Dijkmans, T.F.
Citation
Dijkmans, T. F. (2009, October 14). Doublecortin-like kinase and neuronal differentiation. Retrieved from https://hdl.handle.net/1887/14055
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GENERALDISCUSSION
Partiallypublishedas
DijkmansTF,VanHooijdonkLW,FitzsimonsCP,VreugdenhilE.
Thedoublecortingenefamilyanddisordersofneuronalstructure.(Review).
Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research
andLeidenUniversityMedicalCenter,TheNetherlands
inCentralNervousSystemAgentsinMedicinalChemistry.Inpress.
OUTLINE
1.Introduction
2.NGFresponsiveimmediateearlygenes
3.NGFresponsivedelayedresponsegenes
4.FunctionofDCLKshortinneuronaldifferentiation
5.TranscriptionalregulationofDCLKshort
6.Perspective
7.References
1.INTRODUCTION
The development of the nervous system encompasses the generation of its
constituentcells,theirpositioningwithintheanimalbodyandtheirestablishment
offunctionalconnectionswithothercells.Tounderstandhowthiscomplexprocess
ofneuronaldifferentiationunfolds,cellmodelshavebeenused.ThePC12cellline,
anditscellularmetamorphosisuponNGFaction,hasprovenparticularlyfruitfulin
elucidating the molecular biology of a differentiating neuron [1]. In combination
with ongoing technological advances, new, valuable information has continued to
arise. Recently, DNA microarrays have become available that enable the
measurement of genomewide expression and can thus give an unprecedented
broadperspectiveonneuronaldifferentiation.Atthesametime,eachofthemany
expressed genes in a differentiating cell can have unique functions and shouldbe
characterizedindividually.
The central goal of this thesis was therefore to identify genes important
for NGFinduced neuronal differentiation of Ns1 PC12 cells, and to perform a
specific analysis of DCLKshort. To achieve this, the following experiments were
conductedinNs1PC12cells:
1. a DNA microarray screening to identify previously unrecognized NGF
responsiveIEGs.
2.aDNAmicroarrayscreeningtoidentifyNGFresponsiveDRGs.
3.afunctionalanalysisofDCLKshortinNGFinduceddifferentiation.
4.atranscriptionalregulationstudyonDCLKshort.
Based on these experiments, the results in this thesis have led to the following
conclusions:
x Cited2, Klf4, Maff, Tieg, Atf3 are IEGs induced by NGF in Ns1 PC12 cells
andaretranscriptionfactorsthatarelikelytoplayimportantrolesinNGF
inducedneuronaldifferentiation.
x Neuronal differentiation of Ns1 PC12 cells involves temporally
coordinatedtranscriptionalregulationofgenesassociatedwithchromatin
packagingandremodeling,cellcycleprocesses,cellstructure,intracellular
protein trafficking, intracellular signaling, protein biosynthesis and mRNA
transcription.Neuronaldifferentiationisnotcharacterizedbyprogressive
upordownregulationofgeneexpressionintime.
x In Ns1 PC12 cells, NGF induces DCLKshort expression and activates ERK
1/2mediated phosphorylation. NGFinduced neuritogenesis is mediated
byDCLKshortinaphosphorylationdependentmanner.
x BothbasalandNGF/FSKinducedexpressionofDCLKshortdependonthe
51 to 43 AGACGTCA promoter sequence. NGFinduced expression of
DCLKshort can be suppressed after GRactivation by the synthetic
glucocorticoidDexamethasone.
Below,theresultsderivedfromtheseexperimentswillbediscussedinthelightof
the formulated goal (Section 2. to 5.). Thereafter, I will discuss how the acquired
insightscanbeusedforfutureresearch(Section6.).
2.NGFRESPONSIVEIMMEDIATEEARLYGENES
Classically, the transcriptional response of a cell to extracellular stimuli, such as
NGF,isdividedinIEGsandDRGs[2].Thesegeneclassesaredefinedbygenesthat
eitherrequirenoproteinsynthesispriortotheirtranscriptionalinduction(IEGs)or
genesthatdo(DRGs).BecausemanystudieshavereportedimportantrolesforIEGs
in neuronal differentiation [37], we performed a microarray screeningto identify
new NGFresponsive IEGs in Ns1 PC12 cells (Chapter 2). After measuring 27000
genes across two hours of NGF stimulation, we replicated the induction of many
knownNGFresponsiveIEGs,includingcFosandEgr1.Wealsoidentified5genes,
whichwerenotknowntobeNGFresponsiveinthiscontext,butwhoseexpression
seemed to be substantially increased by NGF treatment. Subsequent qPCR and
Western Blot experiments confirmed the NGFresponsiveness of these genes
(Cited2, Klf4, Maff, Tieg and Atf3). Also, these genes could be classified as IEGs,
since their expression was elevated by NGF in a manner independent of protein
synthesis. Two other characteristics, which may be considered as typical for NGF
responsiveIEGsinPC12cells,appliedtomostofthesegenes[2].First,expression
ofeachofthe5geneswasshowntobepartiallyinducedthroughthePI3KandERK
1/2 pathway. These pathways were previously reported as important for IEG
inductioninPC12cellsbyNGF[2;8].Second,4ofthese5genesshowedexpression
profiles that were strongly and mostly transiently increased by NGF. In contrast,
expression of Cited2 was increased steadily over the twohour period of NGF
treatment.ThesedatatogethershowedthatthetranscriptionfactorsCited2,Klf4,
Maff,TiegandAtf3areactivatedbyNGF,indicatingapossibleroleinNGFinduced
differentiationofNs1PC12cells.
SeveralstudieshavereportedsignificantrolesforthenewlyidentifiedIEGs
in apoptosis, proliferation and/or differentiation in different cell systems. NGF
coordinatesthesecellularprocessesinPC12cells(seeIntroduction§1.3)andthis
action exerted by NGF may be mediated by the new IEGs. For instance, Klf4 was
reported to either inhibit or drive cell proliferation, dependent on the cellular
context it operates [9]. Tieg was shown to mediate cessation ofcell proliferation
prior to differentiation of cerebellar granular neuron precursors [10]. Also, Cited2
regulatesmanydevelopmentalprocessessuchasheartdevelopment,leftrightaxis
formation, neural tube formation, adrenal and placental development [1113]. In
PC12 cells, it was reported that Mafk, which is highly homologous to Maff, is an
NGFresponsive IEG that regulates neurite outgrowth of PC12 cells [6]. Given the
similarityingenesequenceandNGFresponsiveness,similarityoftheirfunctionin
neuritogenesisislikely.Finally,priortoidentificationasaNGFresponsiveIEG,Atf3
was shown to regulate neuritogenesis in PC12 cells [14;15]. In these studies, Atf3
overexpression conferred protection against apoptosis and promoted neurite
outgrowth. Our data extend these observations and suggests that increasing Atf3
expression is part of NGFinduced neurite outgrowth. Together, the data provide
supporttothehypothesisofCited2,Klf4,Maff,TiegandAtf3beingrelevantplayers
inNGFinducedneuronaldifferentiation.
ItwouldbeofinteresttodedicatefuturestudiestotheroleoftheseNGF
responsivetranscriptionfactorsindifferentiationofPC12cells.Aswithpreviously
tested IEGs in PC12 cells, RNAimediated knockdown or over expression of these
genes could be pursued [3;6]. Then, studying their effects on differentiation,
proliferationandcellviabilitycouldgivevaluableinsightsintheirfunction.
Figure 1. NGF-responsive genes in Ns-1 PC12 cells across time. Although NGF induced the expression of IEGs within 2 hours A). in cases their expression lasted for days B). Expression data of each gene is derived from DNA microarrays and normalized against expression of the gene prior to NGF stimulation. Expression data of IEGs after 1 to 4 days are shown, when significantly enhanced by NGF, only (see Chapter 3 for details).
0 30 60 90 120
0 5 10 15 20 500 1000
Time (min)
1 2 3 4
0 5 10 15 20
Time (day) Cited2 Tieg
Maff
Atf3 Klf4 Egr1
Tis11 Fosl1/Fra-1
A B
3.NGFRESPONSIVEDELAYEDRESPONSEGENES
In Chapter 3, we performed a DNA microarray study to investigate the
transcriptomeofNs1PC12cells during 4 subsequent days of differentiation.This
time frame is complementary to the immediateearly period of 2 hours. Hence,
NGFresponsivegenesidentifiedduringthetimeframeofdayswereassumedtobe
DRGs. Here, we identified at least 200 previously unknown NGFresponsive genes
whencomparedwithpreviouslargescalescreenings[6;1618].Moreimportantly,
it was shown that NGFinduced differentiation is not merely paralleled by
progressiveup ordownregulation of a setof genes, but rather involveswavesof
transcriptional responses. Previous expression profiling studies of differentiating
cell types including neurons, myocytes and hair cells also pointed to a high
temporal orchestration of the transcriptome [1921]. Apparently, this is a more
general feature of differentiating cells, but was not shown for NGFinduced
differentiationofNs1PC12cells.
By performing a gene ontology analysis, we determined which types of
genes were specifically regulated by NGF in time. How these gene classes may
affect the transcriptomal development during differentiation was discussed in
Chapter 3. Of particular interest was the observation that NGF changed the
expression in time of 16 genes that mediate MAPK signaling. Spatiotemporal
control of MAPK signaling (including ERK 1/2) is crucial in directing the cell into a
fate of apoptosis, proliferation or differentiation and is employed by a variety of
growth factors in PC12 cells [22;23]. It is known that NGF maintains a prolonged
activationoftheERK1/2pathwayandherebyinducesdifferentiation,whereasEGF
triggers only a transient activation and hereby promotes cell division. Our
observationsindicatethatafeedbackmechanismonNGFinducedMAPKsignaling
exists, because NGF changes the expression of signaling MAPK components. This
observationcanprovideamolecularbasisfortheacquireddependenceonNGFof
differentiatedPC12cellsfortheirsurvival[23;24].PriortoNGFexposure,PC12cells
are not dependent on NGF for their survival. However, after neuronal
differentiation, that is, after a history of NGFinduced transcriptional
reprogramming,PC12cellsdiewhenNGFiswithdrawnfromtheirculturemedium.
Indeed,NGFwithdrawalinducedapoptosisismediatedbyMAPKsignalingandmay
betheresultofalteredexpressionofMAPKrelatedgenes[25].
Moreover, this observation can provide a molecular basis for altered
sensitivity of PC12 cells after differentiation to other growth factors, such as EGF
[1;26]. Alternatively, changing the MAPK pathway within the differentiating cell
may also account for the measured, dynamic transcriptomal changes. As an
example, Elk1 initiates transcription upon NGFdriven phosphorylation by MAPK
ERK 1/2. Changing the expression of MAPKrelated genes by NGF exposure may
change Elkdriven transcription in time, thus explaining part of the temporal
expression profiles. Future experiments that manipulate the MAPK pathway at
different time points during differentiation may reveal such roles. For instance,
using pharmacological inhibitors of specific MAPK components it may be shown
that the transcriptome of the differentiating PC12 cell is susceptible to these
inhibitorsinatimedependentmanner.
The expression data obtained during the 4 days of NGF treatment (Chapter 3)
appear complementary to those obtained during the 2 hour interval (Chapter 2).
Specifically, for the 5 NGFinduced IEGs that were identified in Chapter 2, known
target genes were found induced after days of NGF treatment. Tieg has a target
gene named cyclindependent kinase inhibitor p21 (Cdkn1a) which was induced
after 23 days of NGF treatment. Atf3 has 2 known target genes, Hsp27 and
Gadd153/Chop, which were also upregulated after 12 days of NGF treatment
[14;27;28]. These observations provided additional support for the putative
functionality of the identified IEGs in neuronal differentiation. However, studies
thatsystematicallydelineatewhichgenesaretargetsoftheidentifiedIEGsinNs1
PC12 are required. Chromatinimmunoprecipitation studies of these transcription
factors followed by DNA microarrays or sequencing, would allow a more precise
assessmentoftheirroleintheNGFregulatedtranscriptome.
Comparing the data of both microarray studies also revealed increased
transcription of several IEGs after days of NGF treatment. The IEG definition is
founded on the lack of translationdependence of gene induction and is often
accompaniedbyarapid,strongandtransientgeneinductionprofile[2].Egr1,used
as a positive control for IEG induction during short term NGF treatment, was
amongthemostresponsivegenesduringlongtermNGFtreatment(Fig.1).When
theEgr1inductionprofileisinspectedacross2hoursofNGFtreatment,inductionis
rapid, strong and seemingly transient. However, after 2 hours the expression of
Egr1hassubstantiallydecreased,yetremainsmorethan200foldhigherthanprior
to stimulation. During the 4 following days of NGF treatment, Egr1 expression
slowlydeclines,butremainshigherthanpriortoNGFstimulation.SimilartoEgr1,
alsoanelevatedFosl1expressionwasretainedafter2hours(33fold),whichslowly
decreasesduring4subsequentdays.Moreover,thenewlyidentifiedIEGMaffwas
induced maximally to 8fold after 1 hour of NGF and remained elevated on
subsequentdays.AlsoCited2showedaremarkabletranscriptionprofile:induction
showed a progressive increase during 2 hours of NGF treatment and a decrease
during days of NGF treatment. The other IEGs, Atf3, Tieg and Klf4 were not
significantlyaffectedafterdaysofNGFtreatment.WhereastypicalIEGprofilesare
transient within two hours, such as Atf3 or Tis11, others, such as Egr1 and Maff,
peakafter1hourandslowlydecreasethesubsequentdays(Fig.1).Cited2iseven
less typical by displaying a delayed maximal induction later than 2 hours of NGF
stimulationandadecreaseduringthesubsequentdays.
Inductions of the new IEGs were shown to be translationindependent
withinthetwohourtimeframeandarethusIEGsbydefinition.Itispossiblethat
theincreasedexpressionofsomeoftheseIEGsduringdaysofNGFtreatmenthasa
translationdependent component. Then, these genes would classify as IEGs, but
also as DRGs. The combined microarray studies illustrate that, although the IEG
concept as a gene with a translationindependent induction is unambiguous, the
onsetanddurationoftheirinductiondynamicscanvarysubstantially.Itwouldbe
ofinteresttoinvestigatetowhatextenttheresponsivenessoftheIEGinductionis
specific to NGF. For instance, induction dynamics of the IEGs in response to EGF
mightbedifferentfromNGF.Thismaybereflectedindifferencesinproliferationor
differentiationcausedbythesetwogrowthfactors.
4.FUNCTIONOFDCLKSHORTINNEURONALDIFFERENTIATION
In Chapter 4 we extended the investigations in molecular mechanisms underlying
neuronaldifferentiation.FromChapter3,itbecameclearthatDCLKwasamongthe
most strongly induced genes by days of NGFexposure. Validation of microarray
signalsbyqPCRagainstspecificDCLKsplicevariantsshowedthattheNGFregulated
transcriptwasDCLKshort[2931].BasedonitsinductionbyNGFandonanumber
ofstudiesthatreportimportantrolesfortheDCLKgeneinneuronaldifferentiation,
DCLKshort was hypothesized to play a functional role in NGFinduced neuronal
differentiation of Ns1 PC12 cells [2933]. With the aim of generating a lead for
exploring DCLKshort function, we performed an in silico sequence analysis using
Scansite 2.0 to identify proteins that may interact with DCLKshort
(http://scansite.mit.edu/[34]).WeperformedthisanalysisonthefulllengthDCLK
protein of 740 amino acids, containing the 426 Cterminal amino acids of DCLK
short(Fig.2A).
Fig. 2B shows the surface accessibility as calculated by Scansite 2.0 over
the entire length of the DCLK protein. As can be seen, the SPrich domain has a
relativelyelevatedsurfaceaccessibility,whichisinlinewiththereportedfunction
Figure 2 (next page). Predicted protein interactions with the SP-rich domain of DCLK-short.
A). Schematic representation of DCLK-short. aa is amino acids. B. Visualization based on Scansite analysis from the consensus sequence of human, rat and mouse full length DCLK protein sequence (DCLK-long). B). Calculated surface accessibility from the consensus DCLK protein sequence (Scansite). X-axis represents the 740 amino acid DCLK sequence from N-terminus to C-terminus, whereas the y-axis is an indication of steric availability of DCLK subregions. Note the increased accessibility at the SP-rich domain. C). Quantification of all predicted interactions according to Scansite, which are 107 in total. From N-terminus to C-terminus (740 amino acids), the number of predicted interacting proteins is depicted. The box captures the DCLK sequence from aa 281 to 343, which is largely the SP-rich domain and shows a relative high number of 36 predicted interactions when compared across the entire sequence. D). Consensus sequence from aa 281 to 343. The 2 “X” letters represent two amino acids that are not conserved across human, rat and mouse DCLK. The underlinings indicate sites that were predicted to interact with or be phosphorylated by at least one other protein (in total 36 predicted interactions). The box captures the sequence that is contained by rat DCLK-short protein. E). Alignment of the SP-rich sequence of human DCLK1 with human DCLK2 and DCX, together with 36 predicted interacting proteins.
Conserved sequence has black background, less conserved grey and different white. The protein interactions predicted from the DCLK1 sequence are visualized underneath the alignment. FYN = Fyn kinase; 14-3-3 = 14-3-3 protein; NCK = NCK adaptor protein 1; , , and indicate kinase subunits. CDK = Cyclin-dependent kinase; CDC2 = Cell division cycle 2 (CDK1); GSK3 = Glycogen synthase kinase 3. Grb2 = Growth-receptor Bound 2.
oftheSPrichdomainasaproteininteractiondomain[3539].
Fig.2Cisaquantificationofthenumberofpredictedproteininteractions
per amino acid residue. The Xaxis of Fig. 2C represents the DCLK amino acid
sequence,whereastheYaxisindicatesthenumberofpredictedproteinsforeach
particularaminoacid.WithintheSPrichregion,aminoacids281until343showa
pronounced enrichment of 36 predicted interactions (36 of 107 in total). The
corresponding sequence is given in Fig. 2D. Each underlined amino acid indicates
thatforthataminoacidatleastoneproteinispredictedtointeract.Toincreasethe
likelihoodofgeneratingfunctionallyrelevantpredictions,wedismissedaminoacids
fromtheanalysisthatwerenotconservedacrosshuman,ratandmouse(2amino
acid positions are substituted by “X”). To further assess the degree of amino acid
conservation to which the predictions apply, we aligned the SPrich domains of
humanDCLK,DCLK2andDCX(Fig.2E).
The highest number of predicted phosphorylations on DCLK (and DCLK2
andDCX)appliedtoMAPKERK1:S22,T44,S57,S59,S61andS64(positionsasin
Fig.2E).Thispredictionwasofparticularinterest,becauseofthepivotalroleofERK
B
C
STSYTKIASXSRRXTTKSPGPSRRSKSPASTSSVNGTPGSQLSTPRSGKSPSPSPTSPGSLRK
Kinase
DCX DCX SP
D
Accessibility
281 343
# Predictions
CDK5 CDC2 ERK1
CDK5 CDC2
PKA GSK3
CDC2 CDK5 ERK1
CDC2 CDK5ERK1 p38 PKC FYN
PKC
14-3-3
PKA GRB2 PKC
PKC
CDC2
PKC
PKC
PKC
PKC
PKC CDK5 CDC2 GSK3
ERK1 CDC2
ERK1 NCK
740 aa
281 343
ERK1 GSK3
1.0
A
SP Kinase 433 aaSP
E
Accessibility
1.0
1/2 signaling in NGFinduced differentiation. Moreover, theERK 1/2pathway,like
DCXandDCLK,playsanimportantroleinvivoduringcorticogenesis[40;41].During
this process, growth factors signal through the ERK 1/2 pathway to control cell
proliferation, progression and fate decisions of neural progenitor cells [40;41].
AlterationofERK1/2signalinghasbeenreportedtoabrogatethegenerationofa
fullypopulated,normalsizecortex[40;41].Afterinhibitionofupstreamactivators
or scaffolding proteins of the ERK 1/2, neuronal progenitor cells remain in the
subventricularzoneinanundifferentiatedstate.Moreover,weandothersalready
described a role for splice variant DCL in determining neuronal fate by regulating
mitotic spindle integrity in the proliferative zone [7;42;43]. The exact underlying
mechanisms remain elusive, however, are likely to involve phosphoregulation by
upstream kinases, possibly ERK1. Together, DCLKshort appeared a likely and
relevant ERK 1 substrate. Therefore, we decided to investigate whether NGF
inducesDCLKshortphosphorylationandwhetheranyfunctionalrelevancemaybe
associatedtothisinChapter4.
In Chapter 4, we showed by a number of experiments that NGF
stimulationleadstoERK1/2dependentphosphorylationofDCLKshortatserine30
(correspondingtoserine64ofFig.2E).Tocouplethisphosphorylationtoacellular
functionwithinthedifferentiatingcell,weinvestigatedthesubcellularlocalization
of DCLKshort in Ns1 PC12 cells. Both prior and after stimulation, DCLKshort
resided throughout the cell. After NGF stimulation, DCLK showed statistically
insignificantnucleartranslocation.Ontheotherhand,nuclearphosphorylationwas
evident. A transcriptional role, which may be associated with this nuclear signal,
has been reported by two studies that indicated a function of DCLK(short) in
cAMPdriven transcriptional regulation [31;44]. However, we were not able to
confirmthesefindingsasnoeffectwasobservedforDCLKshortinNGFstimulated
Egr1 or cFosinduction. In the discussion of Chapter 4, anumberofexperimental
differences were brought forward that may explain these findings. Moreover, our
localizationstudiesinNs1PC12cellsindicatedthatDCLKshortwaspredominantly
extranuclearandthusindicatedaroleoutsidethenucleus.
We showed that DCLKshort localizes to Factin in growth cones of
(sprouting) neurites and regulates neuritogenesis in a phosphorylationdependent
manner. Because regulation of the actin cytoskeleton by phosphoproteins is an
important underlying mechanism of neuritogenesis, DCLKshort may be such a
phosphoprotein[4547].Insupport,otherproteinsoftheDCXgenefamilyinteract
with Factin, indicatingthat actin binding is a conserved feature ofthese proteins
[48]. Moreover, DCLKshort homologues CamK1 and CamK2 regulate neuronal
morphologythroughactindynamics[49;50].Forinstance,CamK2isabletodirectly
interact with Factin and mediates spine morphology and neurite extension in
hippocampal neurons [51]. Another study showed phosphorylationdependent
colocalization of CamK2 with Factin [52]. The substrates of CamK2 that are
involved in the actin cytoskeleton remodeling are Guanine nucleotide exchange
factor(GEF)Tiam1,GTPaseactivatingprotein(GAP)SynGAPandAsk1[53].Similar
to CamK2, CamK1 was shown to regulate actin dynamics in growth cones and
consequently neurite outgrowth in hippocampal and cerebellar granule neurons
[54]. In the latter study, it appeared that the effects of CamK1 on growth cone
motilityareinitiatedbyCamKK.CamKKistheupstreamkinaseofCamK1andraises
CamK1kinaseactivitybyphosphorylation,whichwasimportantforaffectingactin
dynamicsingrowthcones.Unfortunately,theresponsiblephysiologicalsubstrates
ofCamK1remainelusive.Proposedsubstrateswithknownrolesinactindynamics
includeGEFsandGAPs[54].Synapsin1wasshowntobephosphorylatedinvitroby
CamK1 and by ERK 1/2 in PC12 cells, which regulated its interaction with actin in
Figure 3. Hypothetical model for DCLK-short function in Ns-1 PC12 cells in NGF- induced neuritogenesis. The growth cone is attracted to the NGF source and leads to neurite extension in this direction. Upon NGF treatment, NGF is internalized with its receptor and activates a number of signaling pathways, including ERK 1/2, and transcriptional programs (see Introduction). DCLK-short is phosphorylated by ERK 1/2 upon NGF treatment, regulates neuritogenesis and colocalizes to F-actin in growth cones (see Chapter 4). Neuritogenesis involves extensive interplay of actin and microtubule dynamics and intracellular transport (V). Actin dynamics are regulated by many phosphoproteins, including GAP43, Synapsin 1 (Syn), Guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and Myosin. Potential substrates and interaction partners of DCLK-short are indicated with dotted lines with arrowheads. Solid lines with arrowheads indicate verified phosphorylations, indicated by "p". Grb2 = Growth Receptor Bound 2.
the latter case [55]. In vivo, Myosin II regulatory light chain is known to be
phosphorylated by CamK1 and induces reorganization of actin filaments. This
protein has been extensively characterized as a substrate of Myosin light chain
kinase (MLCK),andregulates actin dynamics and vesicle transport inconcert with
MLCK [56;57]. Interestingly, MLCK is a Cam Kinase with reasonable homology to
DCLKshort (39.9% aa consensus), suggesting similar function [58]. Together, it
would be of interest to investigate DCLKshort and its substrates as regulators of
the actin cytoskeleton in differentiating Ns1 PC12 cells. A hypotheticalmodel for
DCLKshort and its molecular interactions in the extending neurite is illustrated in
Fig.3.
5.TRANSCRIPTIONALREGULATIONOFDCLKSHORT
InChapter5,thetranscriptionalregulationofDCLKshortwasinvestigated.Insilico
promoter analysis and chromatinimmunoprecipitation studies suggested that the
promoterofDCLKshortharboredaprimaryCREBresponsiveelement,AGACGTCA
[59]. However, stimulusinduced upregulation of DCLKshort expression occurred
laterthan8hoursandsuggestsatranslationdependentmechanism.Althoughthis
translation dependence can imply a secondary or sequential transcriptional
response, Atf3 and cJun were discussed as likely candidates to mediate the
transcriptional induction of DCLKshort. Remarkably, the basal and the delayed
induction of expression of DCLKshort were dependent on the identical promoter
element in Ns1 PC12 cells. A similar case was reported for GnRHII in human
neuronalmedulloblastomacells[60].Alsohere,AGACGTCAintheGrRHIIpromoter
renderedarelativelydelayedtranscriptionalresponsetocAMPafter1224hours.
Moreover, mutation of this promoter element severely impinged upon basal and
induced expression. The AGACGTCA sequence slightly differs from the consensus
CREmotifTGACGTCAandmayholdcluesforitsatypicaltranscriptionalregulation.
DCLKshort was shown to be an NGF/FSKresponsive transcript, which
mediates neuronal differentiation. NGF and FSK activate overlapping pathways in
PC12 cells and both induce neuritogenesis [61]. Likewise, neurotrophins including
NGF activate cAMPresponsive gene expression in vivo and thereby stimulate
neuronal differentiation or modulate neuronal morphology [8;62]. It would be of
interesttoinvestigatetheexpressionandactionsofDCLKshortinresponsetothe
various neurotrophins available in the mammalian nervous system. Moreover, in
vitro and in vivo experimentations that investigate DCLKshort as a convergence
point of glucocorticoids and neurotrophins to modulate neuronal morphology are
goodfutureaims.
6.PERSPECTIVE
One of the findings of this thesis was that 5 transcription factors are robustly
induced by NGF and are therefore hypothesized to regulate transcription in
neuronal differentiation of Ns1 PC12 cells. Future studies should assess the
relevance of their inductions for the differentiating cell by manipulating their
functions in this model, either genetically or, where available, pharmacologically.
Investigating which other proteins interact with the IEGs and which are the IEG
targetgeneswillalsohelptodissecttheprocessofneuronaldifferentiationfurther.
Inaddition,1595ofsuchpotentialtargetgeneswereshowntobeinducedbyNGF
and these may all have their specific contributions in generating a differentiated
neuron.Eachoftheirfunctionsmaybequestionedbyexperimentsusingcommon
cell and molecular biological methods. For many this has already been done or is
ongoing,butforothersthisremainstobedone.
ForoneNGFresponsivetranscript,DCLKshort,apartialfunctionalanswer
was given in this thesis by showing that DCLKshort regulates neuritogenesis. As
expected,thisanswergenerated new questions. Mostpertinent iswhetherDCLK
short regulates neuritogenesis by affecting actin polymerization, or by other
mechanisms. Cosedimentation or coimmunoprecipitation experiments can help
to addressthesequestions.Second, additional investigations on apossiblerolein
thenucleusand/orintranscriptionalregulationarerequiredtobetterdefineDCLK
shortfunctioninneuronaldifferentiation.Also,giventhefactthatDCLKshortisa
kinase, identification of its physiological substrate(s) is likely to provide relevant
functional information. Finally, future research should verify observations done in
theNs1PC12cellmodelonDCLKshort,orotherNGFresponsivegenes,ininvivo
models of neuronal differentiation. Given the expression of DCLKshort in the
mammalian brain, functions may exist here similar to those in Ns1 PC12 cells.
Therefore, it would be of interest to validate DCLKshort in vivo as a
phosphoprotein that regulates neurite outgrowth and dependent phenomena.
Collectively,thedatapresentedinthisthesiscontributetoabetterunderstanding
ofthemolecularmechanismsunderlyingneuronaldifferentiation.
7.REFERENCES
1. L.A.GreeneandJ.M.Angelastro,Youcan'tgohomeagain:transcriptionallydriven
alterationofcellsignalingbyNGF,Neurochem.Res.30(2005)13471352.
2. H.R.Herschman,Primaryresponsegenesinducedbygrowthfactorsandtumor
promoters,Annu.Rev.Biochem.60:281319.(1991)281319.
3. E.Ito,L.A.Sweterlitsch,P.B.Tran,F.J.Rauscher,III,andR.Narayanan,InhibitionofPC
12celldifferentiationbytheimmediateearlygenefra1,Oncogene.5(1990)1755
1760.
4. Y.Levkovitz,K.J.O'Donovan,andJ.M.Baraban,BlockadeofNGFInducedNeurite
OutgrowthbyaDominantNegativeInhibitoroftheEgrFamilyofTranscription
RegulatoryFactors,J.Neurosci.21(2001)4552.
5. K.Maruyama,S.C.Schiavi,W.Huse,G.L.Johnson,andH.E.Ruley,mycandE1A
oncogenesaltertheresponsesofPC12cellstonervegrowthfactorandblock
differentiation,Oncogene.1(1987)361367.
6. B.Torocsik,J.M.Angelastro,andL.A.Greene,Thebasicregionandleucinezipper
transcriptionfactorMafKisanewnervegrowthfactorresponsiveimmediateearly
genethatregulatesneuriteoutgrowth,J.Neurosci.22(2002)89718980.
7. E.Vreugdenhil,S.M.Kolk,K.Boekhoorn,C.P.Fitzsimons,M.Schaaf,T.Schouten,
A.Sarabdjitsingh,R.Sibug,andP.J.Lucassen,Doublecortinlike,amicrotubule
associatedproteinexpressedinradialglia,iscrucialforneuronalprecursordivision
andradialprocessstability,Eur.J.Neurosci.25(2007)635648.
8. D.R.KaplanandF.D.Miller,Neurotrophinsignaltransductioninthenervoussystem,
Curr.Opin.Neurobiol.10(2000)381391.
9. B.D.RowlandandD.S.Peeper,KLF4,p21andcontextdependentopposingforcesin
cancer,Nat.Rev.Cancer.6(2006)1123.
10. R.varezRodriguez,M.Barzi,J.Berenguer,andS.Pons,Bonemorphogeneticprotein2
opposesShhmediatedproliferationincerebellargranulecellsthroughaTIEG1
basedregulationofNmyc,J.Biol.Chem.282(2007)3717037180.
11. S.D.Bamforth,J.Braganca,J.J.Eloranta,J.N.Murdoch,F.I.Marques,K.R.Kranc,H.Farza,
D.J.Henderson,H.C.Hurst,andS.Bhattacharya,Cardiacmalformations,adrenal
agenesis,neuralcrestdefectsandexencephalyinmicelackingCited2,anewTfap2
coactivator,Nat.Genet.29(2001)469474.
12. W.J.Weninger,F.K.Lopes,M.B.Bennett,S.L.Withington,J.I.Preis,J.P.Barbera,
T.J.Mohun,andS.L.Dunwoodie,Cited2isrequiredbothforheartmorphogenesisand
establishmentoftheleftrightaxisinmousedevelopment,Development.132(2005)
13371348.
13. S.L.Withington,A.N.Scott,D.N.Saunders,F.K.Lopes,J.I.Preis,J.Michalicek,K.Maclean,
D.B.Sparrow,J.P.Barbera,andS.L.Dunwoodie,LossofCited2affectstrophoblast
formationandvascularizationofthemouseplacenta,Dev.Biol.294(2006)6782.
14. S.Nakagomi,Y.Suzuki,K.Namikawa,S.KiryuSeo,andH.Kiyama,Expressionofthe
activatingtranscriptionfactor3preventscJunNterminalkinaseinducedneuronal
deathbypromotingheatshockprotein27expressionandAktactivation,J.Neurosci.
23(2003)51875196.
15. A.G.Pearson,C.W.Gray,J.F.Pearson,J.M.Greenwood,M.J.During,andM.Dragunow,
ATF3enhancescJunmediatedneuritesprouting,BrainRes.Mol.BrainRes.120
(2003)3845.
16. J.M.Angelastro,B.Torocsik,andL.A.Greene,Nervegrowthfactorselectively
regulatesexpressionoftranscriptsencodingribosomalproteins,BMC.Neurosci.3
(2002)3.
17. A.J.Brown,C.Hutchings,J.F.Burke,andL.V.Mayne,Applicationofarapidmethod
(targeteddisplay)fortheidentificationofdifferentiallyexpressedmRNAsfollowing
NGFinducedneuronaldifferentiationinPC12cells,Mol.CellNeurosci.13(1999)
119130.
18. L.Marek,V.Levresse,C.Amura,E.Zentrich,P.Van,V,R.A.Nemenoff,andL.E.Heasley,
Multiplesignalingconduitsregulateglobaldifferentiationspecificgeneexpressionin
PC12cells,J.CellPhysiol.201(2004)459469.
19. M.F.Dabrowski,P.AertsSFAUVanHummelen,P.F.VanHummelen,K.F.Craessaerts,
B.F.DeMoor,W.F.Annaert,Y.F.Moreau,andS.B.De,Geneprofilingofhippocampal
neuronalculture.
20. C.F.Peng,Y.Wei,J.M.Levsky,T.V.McDonald,G.Childs,andR.N.Kitsis,Microarray
analysisofglobalchangesingeneexpressionduringcardiacmyocytedifferentiation,
PhysiolGenomics.9(2002)145155.
21. M.N.Rivolta,A.Halsall,C.M.Johnson,M.A.Tones,andM.C.Holley,Transcriptprofiling
offunctionallyrelatedgroupsofgenesduringconditionaldifferentiationofa
mammaliancochlearhaircellline,GenomeRes.12(2002)10911099.
22. D.Vaudry,P.J.S.Stork,P.Lazarovici,andL.E.Eiden,SignalingPathwaysforPC12Cell
Differentiation:MakingtheRightConnections,Science296(2002)16481649.
23. K.Fujita,P.Lazarovici,andG.Guroff,RegulationofthedifferentiationofPC12
pheochromocytomacells,Environ.HealthPerspect.80:12742.(1989)127142.
24. N.F.Lambeng,J.WillaimeMorawekSFAUMariani,J.F.Mariani,M.F.Ruberg,and
B.Brugg,Activationofmitogenactivatedproteinkinasepathwaysduringthedeathof
PC12cellsisdependentonthestateofdifferentiation.
25. C.D.Nobes,J.B.Reppas,A.Markus,andA.M.Tolkovsky,Activep21Rasissufficientfor
rescueofNGFdependentratsympatheticneurons,Neuroscience.70(1996)1067
1079.
26. J.M.Aletta,DifferentialeffectofNGFandEGFonERKinneuronallydifferentiated
PC12cells.
27. K.Tamura,B.Hua,S.Adachi,I.Guney,J.Kawauchi,M.Morioka,M.TamamoriAdachi,
Y.Tanaka,Y.Nakabeppu,M.Sunamori,J.M.Sedivy,andS.Kitajima,Stressresponse
geneATF3isatargetofcmycinseruminducedcellproliferation,EMBOJ.%20;24
(2005)25902601.
28. H.Tsujino,E.Kondo,T.Fukuoka,Y.Dai,A.Tokunaga,K.Miki,K.Yonenobu,T.Ochi,and
K.Noguchi,Activatingtranscriptionfactor3(ATF3)inductionbyaxotomyinsensory
andmotoneurons:Anovelneuronalmarkerofnerveinjury,Mol.CellNeurosci.15
(2000)170182.
29. B.M.Engels,T.G.Schouten,D.J.van,I.Gosens,andE.Vreugdenhil,Functional
differencesbetweentwoDCLKsplicevariants,BrainRes.Mol.BrainRes.120(2004)
103114.
30. D.Hevroni,A.Rattner,M.Bundman,D.Lederfein,A.Gabarah,M.Mangelus,
M.A.Silverman,H.Kedar,C.Naor,M.Kornuc,T.Hanoch,R.Seger,L.E.Theill,E.Nedivi,
G.RichterLevin,andY.Citri,Hippocampalplasticityinvolvesextensivegeneinduction
andmultiplecellularmechanisms,J.Mol.Neurosci.10(1998)7598.
31. M.A.Silverman,O.Benard,H.Jaaro,A.Rattner,Y.Citri,andR.Seger,CPG16,anovel
proteinserine/threoninekinasedownstreamofcAMPdependentproteinkinase,J.
Biol.Chem.274(1999)26312636.
32. H.A.BurgessandO.Reiner,Doublecortinlikekinaseisassociatedwithmicrotubules
inneuronalgrowthcones,Mol.CellNeurosci.16(2000)529541.
33. H.Koizumi,T.Tanaka,andJ.G.Gleeson,Doublecortinlikekinasefunctionswith
doublecortintomediatefibertractdecussationandneuronalmigration,Neuron.49
(2006)5566.
34. J.C.Obenauer,L.C.Cantley,andM.B.Yaffe,Scansite2.0:Proteomewidepredictionof
cellsignalinginteractionsusingshortsequencemotifs,NucleicAcidsRes.31(2003)
36353641.
35. G.J.Schenk,B.Engels,Y.P.Zhang,C.P.Fitzsimons,T.Schouten,M.Kruidering,K.E.Ron
de,andE.Vreugdenhil,Apotentialroleforcalcium/calmodulindependentprotein
kinaserelatedpeptideinneuronalapoptosis:invivoandinvitroevidence,Eur.J.
Neurosci.26(2007)34113420.
36. A.Gdalyahu,I.Ghosh,T.Levy,T.Sapir,S.Sapoznik,Y.Fishler,D.Azoulai,andO.Reiner,
DCX,anewmediatoroftheJNKpathway,EMBOJ.23(2004)823832.
37. B.T.Schaar,K.Kinoshita,andS.K.McConnell,Doublecortinmicrotubuleaffinityis
regulatedbyabalanceofkinaseandphosphataseactivityattheleadingedgeof
migratingneurons,Neuron.41(2004)203213.
38. A.Shmueli,A.Gdalyahu,S.Sapoznik,T.Sapir,M.Tsukada,andO.Reiner,Sitespecific
dephosphorylationofdoublecortin(DCX)byproteinphosphatase1(PP1),Mol.Cell
Neurosci.32(2006)1526.
39. C.P.Fitzsimons,S.Ahmed,C.Wittevrongel,T.G.Schouten,T.F.Dijkmans,W.J.Scheenen,
M.J.Schaaf,E.R.deKloet,andE.Vreugdenhil,Themicrotubuleassociatedprotein
Doublecortinlikeregulatesthetransportoftheglucocorticoidreceptorinneuronal
progenitorcells,Mol.Endocrinol..(2007).
40. F.BarnabeHeiderandF.D.Miller,Endogenouslyproducedneurotrophinsregulate
survivalanddifferentiationofcorticalprogenitorsviadistinctsignalingpathways,J.
Neurosci.23(2003)51495160.
41. R.E.Thomson,F.Pellicano,andT.Iwata,Fibroblastgrowthfactorreceptor3kinase
domainmutationincreasescorticalprogenitorproliferationviamitogenactivated
proteinkinaseactivation,J.Neurochem.100(2007)15651578.
42. K.Boekhoorn,A.Sarabdjitsingh,H.Kommerie,P.K.de,T.Schouten,P.J.Lucassen,and
E.Vreugdenhil,Doublecortin(DCX)anddoublecortinlike(DCL)aredifferentially
expressedintheearlybutnotlatestagesofmurineneocorticaldevelopment,J.
CompNeurol.507(2008)16391652.
43. T.Shu,H.C.Tseng,T.Sapir,P.Stern,Y.Zhou,K.Sanada,A.Fischer,F.M.Coquelle,
O.Reiner,andL.H.Tsai,Doublecortinlikekinasecontrolsneurogenesisbyregulating
mitoticspindlesandMphaseprogression,Neuron.49(2006)2539.
44. S.Ohmae,S.TakemotoKimura,M.Okamura,A.chiMorishima,M.Nonaka,T.Fuse,
S.Kida,M.Tanji,T.Furuyashiki,Y.Arakawa,S.Narumiya,H.Okuno,andH.Bito,
Molecularidentificationandcharacterizationofafamilyofkinaseswithhomologyto
Ca2+/calmodulindependentproteinkinasesI/IV,J.Biol.Chem.281(2006)20427
20439.
45. J.S.daSilvaandC.G.Dotti,Breakingtheneuronalsphere:regulationoftheactin
cytoskeletoninneuritogenesis,Nat.Rev.Neurosci.3(2002)694704.
46. S.OkabeandN.Hirokawa,Actindynamicsingrowthcones,J.Neurosci.11(1991)
19181929.
47. K.PakCWFAUFlynn,J.FlynnKCFAUBamburg,andJ.R.Bamburg,Actinbinding
proteinstakethereinsingrowthcones.
48. F.M.Coquelle,T.Levy,S.Bergmann,S.G.Wolf,D.BarEl,T.Sapir,Y.Brody,I.Orr,
N.Barkai,G.Eichele,andO.Reiner,Commonanddivergentrolesformembersofthe
mouseDCXsuperfamily,CellCycle.5(2006)976983.
49. F.Suizu,Y.Fukuta,K.Ueda,T.Iwasaki,H.Tokumitsu,andH.Hosoya,Characterizationof
Ca2+/calmodulindependentproteinkinaseIasamyosinIIregulatorylightchain
kinaseinvitroandinvivo,Biochem.J.367(2002)335345.
50. P.Penzes,M.E.Cahill,K.A.Jones,andD.P.Srivastava,ConvergentCaMKandRacGEF
signalscontroldendriticstructureandfunction,TrendsCellBiol.18(2008)405413.
51. C.C.Fink,K.U.Bayer,J.W.Myers,J.E.Ferrell,Jr.,H.Schulman,andT.Meyer,Selective
regulationofneuriteextensionandsynapseformationbythebetabutnotthealpha
isoformofCaMKII,Neuron.39(2003)283297.
52. K.F.ShenandT.Meyer,DynamiccontrolofCaMKIItranslocationandlocalizationin
hippocampalneuronsbyNMDAreceptorstimulation.
53. H.KennedyMBFAUBeale,H.BealeHCFAUCarlisle,L.CarlisleHJFAUWashburn,
andL.R.Washburn,Integrationofbiochemicalsignallinginspines.
54. S.WaymanGAFAUKaech,W.KaechSFAUGrant,M.GrantWFFAUDavare,
M.F.Davare,H.ImpeySFAUTokumitsu,H.F.Tokumitsu,N.F.Nozaki,G.F.Banker,and
T.R.Soderling,Regulationofaxonalextensionandgrowthconemotilityby
calmodulindependentproteinkinaseI.
55. J.N.Jovanovic,F.Benfenati,Y.L.Siow,T.S.Sihra,J.S.Sanghera,S.L.Pelech,P.Greengard,
andA.J.Czernik,NeurotrophinsstimulatephosphorylationofsynapsinIbyMAP
kinaseandregulatesynapsinIactininteractions,Proc.Natl.Acad.Sci.U.S.A.93
(1996)36793683.
56. A.R.Means,Regulatorycascadesinvolvingcalmodulindependentproteinkinases,
Mol.Endocrinol.14(2000)413.
57. E.W.DentandF.B.Gertler,Cytoskeletaldynamicsandtransportingrowthcone
motilityandaxonguidance,Neuron.40(2003)209227.
58. S.F.Altschul,W.Gish,W.Miller,E.W.Myers,andD.J.Lipman,Basiclocalalignment
searchtool,J.Mol.Biol.215(1990)403410.
59. S.Impey,S.R.McCorkle,H.ChaMolstad,J.M.Dwyer,G.S.Yochum,J.M.Boss,
S.McWeeney,J.J.Dunn,G.Mandel,andR.H.Goodman,DefiningtheCREBregulon:a
genomewideanalysisoftranscriptionfactorregulatoryregions,Cell.119(2004)
10411054.
60. A.Chen,O.LaskarLevy,N.BenAroya,andY.Koch,Transcriptionalregulationofthe
humanGnRHIIgeneismediatedbyaputativecAMPresponseelement,
Endocrinology.142(2001)34833492.
61. J.W.GysbersandM.P.Rathbone,NeuriteoutgrowthinPC12cellsisenhancedby
guanosinethroughbothcAMPdependentandindependentmechanisms,Neurosci.
Lett.%20;220(1996)175178.
62. H.Thoenen,Neurotrophinsandneuronalplasticity,Science.270(1995)593598.
