SH3SY5Y!cells!!!! 19!#2:# Peptide6Mediated# Inhibition# of# TDP643# Nuclear#Targeting#2.1#Introduction#! The!karyopherin3?!isotypes!involved!in!classical!nuclear!import!within!a!cell!depends! upon! both! the! cell! type! and! the! developmental! stage! of! the! cell! (Kohler,!Ansieau!et!al.!1997;!Yasuhara,!Shibazaki!et!al.!2007).!While!Nishimura!et!al.!(2010)!determined! that! endogenous! TDP343,! derived! from! SH3SY5Y! cell! lysate,! is! able! to!bind! to! recombinant! GST3tagged! KPNA1,! KPNA2,! KPNA3,! KPNA4! and! KPNA6,! in!order!to!fully!investigate!the!mechanisms!underlying!the!nuclear!import!of!TDP343!the!identities!of!the!endogenous!KPNA!isotypes!involved!must!be!elucidated.!Furthermore,!in!order!to!explore!a!cause!and!effect!relationship!between!the!nuclear! import! of! TDP343! and! the! pathogenesis! of! TDP343! proteinopathy,! it! is!essential!that!one!is!able!to!disrupt!the!nuclear!import!of!TDP343!in!such!a!way!that!reduces! the! risk! of! compensatory! cellular!mechanisms! taking! over.! Utilizing! RNA!interference,!Nishimura!et!al.! (2010)!showed!that! the!knockdown!of!expression!of!individual! KPNA! isoforms! was! not! sufficient! to! induce! TDP343! cytoplasmic!mislocalization.! These! results! suggest! that! redundancies! within! the! karyopherin!family!allow!for!nuclear!import!to!continue!in!the!absence!of!a!single!KPNA!isoform,!but! these! redundancies! may! be! a! result! of! compensatory! changes! in! KPNA!expression! rather! than! a! normal! cellular! mechanism;! RNA! interference! has! been!! 20!shown!to!induce!compensatory!changes!in!cellular!physiology!(Villarroya,!Lara!et!al.!2011).!!To! investigate! the! effect! of! inhibiting! the! nuclear! import! of! TDP343,! while!minimizing! the! potential! for! cellular! compensation,! we! set! out! to! develop! small!interference! peptides,! comprised! of! short! stretches! of! amino! acids! derived! from!TDP343,! that! could! be! utilized! to! rapidly! and! specifically! block! the! interaction!between!TDP343!and!relevant!karyopherin3?!isoforms.!!!First,! we! performed! Western! blotting! on! cell! lysate! to! determine! the!identities!of! the!KPNA! isoforms!expressed! in!SH3SY5Y!cells.!We! then!performed!a!co3immunoprecipitation! on! SH3SY5Y! cell! lysate,! utilizing! endogenous! KPNA!isoforms!as!bait,! in!order!to!elucidate!which!isoforms!were!able!to!bind!TDP343! in1vitro.!!Once!we!determined!the!KPNA! isoform!involved! in!TDP343!nuclear! import,!we!utilized!a!high3density!peptide!array!to!identify!putative!regions!of!TDP343!that!were! able! to! directly! bind! to! the! recombinant! KPNA! isoform.! Our! peptide! array!consisted!of!a!series!of!overlapping,!sequential!12314mer!peptides!with!a!frameshift!of!two!amino!acids,!which,!all!together,!encompassed!the!entire!primary!sequence!of!human! TDP343.! From! these! KPNA3binding! regions! of! TDP343! we! designed! six!putative! interference! peptides,! ranging! from! 8311mer,! and! tested! their! blocking!efficacy! in1 vitro.! After! establishing! our! peptides! as! a! useful! tool! for! blocking! the!binding! of! TDP343! and! its! nuclear! import! protein,! we! went! on! to! investigate!! 21!whether! the! inhibition! of! this! interaction! is! sufficient! to! induce! TDP343!proteinopathy!in1vitro.!!!!2.2#Materials#and#Methods#2.2.1#Cell#Culture#! A! human! neuroblastoma! cell! line,! SH3SY5Y,! was! procured! from! American!Type! Culture! Collection! (Manassas,! VA).! The! cell! line! was! maintained! as! per! the!guidelines! of! American! Type! Culture! Collection,! and! cells! were! cultured! in!Dulbecco?s! modified! Eagle?s! medium! (DMEM:! Sigma! Chemical! Co.,! St.! Louis,! MO)!supplemented!with!10%!fetal!bovine!serum!(Gibco3BRL,!Grand!Island,!NY)!and!1%!streptomycin/penicillin!(Gibco3BRL,!Grand!Island,!NY).!Cell!cultures!were!cultivated!in! a! humidified! incubator! (NuAir,! Plymouth,! MN)! with! a! 5%! CO2! environment! at!37oC.!2.2.2#High6Density#Peptide#Array#Synthesis##Blank!cellulose!membranes!were!acquired!from!Intavis!AG!(K?ln,!Germany).!High3density!peptide!arrays!were!synthesized!by!the!UBC!Peptide!Synthesis!facility,!utilizing! cellulose!membranes!as!per!Hilpert,!Winkler! et! al.! (2007).!Peptides!were!synthesized!from!C3terminal!to!N3terminal,!with!the!C3terminal!end!anchored!to!the!membrane!via!an!ester!bond.!First,!pentafluorophenyl!ester!(PFP)!activated!amino!acids!were!loaded!on!their!appropriate!membrane!coordinate,!at!0.11uL!of!a!single!amino! acid! per! spot.! The! unused! portions! of! the! membranes! were! blocked! via!acetylation.! Fluorenylmethyloxycarbonyl! (Fmoc)! protection! groups!were! removed!! 22!by!four!short,!successive!washes!with!dimethylformamide!(DMF),! followed!by!two!five3minute!washes!with!a!solution!of!20%!piperidine!in!DMF,!four!more!quick!DMF!washes! and! two!methanol! washes.! Free! amino! groups!were! stained! using! 0.02%!bromophenol! blue! (BPB)! in!methanol.!Membranes!were! then! subjected! to! a! final!methanol!wash!and!subsequently!air3dried,!after!which!they!were!ready!for!the!next!cycle! of! amino! acid! coupling.! Once! the! final! amino! acids! were! coupled! to! the!membrane3bound! peptides,! side3chain! protecting! groups! were! removed! via!treatment!with!trifluoroacetic!acid!(TFA)!in!a!solution!of!3%!triisopropylsilane,!2%!water!and!1%!phenol.!A!finished!membrane!represented!the!full3length!sequence!of!human! TDP343! and! was! composed! of! multiple! spots.! Each! spot! consisted! of!sequential,! overlapping!peptides!of! either!123mer!or!143mer!with! a! frame! shift! of!two!amino!acids!between!neighboring!spots.!2.2.3#Protein6Protein#Interaction#Assay### The!TDP343!high3density! peptide!membrane!was! activated! by! a! 103minute!ethanol! wash! with! light! rocking.! Membrane! was! washed! three! times! in! TBS3T!(50mM! Tris3base,! 27mM! KCl,! 136mM! NaCl,! 0.2%! Tween320,! pH! 8.0)! for! five!minutes,! and! blocked! at! room! temperature! for! 3! hours! in! blocking! buffer! (5%!sucrose,!4%!non3fat!milk,!TBS3T).!Membrane!was!then!washed!once!in!TBS3T!for!5!minutes! and! probed! with! primary! protein! solution! consisting! of! either! blank!blocking! buffer! (for! negative! control)! or! recombinant! human! KPNA2! protein!(5ug/mL,!Abcam,!Toronto,!ON)! in!blocking!buffer,! overnight! at!4oC.!The! following!day,!membrane!was!washed! three! times! for!20!minutes! in!TBS3T.!Membrane!was!! 23!then! incubated! overnight! at! 4oC! in! Goat! polyclonal! antibody! against! KPNA2!(Abnova,! Taipei,! Taiwan)! diluted! in! blocking! buffer! (0.25ug/mL).! The! peptide!membrane!was!washed!at!room!temperature!three!times!for!20!minutes!in!TBS3T,!and! immediately! incubated! in! donkey! anti3goat! HRP3labeled! secondary! antibody!(1:2000,!Santa!Cruz!Biotechnology,!Dallas,!TX)!in!blocking!solution!for!two!hours!at!room! temperature.! After! three! more! 203minute! washes! in! TBS3T,! the! membrane!was! gently! blotted! dry! with! tissue! and! developed! using! Western! Lightning3ECL!Enhanced! Chemiluminescence! Substrate! (PerkinElmer,! Waltham,! MA).! After!visualization,! the! membrane! was! washed! for! five! minutes! with! TBS3T! and! twice!briefly! with! deionized! water.! If! membrane! was! probed! with! negative! control,!regeneration! procedure! was! performed! (as! per! 2.2.4)! and! above! procedure! was!repeated,!without!initial!ethanol!activation,!using!recombinant!KPNA2.!Membranes!were! unable! to! be! regenerated! for! further! use! after! performing! KPNA2! protein!probe,!so!three!separate!membranes!were!utilized!during!experimentation.!!!2.2.4#Peptide#Array#Regeneration#After!visualization,!peptide!membrane!was!washed!three!times!for!5!minutes!with! TBS3T! followed! by! one! brief! wash! with! deionized! water.! Membrane! was!incubated!in!fresh!Regeneration!Buffer!A!(8M!Urea,!1%!SDS,!0.1%!?3ME,!deionized!water)!overnight! at!37oC.!The! following!day,!membrane!was!briefly!washed! twice!with! deionized! water! and! treated! with! Regeneration! Buffer! B! (10%! Acetic! Acid,!40%! deionized! water,! 50%! ethanol)! for! 90! minutes! at! room! temperature,! with!! 24!gentle!shaking.!Membrane!was!then!rinsed!twice!with!deionized!water!and!washed!three!times!for!five!minutes!with!TBS3T.!!!2.2.5#Peptide#Synthesis## Seven!short!cell3penetrating!peptides!were!synthesized!by!GL!Biochem!Ltd.!(Shanghai,! China)! and! refined! to! greater! than! 90%! purity! by! HPLC.! Purity! was!verified!by!mass!spectrometry.!Six!putative!blocking!peptides!(p13p6),!derived!from!segments!of!TDP343,!and!one!scrambled!control!peptide!(pS)!were!developed!with!a!cell!penetrating,!truncated!N3terminal!TAT!domain.!The!scrambled!control!peptide!contains!the!same!amino!acid!composition!and!charge!density!as!p1.!The!peptides!were!dissolved!in!distilled!water!and!stored!at!380oC.!!2.2.6#Peptide#Treatments## Peptides! were! combined! and! diluted! to! appropriate! composition! and!concentration!in!warm!DMEM,!with!10%!FBS!and!1%!antibiotics.!The!media!of!near3confluent,! 100mm! plates! of! SH3SY5Y! cells! was! replaced! with! peptide3rich!media,!and!plates!were!placed!back!in!incubator.!Cells!were!allowed!to!grow!for!either!four!hours! (Co3immunoprecipitation)! or! 24! hours! (Fractionation! and!Immunocytochemistry)!before!harvesting!for!experimentation.!2.2.7#Protein#Extraction#for#Western#Blotting!# Confluent!63well!plates!of!SH3SY5Y!cells!were!washed!two!times,!on!ice,!with!cold!PBS!and!lysed!with!100uL!of!2x!sample!buffer!(62.5mM!Tris3HCl!at!pH!6.8,!25%!glycerol,! 2%! sodium! dodecyl! sulfate,! 0.01%! Bromophenol! blue,! 5%! ?3! 25!mercaptoethanol).!Lysate!was!collected,!boiled!at!100oC!for!5!minutes,!and!stored!at!380oC.!2.2.8#Co6Immunoprecipitation## Confluent! 100mm! plates! of! SH3SY5Y! cells! were! washed! two! times,! on! ice,!with!cold!PBS.!Cells!were!collected!in!1mL!of!cold!PBS!and!centrifuged!at!3000g!for!5!minutes!at!4oC.!The!supernatant!was!discarded,!and!cell!pellets!were!resuspended!in!1mL!of!iced!lysis!buffer!(0.5%!deoxycholate,!0.5%!Triton!X3100,!10uM!PMSF!and!protease! inhibitor! cocktail! tablet! in! PBS)! and! incubated! on! ice! for! 15! minutes.!Samples! were! then! centrifuged! at! 3000g! for! 5! minutes.! From! each,! 400uL! of!supernatant!was!collected!and!diluted!to!1mL!with!PBS!(supplemented!with!10uM!PMSF!and!protease! inhibitor!cocktail! tablet).!We!added!30uL! from!each!sample! to!10uL!of!4X!sample!buffer,!boiled!for!5!minutes!and!stored!at!320oC!to!serve!as!lysate!controls.! Samples! were! then! precleared! for! one! hour! at! 4oC! with! 10uL! of! a! 1:1!solution!of!Protein!A/G!Sepharose!beads!(GE!Healthcare,!Piscataway!Township,!NJ)!and! lysis!buffer.!Beads!were!pelleted!at!300g! for!30!seconds!and!supernatant!was!collected.!Samples!were!then!incubated!overnight!at!4oC!with!either!4ug!of!normal!goat! IgG! (Santa!Cruz!Biotech,!Dallas,!Texas),!4ug!of!normal! rabbit! IgG! (Santa!Cruz!Biotech,! Dallas,! Texas),! or! 4ug! of! primary! antibody.! Primary! antibodies! utilized:!anti3KPNA1,! anti3KPNA4,! anti3KPNA6! (Abcam,! Toronto,! ON)! and! anti3KPNA2!(Abnova,!Taipei,!Taiwan).!The!following!day,!50uL!of!a!1:1!solution!of!Protein!A/G!Sepharose!beads!and!lysis!buffer!were!added!to!each!sample.!Samples!were!rotated!gently! for! 4! hours! at! 4oC.! Beads! were! pelleted! via! centrifugation! at! 300g! for! 30!! 26!seconds,!supernatant!was!discarded,!and!beads!were!washed!with!1mL!of!cold!PBS.!Centrifugation!and!washes!were!repeated!three!more!times.!Beads!were!dried!using!a!fine!needle!and!syringe,!and!proteins!were!eluted!by!60uL!of!2x!sample!buffer!per!sample.!Samples!were!boiled!at!100oC!for!5!minutes,!and!stored!at!320oC.!!2.2.9#Western#Blot## Samples! were! boiled! at! 100oC! for! 5! minutes! and! briefly! centrifuged! at!13000g.! 30uL! of! each! sample! was! electrophoretically! resolved! on! a! 10%! SDS3polyacrylamide!gel!using!a!Bio3Rad!Gel!electrophoresis!system!(Bio3Rad,!Hercules,!CA)! at! 100V.! Samples! were! run! alongside! PageRulerTM! Plus! Prestained! Protein!Ladder! (Fermentas,! CA)! for!determination!of!molecular!weight.!Resolved!proteins!were! transferred! to! a! nitrocellulose!membrane! (Bio3Rad,! Hercules,! CA)! using! the!Bio3Rad!Wet!Transfer!system!(Bio3Rad,!Hercules,!CA)!at!100V!for!90!minutes!at!4oC.!Nitrocellulose!membranes!were!washed!with!TBST!three!times!before!blocking!for!one!hour!at!room!temperature!in!5%!non3fat!milk,!dissolved!in!TBST.!After!blocking,!membranes!were! briefly!washed! three! times! in! TBST! and! incubated! overnight! at!4oC!in!primary!antibody,!diluted!1:1000!in!a!solution!of!3%!bovine!serum!albumin!(BSA)!and!0.3%!sodium!azide!in!TBST.!Primary!antibodies!used:!anti3KPNA1,!anti3KPNA4,! anti3KPNA6! (1:1000,! Abcam,! Toronto,! Ontario),! anti3KPNA2! (1:1000,!Abnova,! Taipei,! Taiwan)! and! anti3TDP343! (1:1000,! Proteintech,! Chicago,! IL).!Membranes! were! subsequently! washed! three! times! in! TBST! and! incubated! in! an!appropriate! HRP3conjugated! secondary! antibody! at! a! 1:1000! dilution! in! 5%!BSA/TBST,!for!one!hour!at!room!temperature.!Secondary!antibodies!used:!anti3goat!! 27!(Santa!Cruz,!Dallas,!TX)!and!anti3rabbit!(PerkinElmer,!Waltham,!MA).#After!washing!membranes! 3!more! times!with! TBST,!membranes!were! developed! using!Western!Lightning3ECL! Enhanced! Chemiluminescence! Substrate! (PerkinElmer,! Waltham,!MA).! Protein! band! densities! were! analyzed! with! ImageJ! software! (NIH)! and!normalized!against!internal!actin!controls.!2.2.10#Subcellular#Fractionation### Confluent! 100mm! plates! of! SH3SY5Y! cells! were! placed! on! ice! and! washed!twice!with!5mL!of! iced!PBS.!Each!plate!was! incubated! in!1mL!of!Buffer!A! (10mM!HEPES3KOH,! 10mM! KCl,! 10mM! EDTA,! 0.4%! NP340,! 1.5mM! MgCl2,! 1mM! DTT,!Protease!inhibitor!cocktail! tablet,!at!pH!7.9)!for!20!minutes,!with!gentle!rocking!at!4oC.!Cells!were!gently!collected!into!microcentrifuge!tubes!and!centrifuged!at!300g!for!5!minutes,!at!4oC.!Nuclear!pellets!were!set!on!ice!and!supernatant!was!collected,!centrifuged! at! 17000g! to! clear! nuclear! debris,! and! transferred! to! clean! tubes! as!cytoplasmic! fractions.! Nuclear! pellets! were! resuspended! in! 1mL! of! Buffer! A,!vortexed! briefly,! and! centrifuged! at! 300g! for! 5! minutes! at! 4oC.! Supernatant! was!discarded.!Nuclear!pellets!were!resuspended!in!iced!PBS,!spun!down!at!300g!for!5!minutes!at!4!oC,!and!supernatant!was!again!discarded.!Pellets!were!then!subjected!to!150uL! of! Buffer! B! (20mM! HEPES3KOH,! 400mM! NaCl,! 1mM! EDTA,! 10%! glycerol,!1mM!DTT! and! protease! inhibitor! cocktail! tablet,! at! pH! 7.9)! on! ice! for! two! hours,!with! brief! vortexing! every! 30!minutes.! Nuclear! samples!were! then! centrifuged! at!17000g! for! 10!minutes! at! 4oC,! and! supernatant!was! transferred! to! clean! tubes! as!nuclear! fractions.! Both! cytoplasmic! and! nuclear! fractions!were! combined! in! a! 3:1!! 28!ratio! with! 4X! sample! buffer,! boiled! at! 100oC! for! 5! minutes,! and! stored! at! 320oC.!Purity!of!fractions!was!confirmed!by!Western!blotting!for!the!presence!of!Lamin3b1,!a!nuclear!envelope!protein!(Abcam,!Toronto,!ON)!and!heat!shock!protein!90!(HSP390),!a!cytoplasmic3specific!protein!(BD!Biosciences,!Mississauga,!ON)!2.2.11#Immunocytochemistry## SH3SY5Y! cells!were! grown! to! approximately! 60%! confluency! on! untreated!glass! coverslips! in! 123well! plates.! Coverslips!were! placed! directly! into! a! warmed!fixation! solution!of!4%!PFA!and!4%!sucrose! in!PBS.!Coverslips!were! incubated! in!fixation!solution! for!45!minutes!at!37oC,! then! immediately!washed!with!PBS!three!times!for!two!minutes.!Cells!were!permeabilized!in!.25%!TritonX3100!in!PBS!for!five!minutes! at! room! temperature,! and! briefly! washed! once! for! five! minutes! in! PBS.!Coverslips!were!blocked! in! a! solution!of! 10%!BSA! in!PBS! for!30!minutes! at! 37oC.!Blocking! solution! was! carefully! replaced! with! a! primary! antibody! solution!composed!of!3%!BSA!in!PBS!and!mouse!polyclonal!HSP390!(1:150,!BD!Biosciences,!Mississauga,! ON),! and! coverslips! were! left! to! incubate! in! a! sealed! container!overnight! at! 4oC.! The! following! day,! coverslips!were! briefly!washed! five! times! in!PBS.!Secondary!antibody!solution,!consisting!of!3%!BSA!in!PBS!and!Alexa!Fluor!488!goat! anti3mouse! fluorescent! antibody! (1:1000,! Invitrogen,! Burlington,! ON)! was!placed! on! coverslips! for! 45! minutes! at! 37oC.! After! five! brief! washes! in! PBS,!coverslips! were! probed! with! rabbit! polyclonal! antibody! against! TDP343! (1:150,!Proteintech,!Chicago,!IL)!in!3%!BSA/PBS!overnight!at!4oC.!Coverslips!were!washed!five! times! in! PBS,! then! incubated! in! Alexa! Fluor# 633! goat! anti3rabbit! antibody!! 29!(1:1000,! Invitrogen,! Burlington,! ON)! at! 37oC! for! 30! minutes.! After! washing! five!times! in!PBS,! coverslips!were!gently!patted!dry!and!mounted!on!glass! slides!with!Prolong! Gold! Anti3fade! Reagent! with! DAPI! (Invitrogen,! Burlington,! ON).! Samples!were! imaged! with! an! Olympus! Fluoview! FV1000! Confocal! scanning! microscope.!Mean! cytoplasmic! and! nuclear! TDP343! staining! was! quantified! using! ImageJ!software.!2.3#Results#2.3.1#Human#Neuroblastoma#SH6SY5Y#Cells#Express#KPNA2#and#KPNA4#! Our! Western! immunoblot! analysis! of! SH3SY5Y! whole! cell! lysate! revealed!significant! expression! of! two! karyopherin3?! isoforms,! KPNA2! and! KPNA4,! while!KPNA1!and!KPNA6!expression!was!minimal!(data!not!shown).!!2.3.2#Karyopherin6?2#Interacts#with#TDP643#in#SH6SY5Y#Cells## The!interaction!between!TDP343!and!endogenous!KPNA!isoforms!in!SH3SY5Y!cells!was! determined! by! utilizing! one! of! either!KPNA1,!KPNA2,!KPNA4! or!KPNA6!isoforms! as! bait! proteins! in! a! co3immunoprecipitation! assay! against! endogenous!TDP343.!After!performing!an!immunoblot!against!TDP343!we!found!that,!out!of!the!four!isoforms!examined,!only!KPNA2!was!able!to!pull!TDP343!out!of!the!cell!lysate!in!an!appreciable!amount!(Fig.#2A).!The!isoforms!KPNA1,!KPNA4!and!KPNA6!did!not!exhibit!binding!with!TDP343!suggesting!that,!under!normal!conditions,!the!principal!karyopherin3?!isoform!responsible!for!transporting!TDP343!within!SH3SY5Y!cells!is!KPNA2.!The!immunoprecipitation!samples!were!then!probed!for!the!presence!of!the!! 30!individual!KPNA!isoforms.!The!isoform!KPNA2!was!present!in!high!quantities!in!the!cell! lysate! both! before! and! after! immunoprecipitation! (IP),! while! KPNA1,! KPNA4!and!KPNA6!were!barely!detected!(Fig.#2B).!!2.3.3# Recombinant# Karyopherin6?2# Binds#with# Six# Distinct# Regions# of#TDP643#on#High6Density#Peptide#Array## High3density!peptide!array!technology!enables!the!identification!of!putative!interaction!domains!between!two!proteins.!Our!membrane!consisted!of!a!series!of!spots,!each!comprised!of!a!12314mer!peptide!derived!from!TDP343.!After!incubation!with!KPNA2!protein,!peptides!that!exhibited!binding!developed!as!dark!spots,!while!non3binding!peptides! remained!undeveloped.!The!developed!spots!on! the!KPNA23treated! membrane! were! compared! with! those! of! the! KPNA23absent! control!membrane! in! order! to! rule! out! any! non3specific! antibody! interactions.! As! each!sequential! peptide! spot! has! a! frame! shift! of! two! amino! acids,! putative! binding!regions!were!determined!by!the!presence!of!multiple,!consecutive!darkened!spots.!We! found! that! KPNA2! strongly! interacts! with! six! distinct! regions! of! the! TDP343!peptide!array!(Fig.#3),!and!identified!a!unique!sequence!of!8310!amino!acids!within!each! region.! All! six! sequences,! as! well! as! a! scrambled! derivative! of! the! first!sequence,! were! analyzed! via! basic! local! alignment! search! tool! (BLAST)! and!were!found!in!no!other!proteins!within!the!database.!Putative!interference!peptides!were!developed! from!these!sequences,!each!with!a!conjugated!TAT3domain! to!allow! for!cellular!penetration.!! 31!2.3.4#The#Interaction#Between#Endogenous#Karyopherin6?2#and#TDP643#Can#Be#Disrupted#by#Interference#Peptides#In#Vitro## To!determine! the!efficacy!of!our! interference!peptides,!we!performed!a! co3immunoprecipitation! on! peptide! treated! SH3SY5Y! cells,! using! KPNA2! as! bait! and!immunoblotting!for!TDP343.!Briefly,!cells!were!treated!with!all!six!blocking!peptides!(pALL),! an! equal! solution!of!peptides!2,! 5! and!6! (p256),! or! the! scrambled! control!peptide!(pS)!for!four!hours,!at!a!final!concentration!of!either!15uM!or!40uM!in!FBS3supplemented! DMEM.! A! CoIP! was! then! performed! on! cell! lysates! as! previously!described!(2.3.2).!The!TDP343!pull3down!bands!were!quantified!and!normalized!to!TDP343! levels! detected! within! lysate! controls.! The! normalized! TDP343! levels! for!each! sample! were! then! compared! to! the! IgG! control,! the! TDP343! levels! of! which!were!assigned!a!baseline!value!of!one.!At!15uM,!only!peptide!cocktail!pALL!was!able!to! significantly! inhibit! the! interaction!between!KPNA2!and!TDP343! relative! to! the!untreated!control!(PD)!and!the!scrambled!peptide!control!(pS)!(p<0.05,!student?s!t3test).! The! 15uM! scrambled! peptide! control! treatment! did! not! significantly! inhibit!the!protein3protein!interaction!when!compared!with!PD!(Fig.#4).!The!40uM!peptide!cocktails!were!observed!to!have!no!significant!effect!on!the!ability!of!KPNA2!to!pull!TDP343!out!of!solution.!!##! 32!2.3.5# Inhibition# of# the# Interaction# Between#KPNA2# and# TDP643# is# Not#Sufficient#to#Induce#Cytoplasmic#Mislocalization#of#TDP643### To!determine!whether! the! inhibition! of! the!KPNA23TDP343!nuclear! import!complex! is! sufficient! to! induce! TDP343! mislocalization,! we! treated! SH3SY5Y! cells!with!our!peptide!cocktails!for!24!hours!and!examined!the!subcellular!distribution!of!TDP343!via!nuclear!fractionation!(Fig.#5)!and!immunocytochemistry!(ICC)!(Fig.#6).!Here,! peptide! cocktails! pS,! p256,! and! pALL! and! were! tested! at! concentrations! of!either!15uM!or!40uM!on!healthy!SH3SY5Y!cells.!For! immunocytochemical!analysis,!peptide! cocktails! p26! and! p6! were! also! utilized.! For! each! interference! peptide!cocktail! examined,!we! found! that! the! subcellular!distribution!of!TDP343! remained!unchanged! when! compared! to! controls! treated! with! scrambled! peptide!(fractionation!data!for!40uM!peptide!treatment!not!shown).!# ! !2.4#Discussion!! Under!normal! cellular! conditions,!TDP343! is! transported! to! the!nucleus!via!the!classical!nuclear!import!system.!During!classical!import,!cytosolic!TDP343!binds!directly! to!one!of! seven!different!karyopherin3?! isoforms! that!mediate! its!passage!through! the! nuclear! pore! complex.! Although! each! recombinant! karyopherin3?!isoform! has! been! shown! to! bind! to! TDP343! in! vitro,! the! endogenous! isoform(s)!responsible!for!the!nuclear!import!of!TDP343!in!a!given!cell!likely!depends!upon!the!expression!level!of!each!KPNA!isoform,!as!well!as!their!individual!binding!affinities!for!TDP343!(Nishimura,!Zupunski!et!al.!2010).!Here,!we!determined!the!expression!patterns! of! KPNA! isoforms! in! SH3SY5Y! cells! by! performing! immunoblotting! on!! 33!whole3cell!lysates!utilizing!antibodies!directed!against!four!KPNA!isoforms:!KPNA1,!KPNA2,! KPNA4! and! KPNA6.! Our! results! showed! that,! out! of! the! four! isoforms!examined,! only! KPNA2! and! KPNA4!were! expressed! in! detectable! amounts.!While!our! data! confirmed! the! presence! of! these! two! isoforms,! it! does! not! rule! out! the!presence!of!other!KPNA!proteins.!However,!rather!than!complete!inhibition!of!TDP343! nuclear! translocation,! our! goal! is! to! examine! the! functional! consequences! of!deficient!KPNA3mediated!TDP343!nuclear!targeting.!Hence!we!set!out!to!identify!the!major!KPNA!isoforms!responsible!for!TDP343!binding!and!nuclear!targeting.!Using! the! four! aforementioned! KPNA! isoforms! as! bait,! we! sought! to!determine!which,!if!any,!strongly!interacted!with!TDP343!in!our!cell!line.!While!it!is!likely!that!multiple!KPNA!isoforms!are!involved!in!the!nuclear!transport!of!proteins,!only!KPNA2!was!found!to!bind!TDP343!with!such!an!affinity!as!to!be!detectable!via!co3immunoprecipitation!(CoIP).!We! went! on! to! examine! the! specific! interaction! domains! involved! in! the!binding! of! KPNA2! and! TDP343! using! our! TDP3433derived! high3density! peptide!arrays.! High3density! peptide! array! technology! has! been! shown! to! be! an! effective!method! of! investigating! discrete! domains! involved! in! protein3protein! interactions!(Lizcano,!Deak! et! al.! 2002).!We! found! that! recombinant!human!KPNA2! interacted!with!TDP343!at! six! specific! regions,!with! the! strongest!binding!occurring!near! the!glycine3rich!region!at!the!C3terminal!end!of!TDP343.!Interestingly,!one!of!the!regions!of! TDP343! that! exhibited! the! strongest! interaction!with! KPNA2!was! a! region! that!contains!five!documented!point!mutations!that!have!been!observed!to!be!associated!! 34!with! familial! ALS.! If! this! region! is! crucial! for! the! binding! of! KPNA2! to! TDP343,! it!follows! that! genotypes! exhibiting! point! mutations! in! this! region! may! display!deficiencies! in! the! nuclear! import! of! TDP343,! resulting! in! the! subsequent!development!of!TDP343!cytoplasmic!accumulation!and!proteinopathy.!!Although!it!has!been!shown!that!a!functional!NLS!is!necessary!for!the!nuclear!import!of!TDP343!(Nishimura,!Zupunski!et!al.!2010),!we!did!not!observe!any!binding!of!KPNA2!to!the!NLS!region!of!TDP343.!We!suspect!this!may!be!due!to!the!fact!that!we!excluded!KPNB1!from!our!protein3protein!interaction!assay!and,!in!the!absence!of!KPNB1,!the!IBB3domain!of!KPNA2!autoinhibits!its!NLS3binding!domain.!While!it!is!likely!that!KPNA2!would!exhibit!strong!binding!at!the!NLS!of!TDP343!in!the!presence!of! KPNB1,! the! NLS! would! not! provide! a! suitable! target! for! blocking! the! specific!nuclear!import!of!TDP343!due!to!the!ubiquity!of!this!signal!within!nuclear3destined!proteins.!The!strength!of!binding!observed!between!KPNA2!and!non3NLS!regions!of!TDP343! indicate! that!other!binding! regions!are! important! for! this! interaction,! and!these!regions!may!prove!to!be!more!suitable!targets!for!the!inhibition!of!this!specific!protein!interaction.!!! We! analyzed! the! putative! KPNA23interaction! regions! of! TDP343! and,! from!these! sequences,! designed! six! short,! 8310mer! TAT3conjugated! blocking! peptides:!p13p6.!A!TAT3conjugated,!scrambled3sequence!peptide,!containing!the!same!amino!acid!composition!as!p1,#was!designed!to!serve!as!a!control.!To!determine!the!efficacy!of!our!peptides,!we!examined!their!ability!to!interfere!with!the!binding!of!KPNA2!to!TDP343! in1vitro!using!our!established!CoIP!assay.!Our!data!indicates!that,!at!a!final!! 35!peptide!concentration!of!15uM,!the!combination!of!all!six!peptides!(pALL)!was!able!to! significantly! inhibit! the! binding! of! TDP343! to! KPNA2! within! four! hours! of!application!when! compared! to! both! the!mock! treatment! (PD)! and! the! scrambled!control! peptide! (Fig.# 4).! The! peptide! cocktail! p256! appeared! to! disrupt! the!interaction!at!a!15uM!concentration,!but!it!did!not!do!so!to!a!significant!amount.!At!the!same!concentration,!the!scrambled!control!did!not!appear!to!significantly!inhibit!this! protein3protein! interaction! relative! to! the! mock! treatment.! At! a! final!concentration!of!40uM,!our!peptide!cocktails!did!not!appear!to!block!the!interaction!between! TDP343! and!KPNA2.! It! is! unclear! as! to!why! the! peptides! did! not! exhibit!interference!at!higher!concentrations.!One!reason! for! this! finding!could!be! that,!at!40uM,!the!peptide!cocktails!may!have!become!toxic!to!the!cells.!If!the!cells!that!were!heavily! affected! by! the! 40uM! cocktails! merely! succumbed! to! apoptosis,! the! co3immunoprecipitation! performed! would! be! sampling! from! the! surviving! cellular!population.! This! incomplete! sampling! may! have! masked! any! minor! peptide3mediated!interference.!To! determine! the! physiological! consequences! of! blocking! the! interaction!between!KPNA2!and!TDP343,!we! treated!SH3SY5Y!cells!with!our!peptide! cocktails!for!24!hours.!This!timeframe!was!chosen!to!allow!for!sufficient!cytoplasmic!TDP343!to!accumulate!before!substantial!compensatory!changes!in!isoform!expression!could!take!effect.!Our!data! from!both!subcellular! fractionation!and! immunocytochemical!staining! showed! that,! in! healthy! cells,! the! inhibition! of! this! protein3protein!interaction!was!not!sufficient!to!induce!the!cytoplasmic!sequestration!of!TDP343.!!! 36!There!are!a!number!of!potential!reasons!as!to!why!our!peptides!were!unable!to! induce! TDP343!mislocalization.! First,! peptides! are! rapidly! degraded! by! cellular!proteases! and! degradation! systems! once! they! penetrate! the! cell.! It! may! be! that!although!our!peptides!induced!TDP343!mislocalization!initially,!they!were!no!longer!effective! at! the! time! of! assessment! (24! hours)! due! to! degradation.! However,! this!scenario! is! unlikely,! as! both! sequestration! and! clearance! of! cytoplasmic! TDP343!would!have!to!occur!within!24!hours,!a!relatively!short!time!frame.!It!is!much!more!feasible! that! the! constitutive! expression! of! other!KPNA! isoforms!was! sufficient! to!maintain! normal! subcellular! TDP343! distribution! in! a! compensatory! manner.!Although! our! co3immunoprecipitation! data! showed! that,! of! the! KPNA! proteins!examined,! KPNA2! exhibited! the! highest! affinity! for! TDP343! under! healthy!conditions,!the!affinities!of!any!other!isoforms!present!may!be!such!that!their!effects!become!more!relevant!as!the!level!of!cytoplasmic!TDP343!increases.!!Another! possible! reason! we! observed! no! mislocalization! of! TDP343! in!response!to!our!peptide!treatments!is!that!we!performed!our!treatments!on!healthy!cells.! Under! normal! physiological! conditions,! TDP343! resides! primarily!within! the!nucleus.!In!response!to!insults,!such!as!oxidative!stress,!TDP343!translocates!to!the!cytoplasm!and!associate!with! stress!granules! (Colombrita,!Zennaro!et!al.!2009).! It!follows! that! pathological! TDP343! cytoplasmic!mislocalization!may!be! the! result! of!deficiencies!in!the!nuclear!import!system!paired!with!mild!cellular!insult.!!!!! 37!##Figure#1.#Schematic!representation!of!classical!nuclear!import!cycle.!1:!Cytoplasmic!karyopherin3?! (KPNB)! binds! karyopherin3?! (KPNA),! allowing! KPNA! to! bind! a!nuclear3targeted!cargo!protein!(cNLS);!this!protein!complex!enters!the!nucleus!via!the! nuclear! pore! complex! (Nup).! 2:! Nuclear! Ran3GTP! binds! KPNB,! causing!dissociation! of! the! protein! trimer! and! release! of! the! cargo! protein.! 3:! Cellular!apoptosis! susceptibility3RanGTP! dimer! (CAS3RanGTP)! binds! and! transports! KPNA!to! the! cytoplasm! while! KPNB! facilitates! its! own! cytoplasmic! translocation;! both!mechanisms! utilize! the! Nup.! 4:! Cytoplasmic! hydrolysis! of! RanGTP! to! RanGDP!induces!the!release!of!KPNA!from!CAS3RanGDP,!completing!the!cycle.####! 38!#Figure# 2:# Karyopherin3?2! interacts! with! TDP343! in! SH3SY5Y! cells.! A:# Western!immunoblotting! of! TDP343! after! co3immunoprecipitation! of! SH3SY5Y! cell! lysate!utilizing!karyopherin3?!isoforms!1,!2,!4!and!6!as!bait!proteins.!TDP343!is!pulled3out!of!SH3SY5Y!cell!lysate!by!KPNA2,!but!not!by!KPNA1,!KPNA4,!or!KPNA6.!B:!Western!immunoblotting! against! KPNA! isoforms.! Cell! lysates! and! immunoprecipitation!samples!from!(A)!were!probed!for!the!presence!of!individual!KPNA!isoforms.!Strong!bands! located! at! 50kDa! correspond! to! either! rabbit! (KPNA1,! KPNA4,! KPNA6)! or!goat!(KPNA2)!heavy3chain!IgG.!#! 39!#Figure#3:!TDP3433derived!high3density!peptide!array.!A:!Control!membrane,!probed!with! anti3KPNA2! antibody! in! the! absence! of! KPNA2! protein.! B:! Interaction!membrane,! probed! with! anti3KPNA2! antibody! after! incubation! with! recombinant!KPNA2! protein.! Dark! spots! appearing! on! (B)! that! are! absent! from! (A)! indicate!putative!KPNA23binding!regions!of!TDP343.!C:!Full3length!human!TDP343!schematic!showing! known! clinical!mutations! associated!with!ALS! pathogenesis.! Six! peptides!(p13p6)! were! derived! from! KPNA23binding! regions! of! TDP343! as! indicated,! with!their! respective! amino! acids! in! brackets.! Five! residues,! whose! mutations! are!thought! to! be! associated! with! ALS,! are! encompassed! by! peptide! 6! (p6)! and! are!displayed!on!the!lower!right!corner.!! 40!#Figure#4:!Peptides!interfere!with!the!interaction!between!KPNA2!and!TDP343.!SH3SY5Y!cells!were!treated!with!a!specific!peptide!cocktail,!at!a!concentration!of!either!15uM! or! 40uM,! for! four! hours! before! harvesting! for! co3immunoprecipitation:!scrambled!peptide!(pS),!all!six!peptides!(pALL),!or!peptides!2,!5!and!6!(p256).!TDP343! immunostaining!was!quantified!and!normalized!to! total!TDP343!content!within!the! lysate! of! each! sample.! Each! sample! was! then! normalized! to! TDP343! staining!observed! in! the! IgG! control.! [? compared! with! PD,! ?! compared! with! pS315;!student?s!t3test.!Error!bars!represent!mean!?!SEM!from!n!=!3]!##! 41!#Figure# 5:! Subcellular! localization! of! TDP343! following! 243hour! in1 vitro! peptide!treatment!and!nuclear!fractionation.!SH3SY5Y!cells!were!treated!for!24!hours!with!a!15uM!peptide!cocktail!comprised!of!either!a!scrambled!control!peptide!(pS),!all!six!peptides!(pALL),!or!peptides!2,!5,!and!6!(p256).!A:!Cytoplasmic!(C)!and!nuclear!(N)!fractions! were! resolved! via! SDS3PAGE! and! Western! immunoblotted! for! TDP343,!LMNB1!and!HSP390.!B:!The! ratio!of!mean! cytoplasmic! to!nuclear!TDP343! staining!was!plotted!for!each!peptide!treatment.!Error!bars!represent!the!mean!?!SEM!from!n!=!3.!!##! 42!#Figure# 6:! Immunocytochemical! analysis! of! the! subcellular! distribution! of! TDP343!following! 243hour! in1 vitro! peptide! treatment.! Four! combinations! of! interference!peptides,! and! one! scrambled! control! peptide,!were! utilized! on! SH3SY5Y! cells! at! a!final!concentration!of!either!15uM!(A)!or!40uM!(B):!scrambled!control!peptide!(pS),!all! six! peptides! (pALL),! peptides! 2,! 5,! and! 6! (p256),! peptides! 2! and! 6! (p26)! and!peptide!6!(p6).!The!ratio!of!mean!cytoplasmic!to!nuclear!TDP343!staining!was!found!to! be! unaffected! by! the! interference! peptides! when! compared! to! the! scrambled!control!peptide.!Error!bars!represent!the!mean!?!SEM!from!n!=!3.!##! 43!3:#Conclusions#and#Future#Directions#In! this! study,! we! set! out! to! determine! if! blocking! the! specific! interaction!between! TDP343! and! its! nuclear! import! proteins! is! sufficient! to! induce! TDP343!cytoplasmic! mislocalization,! as! seen! in! diseases! such! as! FTLD3U! and! ALS.! The!following!is!a!summary!of!what!we!found:!1) Through! the! use! of! co3immunoprecipitation,! we! found! karyopherin3?2! to!exhibit! the! highest! binding! affinity! for! TDP343! of! the! KPNA! isoforms!examined!in!SH3SY5Y!cells.!2) We! identified! six!putative!KPNA23binding!sites!on!TDP343,!utilizing!a!high3density! peptide! array,! and! developed! six! short! interference! peptides! from!these!regions.!3) The! specific! interaction!between!KPNA2!and!TDP343!was! impaired! in1 vitro!using!low!concentrations!(15uM)!of!all!six!interference!peptides!as!assessed!by! Co3IP.! At! higher! concentrations! (40uM)! the! peptides! did! not! appear! to!interfere!with!the!formation!of!the!KPNA23TDP343!protein!complex.!4) Blocking! the! interaction! between!TDP343! and!KPNA2! for! 24! hours! in1 vitro!was! not! sufficient! to! induce! TDP343! cytoplasmic!mislocalization! in! healthy!SH3SY5Y!cells.!Our! investigation!of! the!relationship!between! impaired!TDP343!nuclear! import!and! TDP343! proteinopathy! was! performed! on! healthy! SH3SY5Y! cells.! A! previous!study! by! Columbrita! et! al.! (2009)! found! that! TDP343! is! retained! primarily! in! the!nucleus!under!normal!cellular!conditions;!only! in! the! face!of!cellular! insult!does! it!! 44!migrate! to! the! cytoplasm! and! accumulate! within! stress! granules.! TDP343!proteinopathies,!such!as!ALS!and!FTLD3U,!typically!do!not!present!until!later!in!life,!despite!the!fact!that!any!associated!mutations!would!have!been!present!from!birth.!It! follows! that! the! pathogenesis! of! TDP343! proteinopathies! may! require! an!underlying! genetic! component! coupled! with! the! progressive! accumulation! of!cellular! insults! over! one?s! lifetime.! For! future! studies,! it!may!be!more!biologically!relevant! to! study! the! effects! of! nuclear! import! impairment! alongside! a! cellular!stressor,!such!as!oxidative!stress.!We!plan!to!utilize!our!interference!peptides!on!SH3SY5Y! cells! that! have! been! subjected! to! sodium! arsenite,! which! is! an! established!oxidative!stressor!(Colombrita,!Zennaro!et!al.!2009),!and! investigate!the!effects!on!the!subcellular!localization!of!TDP343.!!As!it!is!likely!that!multiple!KPNA!isoforms!are!involved!in!the!nuclear!targeting!of!TDP343,! in! future!studies!we!will!use!high3density!peptide!arrays! to! investigate!the!specific!regions!of!TDP343!that!interact!with!each!individual!KPNA!isoform.!By!comparing! these!putative! interaction!regions,!we!aim! to! identify!common!sites!on!TDP343! that! bind! to! all! KPNA! isoforms.! 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