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Dijkmans, T. F. (2009, October 14). Doublecortin-like kinase and neuronal differentiation. Retrieved from https://hdl.handle.net/1887/14055
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Chapter1
GENERALINTRODUCTION
OUTLINE
1. Neuronaldifferentiation
1.1. Neuronaldifferentiation
1.2. NerveGrowthFactor
1.3. PC12cellsandNGF
1.4 Molecularmechanismsofneuronaldifferentiation
2. TheDCLKgeneandneuronaldifferentiation
2.1. MembersoftheDCXgenefamily
2.2. TheDCLKgeneanditssplicevariants
2.3. MolecularmechanismsofDCXandDCLK
3. Glucocorticoidsandneuronaldifferentiation
4. Genomics
5. Scopeofthethesis
5.1. Objectives
5.2. Experimentalapproach
6. References
1.NEURONALDIFFERENTIATION
1.1Neuronaldifferentiation
The nervous system senses, processes and converts information relevant for the
animal into a behavioral response. Functioning of the nervous system is
determined by how its hundred billion neurons are connected and how those
connections are used. Therefore, the correct establishment of the neuronal
network of the central and peripheral nervous systems is essential for proper
function of the organism. During development, and to some extent in adult life,
neuronsarebornandintegratedintothisnetwork,aprocesstermedneurogenesis.
Each cell that will ultimately become a neuron was produced by cell proliferation
and given a neuronal identity by a process called neuronal differentiation. After
arrestincellproliferation,thedifferentiatingcellwillmigrate,elaborateatreelike
morphology,formsynapticconnectionsandacquiretheabilitytoreceiveandsend
electric signals using neurotransmitters. It is evident that the neuron in its end
stageistheresultofintricateinteractionswithinthedifferentiatingcell,withother
cells and with their products. However, detailed knowledge of the underlying
molecular biology of neuronal differentiation is far from complete and should be
pursued.
1.2NerveGrowthFactor
Nerve Growth Factor (NGF) is known to be an important regulator of neuronal
differentiation. Originally, NGF was discovered by Rita LeviMontalcini and Viktor
Hamburgerintheearly1950s[1].Theseresearchersinvestigatedhowtherelatively
unspecialized cells of an embryo develop into a neuron of the spinal cord, which
connects to a muscle cell in the limb. Experiments indicated that removal of limb
budsfromchickenembryosresultedindepletionofmotorneuronsinthechicken
spinal cord, which would have innervated the limb bud otherwise. Inversely,
transplantinganextralimbbudontotheembryoresultedinanincreaseofmotor
neuronsinthespinalcord.Therefore,thelimbbudseemedtoprovidetheneurons
with some signal that was important for their survival. In additional experiments,
thelimbbudwasremovedandreplacedwithamousesarcomatumor.Surprisingly,
thetumorwasheavilyinnervatedevenmorethananormallimbbudwouldhave
been.Itturnedoutthatthesetumorcellssecretedonespecific,solublefactorthat
explained their observations. This factor was Nerve Growth Factor (NGF) and its
investigatorsRitaLeviMontalciniandStanleyCohenwereawardedtheNobelPrize
forPhysiologyorMedicinein1986[2].
Today, it is known that NGF regulates the survival and differentiation of
neuronalpopulationsintheperipheralaswellasinthecentralnervoussystem[3
6]. During outgrowth, NGF acts as aneurotropin, meaning that NGF directs the
growth cones of extending neurites towards the NGFsecreting source (<Greek,
trop: "turn").In vivo, this source is the target cell which will be consequently
innervatedbythedifferentiatingneuron.NGFalsoactsasaneurotrophin(<Greek,
troph:"food,nutrition").Duringdevelopment,apopulationofneuronscompetes
to innervate thetargetcell. However, NGF 'nourishes' a subset ofneuronsonly
for which NGF acts as a survival factor while the remaining neurons 'starve' and
disappear through neuronal death by apoptosis. NGF finalizes innervations by the
formation and the maintenance of synaptic connections of the neuron with its
targetcell.Afterdifferentiation,NGFretainstheabilitytoregulatevariousaspects
of neuronal function and is thus considered as a modulator of neuronal plasticity
[5].
1.3PC12cellsandNGF
The PC12 cell line has proven invaluable to elucidate the role of NGF in neuronal
differentiation [7]. This cell line was isolated by Greene and Tischler from a
pheochromocytoma that arose in the adrenal medulla of a rat in 1974 [8]. When
PC12cells aretreated with NGF, they model both neuronal survival and neuronal
differentiation [9]. First, PC12 cells serve as a model for neurotrophic actions of
NGF by showing dependence for their survival on NGF. In general, growing of
(undifferentiated) PC12 cells in culture for subsequent experiments is performed
with medium containing serum. This serum supplies PC12 cells with necessary
growthfactorsforproliferationandviability.WhenPC12cellsaredifferentiatedby
NGF in serumcontaining medium, NGF withdrawal will dedifferentiate the PC12
cells,butnotcausecelldeath.However,whenculturedinserumfreemedium,no
growth factors are available for PC12 cells and they will undergo apoptosis.
Nonetheless, when NGF is present in the medium, cells will differentiate and
apoptosisisprevented.WhenNGFiswithdrawnfromtheserumfreemediumonce
thecellshavecompleteddifferentiation,theneuronlikecellswilldiebyapoptosis.
Thus, maintaining PC12 cells in serumfree medium with NGF mimics the NGF
dependenceobservedininvivoneurons.
Second, PC12 cells serve as a model for neuronal differentiation in
responsetoNGF.Inculture,PC12cellsareactivelydividingcells.Whentreated,the
cells stop dividing and develop long, branched processes typical for neurons (Fig.
1A). In line with NGF as a neurotropin, these processes grow towards the NGF
source.WhenNGFisaddedtotheculturemediumofgrowingPC12cells,however,
no discrete source of NGF is present and the processes extend, with no readily
discernible direction. To achieve directed growth, when required, specialized
culture dishes have been developed that contain two compartments. In the first
compartment,thePC12cellsgrowuntilinthesecondcompartmentNGFisadded.
Inresponse,theNGFcontainingcompartmentbecomesinvadedbyneuritesofthe
PC12 cells. Thesein vitro assays have been informative about how NGF guides
outgrowth, but also about how NGF signals across the long distances from the
neurite tip to the nucleus. In addition to cessation of proliferation and
morphologicalchange,differentiatingPC12cellsdevelopelectricalexcitabilityand
increasedsensitivitytoacetylcholine.Therefore,thecelllineisasuitablemodelfor
electrophysiological properties of neurons. Moreover, when NGFstimulated PC12
cellsarecoculturedwiththeskeletalmusclelineL6,theywillconnectastotarget
Figure 1. Schematic overview of NGF-induced neuronal differentiation in PC12 cells. A). Ns-1 PC12 cells before and after 24 hours of NGF-stimulation. B). Upon NGF stimulation, the TrkA receptor dimerizes and activates 3 central signaling pathways (PI3K, PLC and MAPK).The MAPK pathway transduces the NGF signal into nucleus and activates a number of transcription factors, including CREB, Elk-1 and SRF. Within minutes, immediate-early gene transcription occurs as a result of their activation. After this first wave of gene expression, others follow (delayed-response genes). "P" indicates phosphorylation-dependent activation.
cellswithfunctionalcholinergicsynapses.
NGF induces the PC12 pheochromocytoma cells to obtain a neuronal
phenotype that resembles catecholaminergic, sympathetic neurons [9]. Also the
normalcell counterparts of undifferentiated PC12 cells, chromaffin cells of the
adrenal medulla, respond to NGF treatment by converting into sympathetic
neurons.However,thisresponseremainssuppressedinvivobylocalhighlevelsof
glucocorticoids [10]. Both sympathetic neurons and chromaffin cells derive from
stem cells of the neural crest, from which NGF differentiates the neurons.
Therefore, PC12 cells appear to possess the pluripotency of such stem cells,
allowingtheadoptionofasympatheticneuronlikephenotypeinresponsetoNGF
treatment [9]. Evidently, observed cellular or molecular mechanisms in
differentiating PC12 cells cannot be extrapolated to occur ininvivosystemsorin
differentiation of other types of neurons per se. However, much insight retrieved
fromthePC12cellmodelhasprovenmeaningfulinbroadercellularcontexts[4].In
addition,thereadilycontrolledexperimentalconditions,thehomogeneityofPC12
cell culture versus primary cell culture and the availability of sufficient cellular
materialhaveoftenmadePC12cellsthemodelofchoicetoinvestigatemolecular
mechanismsofneuronaldifferentiation.
1.4Molecularmechanismsofneuronaldifferentiation
When presented to the PC12 cell, NGF binds to the Tropomyosinrelated Kinase
receptorA(TrkA;Fig.1B;[3]).TheTrkAreceptorisareceptorthatspansacrossthe
cell membrane, having an extracellular domain, which can bind NGF, and an
intracellulartyrosinekinasedomain.WhenNGFbindstoTrkAreceptors,oneNGF
complex will bind two TrkA receptors and thus generate a TrkA dimer. The two
intracellular kinase domains are now in such vicinity of each other that the TrkA
proteins will crossphosphorylate each other and initiate intracellular signaling of
NGF. In theory, this mode of signaling does not necessitate entrance of NGF into
the cell; however, it appears that at least some signaling is dependent on
internalization of the NGF complex by endocytosis [11]. This complex contains a
number of proteins, including NGF, TrkA receptors, kinases and adaptor proteins
[12]. Disregarding endocytosis, NGF activates a number of downstream signaling
cascades,whichareoftencomplexandinterconnected.However,ingeneralthree
major signaling pathways are discriminated (Fig 2B; [4]). First, the
Phosphatidylinositol 3Kinase (PI3K) pathway is induced by NGF and leads to
activation of Akt kinase. Second, NGF activates Ras after which the Mitogen
ActivatedProteinKinase(MAPK)ExtracellularsignalRegulatedKinase1/2(ERK1/2)
pathway is induced. And third, the Phospholipase C (PLC ) pathway is induced,
leadingtothereleaseofintracellularCa2+andactivationofProteinKinaseC(PKC).
It is thought that the ERK 1/2 and PLC pathways primarily stimulate processes
responsible for neuronal differentiation and survival, while the PI3K pathway is
primarilyinvolvedinsurvival.
The MAPK ERK 1/2 pathway plays a central role in mediating the cellular
responseinPC12cellsupondifferentstimuliincludingNGF.Thispathwayinvolves
the sequential phosphorylation chain of the kinases RasRafMEKERK 1/2. When
signaling through this cascade is inhibited, NGF is unable to achieve neuronal
differentiation.ItappearsthatdurationofthesignalingeventsoftheactivatedERK
1/2 pathway is of particular importance to the cell [13]. This is illustrated by the
fact that Epidermal Growth Factor (EGF) and NGF both activate the ERK 1/2
cascade, but promote proliferation and differentiation, respectively. It has been
shownthat thesustained activation by NGF versus a more transientactivationby
EGF of this pathway, determines the commitment of PC12 cells to a program of
differentiation. Typically, the activation of ERK 1/2 by NGF lasts several hours,
whereas activation by EGF barely lasts one hour. These temporal differences in
activationoftheERK1/2pathwaymaybeexplainedbydifferentialrecruitmentof
several proteins, including PI3K, to activated TrkA, but not to the EGF receptor
complex[14].
After minutes of NGF exposure, signaling of the ERK 1/2 pathway and
others induce the expression of a class of genes termed immediateearly genes
(IEGs; Fig. 1B and 2A; [15]). Upon phosphorylation, ERK 1/2 directly or indirectly
phosphorylates a number of transcription factors present in PC12 cells. For
instance, ERK 1/2 uses p90RSK as a substrate, which in its turn phosphorylates
transcription factor CREB [16]. Alternatively, ERK 1/2 directly phosphorylates
transcription factor Elk1. Other transcription factors that are activated after NGF
treatment include SRF, cMyc and CBP. Then, the activated transcription factors
bind to specific response elements in the promoters of their target genes and
induceafirstwaveofgeneexpression.WiththeinductionoftheseIEGs,orprimary
response genes, the cell has commenced the transcriptional program of neuronal
differentiation [17]. IEGs have a number of typical characteristics. Above all, IEGs
are defined by genes of which the expression can be increased without protein
synthesisofothergenesinthestimulatedcell.Asanexample,prototypicalIEGc
FosexpressionisinducedinPC12cellsbyNGFasadirectresultfromtranscription
drivenbyendogenousCREB,after itsphosphorylation [18].Anothercharacteristic
is that IEG expression is in general low prior to stimulation, strongly increases
within1hour(101102foldinmanycelllines)andreturnstobasallevelswithintwo
hours after stimulation. IEGs appear to comprise a relatively small set of ~100
genes, which are induced by a variety of different stimuli in many different cell
lines[17].Specificityofthecellularresponseinagivencontextcanbederivedby
IEG induction that is differential in the exact set of responsive genes, in their
induction dynamics and in their posttranscriptional or posttranslational
modifications.Asanexample,NGFinducesneuronaldifferentiationinPC12cellsby
ERK1/2mediatedexpressionandphosphorylationofcJun[19].Botheventshave
been shown to be required for neuronal differentiation and thus illustrate two
equally important levels of regulation [20]. Also, cFos induction in PC12 cells is
importantforneuronaldifferentiation;however,cFosisinducedbybothEGFand
NGF. NGF, and not EGF, also induces cJun expression, which cooperates with c
Fostopromotespecificallythedifferentiationprogram[21].Afinalcharacteristicof
IEGs is that many, yet not all, encode transcription factors [17]. Therefore, IEG
transcriptionfactorsarethoughttosteerthetranscriptionalstatusofthecellinto
its next phase of differentiation. Genes that are regulated by IEGs are termed
delayedresponsegenes(DRGs;Fig.1Band2B).
Figure 2. Overview of NGF-induced transcriptional regulation in PC12 cells. When PC12 cells are treated with NGF, typically 2 phases of transcriptional regulation are discriminated. A). Within 2 hours, NGF induces immediate-early gene (IEG) transcription, which involves a typical strong and transient induction of ~100 genes. These are often, yet not always, transcription factors. Across this time period, no clear morphological changes are observed. B). During days of NGF- treatment, PC12 cells adopt the neuronal identity, which is reflected by the transcriptome. The delayed-response genes mechanistically follow the immediate-early genes and encode the more typical neuronal constituents.
1 2 3 4
Time (days)
0 15 30 60 120
Time (min)
+ NGF
Gene expression (arbitrary)
Immediate-early genes Delayed-response genes
A B
Morphology
In contrast to IEGs, DRGs are genes whose expression is dependent on
protein synthesis of other genes [22]. Also, regulation of expression of DRGs is
typicallyobservedafterdaysofNGFtreatment,ratherthanminutesorhours.And
where IEGs may appear to reflect a more generalized transcriptional response,
NGFresponsive DRGs in PC12 cells encode the more typical constituents of
neurons [22]. Considering the properties of a neuron, regulated genes may be
anticipatedtoincorporatecytoskeletalproteins,vesicleproteins,neurotransmitter
enzymes, neurotransmitter receptors, and voltage dependent channels. Indeed,
many such genes have been found. Several tubulin types and microtubule
associated proteins (MAPs), such as Tau and MAP1, are increased upon NGF
treatment, correlating well with the observed neuritogenesis [23]. Also the actin
cytoskeletonishighlydynamicduringoutgrowthandexpressionofrelatedgenesis
regulated after NGF exposure [22]. Actin dynamics plays a particularly important
roleinneuronaloutgrowthasactinisakeyconstituentofthegrowthcone,which
leads the extending neurite. In support, Growth associated protein 43 (GAP43),
which regulates growth cone dynamics through actin is increasingly expressed by
NGF in PC12 cells [24;25]. In addition, neurotransmitter enzymes like tyrosine
hydroxylase, acetylcholinesterase and choline acetyl transferase have been
reported to be increased by NGF. The ability for neuronal transmission is further
induced, by increasing the expression of calcium, sodium and potassium channels
byNGF[9;26;27].
2.THEDCLKGENEANDNEURONALDIFFERENTIATION
2.1MembersoftheDCXgenefamily
IncreasingevidenceisavailablethatshowsthatmembersoftheDoublecortin(DCX;
[28])genefamilyareimportantfortheinvivodifferentiatingneuron.Inhumanand
mouse,theDCXgenefamilyencompasses11paralogues(Fig.3;[29]).Amongthe
DCX family proteins, at least four conserved domains are found. By definition, all
DCXfamilyproteinshaveaDCXdomain,throughwhichtheycanbindmicrotubules
and which classifies them as MAPs [30]. Second, a ricintype betatrefoil lectin
domainisfoundinFLJ46154,whichmayconferbindingtocarbohydrates.Third,a
serine/threonine protein kinase domain is found in both human and mouse
proteins DCLK, DCLK2 and DCLK3. Fourth, DCX and the DCLK proteins have in
between their DCX and kinase domain a serine, threonine and proline (SP)rich
domain,knowntobeaproteininteractiondomain[3133].
In human, mutations in the prototypical, Xlinked DCX gene result in
mental retardation and epilepsy (lissencephaly or subcortical band heterotopia
(SBH)), whereas mutations in RP1 are associated with blindness (retinitis
pigmentosa)andmutationsinDCDC2areassociatedwithdyslexia[3436].Eachof
theseneuropathologiesappearstobecausedbyaberrantneuronaldifferentiation.
In the retinal photoreceptor, membrane stacks that contain photopigment are
continuously regenerated to maintain the eye's visual function [37]. To achieve
Figure 3. Overview of the DCX gene family. Schematic representation of proteins produced by the DCX gene family. Protein variants are known for the different genes, however, full-length mouse and human proteins are depicted in order to show all relevant domains. The lower scale bar indicates protein size in number of amino acids. Parentheses enclosing DCX-domains indicate that in the murine orthologue, a DCX-domain has not been found. Two vertical lines (| |) are used to indicate variable proteins lengths to match differences in human and mouse orthologues. Right-pointing arrows indicate protein lengths over 800 amino acids. Positions of DCX-domains have been aligned vertically for clarity, while exact positions within the proteins show more variation. DCX: DCX domain, SP: serine, threonine and proline-rich domain, RLD: Ricin-type beta-trefoil lectin domain, Kinase:
Kinase-domain.
( )
1100200300500400600700800aaDCX DCLK DCLK2 RP1 RP1L
DCLK3 DCDC2A DCDC2B DCDC2C FLJ46154
DCDC1 Bac26042
Kinase Kinase Kinase
DCXDCXSP DCXDCXSP DCXDCXSP DCX DCXDCX DCXDCX DCX DCXDCX DCXDCX DCX DCX
RLDDCX
( )
DCX
humanonly mouseonly
this, there is a continuous flow of membrane stack constituents from one
subcellular compartment into the membrane stack containing compartment. This
intracellular flow relies on microtubule based transport and is regulated by MAPs
[37]. Mutations in RP1 cause the photoreceptor to degenerate and leads to
blindness, seemingly due to faulty regulation of microtubule based transport or
cytoskeletal structure in these photoreceptors [38;39]. Interestingly, NGF and
closelyrelatedneurotrophinsregulatetheintegrityofphotoreceptors,suggestinga
mechanisticlinkbetweenRP1andneurotrophins[40;41].Inthedevelopingcortex,
neurons areproducedby proliferation and subsequent differentiation,which is in
part regulated by neurotrophins [42;43]. After proliferation, the neuron migrates
by repeatedly extending a neurite, 'pulling' its nucleus into the neurite and
retracting its trailing process [44]. Clearly, the cytoskeleton is criticallyinvolved in
theseprocessesandinvolvesboththeactinandthemicrotubulecytoskeleton[45].
The microtubules provide stability to the extending protrusions, are important in
'pulling' the nucleus and form the basis of the mitotic spindle. It is thought that
mutations in DCX and DCDC2 produce associated pathologies by impinging on
these microtubule dynamics and thus affecting proper neuronal differentiation
[36;46;47].
Also DCLK is involved in neuronal differentiation. As yet, no
neuropathologies have been directly linked to DCLK function; however, DCLK and
DCX appear to be implicated in similar processes. For instance, where point
mutations in the human DCX gene cause lissencephaly or SBH, DCX knockout in
mousemodelshaslittleeffectoncortexarchitecture[4850].However,whenDCLK
isknockedoutinadditiontoDCX,aphenotypedevelopsthatresemblesthehuman
corticaldisordermoreclosely[51;52].Therefore,itappearsthatthereisadegree
ofredundancybetweenDCXandDCLKfunction.Thelattermaybeinlinewiththe
fact that DCLK is a close paralogue of DCX. However, DCLK also has a number of
properties that are distinct from DCX, which may underlie distinct functionalities.
First, the DCX domains in DCLK are not identical to those in DCX and may entail
differentmicrotubulebindingproperties.Second,DCLKisalargerproteinthanDCX
and additional domains, such as the kinase domain, can provide additional
functionality (Fig. 3). Moreover, DCLK produces several different splice variants,
someofwhichlackDCXdomains.Theselatterproteinsarelikelytohavefunctions
that are more independent of the microtubule cytoskeleton. This thesis will
address the function of such a type of splice variant, named DCLKshort, in NGF
induced neuronal differentiation of Ns1 PC12 cells. In the next section (2.2), the
known different DCLK splice variants will be introduced. Also, knowledge on
associatedin vivo expression data will be summarized. Then, a brief, more
molecularperspectivewillbegivenonhowDCXandDCLKproteinsareregulatedin
thecell(2.3and2.4).ThisaimstocreateaframeworkfromwhichpossibleDCLK
shortfunctionmaybeviewedandinvestigated.
2.2TheDCLKgeneanditssplicevariants
The DCLK gene produces multiple transcripts from 20 exons, making use of two
alternativepromotersandadditionalsplicingroutes(seeFig.4;[5356]).According
tocurrentknowledge,thisisnotthecaseforitsclosestparalogueDCLK2,orother
DCXfamilygenes[57;58].Afirstsplicevarianttype,calledDCLKlongA(alsocalled
KIAA0369)isproducedfromtheupstreampromoter[59].DCLKlongisthelongest
transcripttypeandthereforecontainsthemostfunctionaldomainsencodedbythe
DCLK gene. Beside the kinase and SPrich domain, it contains DCX domains that
enable the protein to bind to microtubules [60]. Two similar splice variants in
additiontoDCLKlongAhavebeenidentified.OnecontainsadifferentCterminus
(DCLKlongB)andasecondcontainsashort16aminoacidinsert(DCLKlongC)[61
63].Asecondsplicevariant type is also derived from theupstreampromoter and
resemblestheDCXprotein,forwhichitistermedDoublecortinlike(DCL;[6466]).
The mouse DCL mRNA encodes a protein of 362 amino acids that shares 73%
sequenceidentitywithDCXoveritsentire length.Athirdtypeofsplicevariantof
theDCLKgeneisderivedfromthedownstreampromoterandcalledCaMKrelated
peptide (CARP; also called Ania4). CARP encodes a 56 amino acid long peptide,
which largely overlaps with the Nterminal part of DCL and therefore mainly
consists of the SPrich domain [67;68]. A final splice variant of the DCLK gene is
DCLKshort A (or cpg16; [62;69;70]). This splice variant also originates from the
downstream promoter. DCLKshort encodes the SPrich domain and the kinase
domain.AsforDCLKlong,twosimilarsplicevariantsinadditiontoDCLKshortA(B
andC)havebeenidentified[63].
Figure 4. Splice variants of the DCLK gene. Schematic representation of different proteins derived from the DCLK gene. Four types of DCLK proteins exist in man, rat and human, that consist of combinations of the DCX-domain, the SP-rich domain and the kinase-domain. The 2 promoters that give rise to the different gene products are indicated relative to the derived proteins. Sizes are indicative.
DCLKlong
DCL
CARP
Kinase
DCX DCX SP
DCX DCX SP
SP
Kinase
SP DCLKshort
100 200 300 400 500 600 700aa
1
Upstream
promoter
Downstream
promoter
The most comprehensive expression studies have been performed in
murinetissue.Here,expressionofDCLKtranscriptsispredominantly,althoughnot
exclusively, found in the developing and adult nervous system [29;59;67]. DCL
expressionisdetectedasearlyasembryonicday8inthedevelopingcortex,before
DCX expression and ceases after development [66;71]. DCL expression was
observed in mitotic cells, radial glia cells and radial processes. DCLKlong variants
expressionoccursonday12to15inthecortex,seeminglyinpostmitoticneurons
[55;60;64]. CARP was detected as a kainate and dopamineinduced transcript in
the adult rat hippocampus and striatum, respectively [67;68;72]. Within the
hippocampus of adrenalectomized rats, CARP specifically localizes to apoptotic
neurons,andhasbeenreportedtofunctioninneuronalapoptosis[73].DCLKshort
variant expression is mostly observed in the adult brain and is associated with
neuronalplasticity[59;63;64;69;70].
2.3MolecularmechanismsofDCXandDCLK
Interaction of DCX with other proteins involves extensive phosphoregulation.
Phosphorylation of DCX has been reported to be mediated by kinases CDK5, JNK,
PKA and MARK, whereas dephosphorylation involves phosphatases PP1 and PP2.
DCX has been reported to specifically colocalize with microtubules in leading
processes of migrating neurons and in growth cones of stationary neurons, while
microtubules are in principle found throughout the cell [74;75]. The specific
interactionofDCXwithmicrotubulesinneuritetipshasshowntobemaintainedby
phosphatase activity of PP2. When okadaic acid is applied to inhibit PP2 activity,
DCX translocates towards the cell body, where microtubules are also present.
MARKandPKAseemtocounteractthisinteractionofDCX,asa.o.theirmediated
phosphorylation of DCX reduces microtubule affinityin vitro [74;75]. In a similar
fashion, CDK5 phosphorylates DCX in its SPrich domain and thereby affects
neuronal migration. Using a neuron migration assay, overexpression of DCX
resulted in enhanced migration, whereas mutation of a CDK5 phosphosite, or
pharmacologicalCDK5inhibition,neutralizedthiseffect.Alsohere,phosphorylation
decreasedmicrotubuleaffinity[31].
Where phosphorylation may drive DCX away from the microtubule
cytoskeleton, it appears that it also increases affinity of DCX for the actin
cytoskeleton.PhosphorylationofDCXbyJNKoccursatitsSPrichdomain[32].DCX
phosphomutants for these residues differentially affected neurite outgrowth of
NGFstimulatedPC12cellsandmotilityofprimarycerebellarneurons.Interestingly,
JNKphosphorylatedDCXwasenrichedintheactinrichregionofgrowthcones.In
line with this, DCX has been reported to interact with actin proteins, directly and
indirectly through Neurabin II [7679]. In addition toin vitro evidence, it was
demonstrated that human DCX mutations can cause loss of interaction with
NeurabinII,andthatNeurabinIIandDCXarecoexpressedinmanyregionsofthe
developing brain [77]. Moreover, use of phosphomutants suggests that also
phosphorylation of DCX by PKA/MARK decreases microtubule binding and
increases Factin binding, in the presence of Neurabin II [78]. In addition, data
suggest that Neurabin II mediates dephosphorylation of JNK and CDK5
phosphorylation sites in the SPrich domain of DCX inin vivo migrating neurons
[74;80]. Again, phosphorylation correlated with increased Factin binding and
dephosphorylationwithincreasedmicrotubulepolymerization.Thus,regulationof
phosphorylation and dephosphorylation of DCX is an important mode to govern
cytoskeletalorganization[45].
In extension of previous examples, the SPrich domain appears to be an
important interaction platform for multiple proteins. The SPrich domain of DCX
andDCLhasbeenshowntointeractwiththe1and2subunitsofadaptorprotein
complexes, which are components of clathrin coated vesicles [81]. Also, DCL has
been shown transport the glucocorticoid receptor, dependent on the SPrich
domain [82]. In addition, CARP was shown to interactin vitro with Growth
ReceptorBound2(Grb2),mostlikelythroughitsSPrichdomain[73].Typically,this
adaptor protein is implicated in retrograde transport of growth factor complexes
over microtubules [4]. Finally, the SPrich domain of DCLKshort contains a PEST
domain, which is subject to cleavage by calpains and caspases during neuronal
apoptosis [83;84]. Where the Nterminal cleavage fragment of DCLKshort (a
peptide similar to CARP) exacerbates apoptosis; an uncleavable DCLK mutant
protectsagainstapoptosis[83].
The function of the DCLK kinase domain remains largely elusive, due to
limitedinformationonhowkinaseactivityisregulatedandontheexactidentityof
physiologicalsubstrates.DCLKkinasesshowsubstantialhomologytoCamKinases2
and1/4andappeartohavesubstratepreferencessimilartoCamKinases[63;69].
Notonlythegeneralkinasesubstratemyelinbasicprotein,butalsothetypicalCam
Kinasesubstratesautocamtide2andsyntidehavebeenreportedtoserveasDCLK
substratesin vitro [63;64;69]. Using synthetic peptide substrates modeled on
SynapsinI,asubstraterecognitionmotifforDCLKwasderived[85].
Overexpression of DCLKshort was reported to partially inhibit cAMP
stimulated transcriptional activity of cAMP responsive element binding protein 1
(CREB; [69]). Potentially, this is a more conserved regulatory feature among DCLK
kinases:activatedDCLK,DCLK2and3,atleastbytruncationofitsautoinhibitoryC
terminal domain, inhibits CREBmediated gene transcription while preventing
nuclear import of Transducer Of Regulated CREB Activity 2 (TORC2) in COS7 cells
[86].Remarkably,CREBinhibitionisincontrastwithclassic,activatedCamKinases,
which are known to phosphorylate CREB and consequently enhance CREB
mediated gene transcription. In addition to the mentioned substrates, DCLK
displayssubstantialautophosphorylation[62;63;86].Aninterestingobservationhas
been made by Edelman and colleagues, where they provideinvitroexperimental
supportforaconnectionbetweenautophosphorylationofDCLK2andmicrotubule
interaction [57]. Enzymatic removal of autophosphorylation groups from DCLK2
greatly increased microtubule affinity, whereas selfphosphorylation seemed to
produce DCLK2 protein of lesser affinity. In principle, this type of
phosphoregulation is very similar to the earlier discussed CDK5, JNK, MARK and
PKAandphosphatasePP1andPP2.
Upstreamregulatory events of kinase activity ofDCLKproteinsremainso
far unknown. Catalytic activity can be artificially increased by mutagenesis of a
DCLKsite,correspondingtotheactivationloopsiteforregulationofCamKinase1
by CamKinase Kinase [85]. Further activation of DCLK by Cterminaltruncationof
anautohibitorydomainresultsin~6foldincrease,similartoCamKinases[69;86].
UnlikeCamKinases,activationofDCLKkinasesseemstobecalciumandcalmodulin
insensitive.IthasbeenreportedthatspecificallycAMPraisingagentsaffectDCLK
short kinase activity, however other studies could not reproduce this observation
[60;69;85].
3.GLUCOCORTICOIDSANDNEURONALDIFFERENTIATION
Glucocorticoid hormones are important regulators of neuronal function [87]. In
mammals, glucocorticoid hormones can affect behavior, electrophysiology,
neuronal morphology and neurogenesis [8891]. However, chronic high levels of
glucorticoids cause nonadaptive behavior, which is associated with impaired
neuronal function. Neurogenesis, which may be divided in proliferation, survival
and differentiation, is suppressed by lasting high levels of glucocorticoids [9295].
Moreover, it has been shown that raised glucocorticoids inhibit differentiation of
progenitorcellsintoneuronsinthehippocampus[95].
Underlyingmolecularmechanismsofmaladaptiveglucocorticoidsignaling
appear to involve altered gene expression. When released glucocorticoids enter
neurons,theycanbindthemineralocorticoidreceptor(MR)andtheglucocorticoid
receptor (GR; [96]). Typically, the MR has a higher affinity for its glucocorticoid
ligandthantheGR.Consequently,atlowerconcentrations,onlytheMRisoccupied
bytheligand.However,athigherintracellularconcentrations,boththeGRandthe
MRareoccupied.Duringchronicstress,glucocorticoidlevelsarehighandresultin
prolonged GR activation. In general, two modes of genomic actions are
discriminated for the GR. First, transactivation is binding of the activated GR to
regulatoryDNAregionsoftargetgenesanddirectactivationofgenetranscription
by recruitment of components of the transcription machinery. Second,
transrepressionisbindingoftheactivatedGRtoothertranscriptionfactors(suchas
NFB and CREB) and inhibiting their ability to drive gene transcription. As a
transcription factor, the GR can thus change expression of genes and provide a
mechanistic connectionbetween (chronic) glucocorticoid signaling andchanges in
neuronalfunction[87;97].
Previously, we and others identified DCLK as a candidate gene that is
repressedinthehippocampusbyglucocorticoidsandhasthepotentialtoregulate
neuronaldifferentiation[9799].However,littleisknownaboutthetranscriptional
regulationofDCLKandwillbeaddressedinthisthesis.
4.GENOMICS
In the past two decades, technologies have emerged that allow genomewide
measurements of gene expression. Particularly Serial Analysis of Gene Expression
(SAGE) and DNA microarrays have paved the way for inspecting the cell's
transcriptome at once [100]. Coming from the genome sequencing projects that
made these technologies possible, such an approach appears to do justice to the
complexity of a living cell. Taking into account the 20 30 000 genes of a
mammalian genome,it seems important to assay many genes atonce. Inevitably,
data sets retrieved from this type of experiments are large and require a
meaningful experimental design. In principle, gene expression profiling reveals no
morethantowhatextent,whichgenesareexpressed.However,thesedatacanbe
usefulwhenstudyingNGFinducedneuronaldifferentiation[22;101].
First, DNA microarray experiments can be used as a screening tool. As described,
onset of NGFinduced neuronal differentiation of PC12 cells is reflected on a
transcriptional level by the induction of IEGs. Although such genes have been
identified, the complexity of the biological process and the availability of modern
DNAmicroarrayssuggestthatnewNGFresponsiveIEGsmaybediscovered.Given
the typical strong and transient response profiles of IEGs, genomewide
quantification of expression after NGF stimulation may yield important new NGF
responsivegenes.
Second, DNA microarray experiments together with current bioinformatics can
providequitedetailedfunctionalinsights[102].Ofmanygenes,biologicalfunctions
areknown,basedonresearchorpredictedfromsequencehomologieswithother
genes.WhenNGFinducesneuriteoutgrowthofPC12cells,thisislikelytoreflectin
thetranscriptomebyincreasedregulationofcytoskeletonrelatedDRGs.Moreover,
other gene classes, which are less obvious may also be identified bethese means
anduncoverunrecognizedaspectsofdifferentiation.
5.SCOPEOFTHETHESIS
5.1Objectives
ThecentralgoalofthisthesiswastoidentifyandanalyzegenesimportantforNGF
induced neuronal differentiation of Ns1 PC12 cells with a particular focus on
DCLKshort. The first objective was therefore to perform a DNA microarray
screening assay to identify previously unrecognized NGFresponsive IEGs. The
secondobjectivewastoperformaDNAmicroarrayexperimentthatcharacterized
andanalyzedNGFresponsiveDRGs.Thethirdobjectivewastoanalyzethefunction
of the DCLKshort in NGFinduced differentiation. The fourth and final objective
wastostudytranscriptionalregulationofDCLKshortinNs1PC12cells.
5.2Experimentalapproach
InordertostudyNGFinducedIEGswithapotentialroleinneuronaldifferentiation,
the Ns1 PC12 cells were stimulated with NGF for a maximum of 2 hours, after
which transcriptional changes were measured using the Expression Array System
from Applied Biosystems. Chapter 2 describes the exact experimental setup,
performed analysis and obtained results. Chapter 3 extends this approach by
stimulating for a maximum of 4 days, measuring and analyzing genomewide
expression,usingthesametypeofDNAmicroarray.Onetranscript,DCLKshortwas
found to be strongly and persistently induced in response to NGF treatment.
Chapter4analyzestheroleofDCLKshortinneuronaldifferentiationbyi)validating
DCLKshort upregulation at protein level, ii) characterizing ERK 1/2mediated
phosphorylationofDCLKshortproteinandiii)showingthatthisphosphorylationis
involvedinneuritogenesisbyusingoverexpressionofDCLKshortphosphomutants.
Chapter5isdedicatedtoidentifytranscriptionfactorsresponsibleforregulationof
DCLKshortexpressioninNs1PC12cells.Here,insilicopromoteranalysiswasused
toidentifycandidatetranscriptionfactors,afterwhichaputativecAMPresponsive
element and different signaling pathways were validated by quantitative PCR,
luciferaseassaysandtheuseofpharmacologicalinhibitorsandstimulants.Chapter
6discussesthegenerateddataandplacestheseinbroadercontext.
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