University of Groningen
Myelin biogenesis
Ozgen, Hande
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Chapter 7
Summary and Future Perspectives &
Nederlandse Samenvatting
Hande Ozgen, Dick Hoekstra, Nicoletta Kahya and Wia Baron
Department of Cell Biology, University of Groningen, University Medical Center Groningen,
Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands
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Chapter 7
Summary
Oligodendrocytes (OLGs) are specialized, myelin forming cells of the central
nervous system . One OLG can myelinate more than 50 axons at a time, thereby
providing rapid and efficient saltatory nerve conduction. To reach a myelin forming
stage, oligodendrocyte progenitor cells (OPCs) need to undergo a series of tightly
regulated developmental and morphological changes. During early development
OPCs proliferate and migrate, properties that cease to function upon further
differentiation, which leads to myelin membrane biosynthesis [1]. Signaling molecules
present in the extracellular environment contribute to OLG development in a spatial,
sequential and transient manner [2]. Upon demyelination of an axon, this myelination
machinery resumes its function, as only adult OPCs, and not mature OLGs, are able
to enwrap denuded axons by functional myelin sheaths [3,4]. However, in the
case of demyelinating diseases, such as multiple sclerosis (MS), the myelination
machinery malfunctions, and loss of OLGs or the presence of quiescent OPCs, unable
to differentiate and synthesize myelin sheaths, results in severe demyelination,
ultimately leading to secondary axonal loss [3,5]. Remyelination failure in MS is
likely a result of dramatic changes in the external environment, among others due
to the persistent presence of signaling molecules, like pro-inflammatory cytokines
such as TNFα [6,7], and the presence of deleterious extracellular matrix (ECM)
molecules, such as fibronectin [8,9] (chapter 1). In order to elucidate the underlying
mechanism(s) of remyelination failure, extensive knowledge of the functioning of the
myelination machinery is required. Therefore, to improve our knowledge of OLGs and
myelination, it is crucial to comprehend the exact role of structural myelin lipids and
proteins, as well as the functioning of the extracellular environment such as ECM and
soluble signals, which was the focus of the studies described in this thesis.
Along their differentiation, OLGs synthesize myelin specific proteins and lipids in a
sequential manner. When compared to other membranes, the composition of myelin
membranes is unique with its high lipid to protein ratio (70:30) [10,11]. Almost one
third of the myelin lipid pool consists of the galactolipids, galactosylceramide (GalC)
and sulfatide. Furthermore, OLGs express a specific repertoire of myelin proteins of
which proteolipid protein (PLP) and myelin basic protein (MBP) are the major ones.
Due to their polarized nature, OLGs exploit a polarized trafficking machinery to
transport their cargo to the final destination, myelin membranes being served by
a basolateral route [10,12]. Besides the trafficking machinery, the organization of
myelin lipids and proteins within the membrane is also crucial for myelin to enable
salutatory nerve conduction. For example, galactolipids are important constituents
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Summary and Future Perspectives
of membrane microdomains, so called ‘lipid rafts’, which play an important role
in OLG development and differentiation [13]. In addition to that, the localization
behavior of myelin proteins within these membrane microdomains is also essential
for myelin assembly (chapters 3 and 4, [10]) . All these aspects and the continuous
reorganization of myelin suggest that myelin membranes display a dynamic structure.
For example, the sequential surface expression of myelin lipids might alter the fluidity
of myelin membranes, which leads to changes in the lateral organization and mobility
of the myelin proteins. In order to obtain more detailed insight into the dynamics of
myelin proteins in the cell body plasma membrane or myelin membranes, different
methodologies such as non-invasive optical microscopic biophysical techniques
like fluorescence correlation spectroscopy (FCS), and raster image correlation
spectroscopy (RISC) might be of use [15–17]. In chapter 2, we provided insight into
how these biophysical methodologies can be applied in the myelin field. Furthermore,
we provided information on the availability of different model systems such as OLG
cell lines and model membrane systems like large and giant unilamellar vesicles (LUVs/
GUVs) [18,19]. These kinds of model membranes can be composed with a minimum
number of elements such as lipids only or a joint set of lipids and proteins, taking into
account the appropriate physiological ratios. In this manner, answers to lipid specific
questions, i.e., the effect of fatty acyl chain length of galactolipids on membrane
domain formation [20] can be investigated, or alternatively, provide information about
specific myelin lipid-protein interactions [21].
The major myelin protein PLP is very important for the integrity of the myelin
membrane as it regulates the close apposition of the extracellular leaflet of the
myelin membranes [22,23]. PLP employs a vesicular transport machinery to reach the
myelin membrane and as shown in chapter 3, vesicular transport of PLP is regulated
in line with the polarized nature of OLGs; i.e., the myelin membrane is the target of
a basolateral-like trafficking mechanism, whereas the cell body plasma membrane
is a target of an apical-like mechanism [10,24,25]. We showed that PLP, prior to its
incorporation into CHAPS-resistant membrane microdomains in the basolateral myelin
membrane, is transported to the apical-like plasma membrane of the OLG cell body in
a syntaxin-3 (t-SNARE) dependent manner, displaying resistance to the detergent TX-
100. Our data further revealed a sulfatide-mediated shift of PLP from TX-100-resistant
microdomains to CHAPS-resistant-microdomains at the cell surface. This apical-to
basolateral transcytotic transport route of PLP to the myelin membrane could be
mimicked in the polarized liver cell line, HepG2 cells. Interestingly, upon transcytotic
transport of PLP to the myelin membranes, we showed that the conformation of the
second extracellular loop of PLP is altered in a sulfatide-dependent manner.
After pinpointing the role of sulfatide in transcytotic PLP transport and the
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Chapter 7
role of PLP’s lateral organization within the membrane, we further examined the
link between this lateral organization and dynamic behavior (chapter 4). A CHAPS
extraction study performed in an OLN-93 cell line that allows selective expression of
GalC alone or GalC and sulfatide, revealed that upon transient transfection with PLP,
the presence of sulfatide increases PLP’s CHAPS insoluble membrane association.
In relation to this observation, a pronounced sulfatide-mediated decrease in the
lateral mobility of PLP was detected. A similar sulfatide-induced increase in the
association with CHAPS-resistant membrane microdomains, along with a decreased
lateral mobility of PLP in the presence of sulfatide was observed, when the cells
were grown on laminin-2, a physiological substrate that promotes myelin membrane
formation and harbors binding sites for sulfatide [26–29]. In contrast, on fibronectin,
a pathological substrate, which inhibits myelin membrane formation and impairs
remyelination in MS lesions [8,9,30], PLP was not present in these CHAPS-resistant
membrane microdomains, which was accompanied by a dramatic increase in its
lateral mobility.
In parallel, the lateral membrane organization and mobility of 18.5-kDa MBP, the
other major myelin protein, was examined in chapter 4. The presence of GalC, but
not sulfatide, increased the CHAPS-resistant microdomain association of MBP, which
correlated with an increase in the lateral mobility of the protein. Unlike PLP, MBP’s
lateral mobility did not display differences in relation to different ECM coatings.
Indeed, previous studies showed that the lateral organization of myelin-localized
MBP is likely driven by secreted neuronal signals [31]. Therefore, under pathological
conditions, rather than ECM proteins, soluble signals in the extracellular milieu might
affect the lateral organization of MBP.
In MS lesions the expression of pro-inflammatory cytokines such as TNFα are
upregulated [32]. In this context, in chapter 5, the effect of TNFα on MBP in myelin
forming OLGs was examined. Rather surprisingly, exposure to relatively low levels of
TNFα reduced the length of myelin segments, produced in myelinated cultures. In
vitro, this was reflected by a marked and reversible redistribution in the localization
of MBP protein, i.e., from myelin sheets towards primary processes, mediated by
activation of TNF receptor 1 (TNFR1) . Notably, cell survival, MBP protein and mRNA
levels, and the localization of MBP mRNA remained unaltered upon TNFα treatment.
Similar changes in the localization of the phosphorylated form of MBP were observed,
with a concomitant decrease of phosphorylation. Interestingly, the underlying
mechanism for the TNF α-mediated redistribution of MBP appeared to be related
to a disorganized actin cytoskeleton, which likely induced a shift of MBP from actin-
dependent to actin-independent membrane microdomains. This might interfere with
the barrier function of MBP, given a similar redistribution of PLP towards primary
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Summary and Future Perspectives
processes, and a relocalization of CNP towards the myelin sheets. Hence, these
findings suggest that TNFα might influence the maintenance of myelin membranes in
an MBP- and actin-dependent manner.
The MBP protein family consists of different isoforms [33], as a result of an
alternative splicing of one MBP transcript, which is generated from a gene complex
called Golli (Gene in the Oligodendrocyte Lineage) consisting of 11 exons [34]. Among
these isoforms, the postnatal MBP isoforms of 14 and 18.5 kDa are lacking exon-II,
and localize to the myelin membrane, playing a role in myelin membrane compaction
and acting as a molecular barrier for the entry of proteins with large cytoplasmic
tails to the myelin sheath [34,35]. In contrast, the postnatal exon-II positive MBP
isoforms, i.e., 17 and 21.5 kDa in rat, localize to the nucleus and cytoplasm and their
expression peaks during early OLG development [36,37]. Their function is however
less clear. Therefore, in chapter 6, we investigated the function of exon-II positive
MBP isoforms. We employed galactolipid deficient OLN-93 cells, which are not able
to synthesize the major postnatal MBP isoforms. Interestingly, our results revealed
that OLN-93 cells endogenously express an MBP isoform with an apparent Mw of 16
kDa, which we identified as the exon-II-positive embryonic isoform of MBP (e-MBP).
e-MBP displayed a similar nuclear and cytoplasmic localization pattern as postnatal
exon-II positive MBPs. When cell proliferation was inhibited, e-MBP was excluded
from the nucleus, whereas upon reestablishment of the proliferative conditions,
e-MBP relocalizes to the nucleus, suggesting active nucleocytoplasmic shuttling
in response to proliferation. Interestingly, e-MBP was also expressed in non-CNS
cell lines, including HepG2, HeLa and HEK293 cells. Direct evidence for a role of
e-MBP in proliferation was obtained upon down regulation of MBP, which markedly
decreased the proliferation of the cell lines. Furthermore, live cell imaging and FRAP
(Fluorescensce recovery after photobleaching) analysis with RFP-tagged exon-II
positive postnatal 21.5-kDa MBP revealed that at proliferative conditions, 21.5-kDa
MBP-RFP localized mainly in the nucleus, whereas it was excluded from the nucleus
when proliferation was inhibited. Interestingly, the nuclear export blocking agent
Leptomycin-B (LMB) prevented nuclear export of MBP. Thus, 21.5-kDa MBP actively
shuttles between cytoplasm and nucleus upon mitogenic modulation. Hence, the
exon-II containing MBP isoforms might be crucial players in cell proliferation during
embryonic development and, after birth in OPC proliferation.
In conclusion, the work presented in this thesis provided new insight into
mechanisms related to myelin biogenesis, highlighting major roles of the myelin
proteins PLP and MBP and the major myelin lipids GalC, sulfatide. Furthermore, we
provided evidence that detailed knowledge of membrane microdomain association
of myelin proteins improve our understanding on myelin biogenesis in both health
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Chapter 7
and disease. The obtained knowledge will help to better understand why myelin
biogenesis or myelin maintenance fails in MS, adding to the elucidation of potential
repair mechanisms, which are still largely unknown in MS.
Future Perspectives
In MS, de novo myelin membrane synthesis fails, which is likely due to an altered
extracellular environment [8,38]. Interestingly, previous studies suggest that the
biochemical structure of myelin galactolipids and myelin proteins are altered in
MS, e.g., the extent of hydroxylation of sulfatide and deamination of MBP are
increased[34,39]. Given the importance of sulfatide in the timing of PLP transport, and
MBP in myelin biogenesis and maintenance (this thesis), these changes in structural
myelin elements might provide novel insight as to why myelin biogenesis fails in MS.
Thus, it will be interesting to further investigate the effect of the biochemical structure
of myelin lipids in model membranes such as GUVs (see chapter 2). For example,
one can make use of myelin membranes obtained from MS patients or alternatively
synthetic lipids where hydroxylation levels are different, to reconstitute GUVs that
mimic the conditions in MS. Furthermore, the data described in chapters 3 and 4
provide a better understanding of the role of myelin galactolipids, especially sulfatide
on transcytotic transport, localization and dynamics of the major myelin protein PLP.
The work presented here would benefit from further investigations; i.e., it is not
known yet how PLP transport, localization and dynamics are affected in MS. In that
regard, dynamics studies relying on bleaching experiments, such as FRAP analyses,
may provide detailed insight on the direction of PLP transport in the presence of
different extracellular signals; i.e., the presence of neuronal-conditioned medium or
the presence of fibronectin to elucidate whether PLP transport is perturbed in MS.
Chapter 4 investigated the lateral mobility and membrane microdomain association of
PLP and MBP as a function of the lipid environment, i.e., by selectively expressing GalC
and/or sulfatide in OLN-93 cells. As galactolipids together with cholesterol play a role
in membrane domain formation and since cholesterol levels are altered in MS [40,41],
it will be interesting to analyze the effect of the cholesterol pool on raft partitioning
and dynamic behavior of PLP and MBP, and their relevance to myelin biogenesis. To
determine the lateral mobility of PLP and MBP, we made use of biophysical techniques
FCS and RICS; however more advanced biophysical techniques such as spot
variation FCS (discussed in chapter 2) enable to directly evaluate whether changes
in the mobility of PLP are a consequence of its localization in distinct membrane
microdomains.
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Summary and Future Perspectives
Similarly, the persistent presence of pro-inflammatory cytokines in MS may
interfere with OLG polarity, myelin biogenesis and maintenance. In that context, our
investigations on the cellular localization of MBP and its membrane microdomain
association, in response to TNFα and a disorganized actin cytoskeleton, provided
some insight as to why myelin segments in myelinated cultures are shortened as
compared to those in untreated cultures. However, we still do not know the exact
underlying mechanism of this myelin segment shortening and its pathological and/
or physiological relevance, if any. Additional experiments on the TNFR1 might provide
some clues. TNFR1 localizes to membrane microdomains upon TNFα stimulation
[42,43]. Therefore, it would be of interest to determine whether the effect of TNFα
is mediated by TNFR1 in membrane microdomains, and whether TNFR1 membrane
microdomain association is altered in MS. In addition, further investigations are
necessary to elucidate why the actin cytoskeleton disorganizes and how this is linked
to MBP and microdomain association. Proteins potentially involved might be Rac and
Rho, since these proteins are known to regulate cytoskeletal dynamics. Besides, in
MS, the post-translational modifications of MBP change dramatically. For example,
MS patients have increased levels of deiminated MBP, and decreased levels of
phosphorylated MBP [33]. Therefore, it will be of further interest to study the effect of
TNFα on MBP post-transitional modifications.
In Chapter 6, we proposed a novel function for exon-II containing MBP isoforms
in cell proliferation. As these MBP isoforms regulate cell proliferation, it will be of
interest to investigate whether these isoforms are re-expressed upon demyelination
and contribute to remyelination, and whether this is valid in MS lesions. In addition,
it is important to clarify the underlying mechanism of how exon-II containing MBPs
regulate cell proliferation. By nuclear export blocking experiments with LMB, we
suggested that the shuttling between nucleus and cytoplasm of MBP can be nuclear
export signal (NES) dependent. However, to our knowledge, MBP does not contain
NES, indicating LMB affects MBP indirectly, i.e., via another protein. In that regard, it
is crucial to identify the interaction partners of nuclear MBP to unravel the underlying
mechanism of the shuttling process. Our preliminary data suggest a direct interaction
between e-MBP and p27 under proliferating conditions, whereas e-MBP exits the
nucleus before p27. Furthermore, the involvement of post-translational modifications
of MBP, such as phosphorylation, in the shuttling , as has been reported for other
nuclear proteins [44], will also be of interest for future research.
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Chapter 7
Netherlandse Samenvatting
Oligodendrocyten (OLGs) zijn cellen van het centrale zenuwstelsel die
gespecialiseerd zijn in het maken van myeline, een vetachtig isolerend laagje van
membranen rondom axonen. Dit myeline maakt een sprongsgewijze, en daardoor
snelle en efficiënte, zenuwgeleiding mogelijk. Om het myeline-vormend stadium
te bereiken moeten voorlopercellen van oligodendrocyten (OPCs) een reeks van
nauwkeurig gereguleerde ontwikkelingsstadia en morfologische veranderingen
ondergaan. Tijdens de vroege ontwikkeling vermenigvuldigen (‘prolifereren’) en
verplaatsen (‘migreren’) de OPCs zich, eigenschappen die verdwijnen tijdens de
verdere ontwikkeling (‘differentiatie’), welke uiteindelijk zal leiden tot de vorming
(‘biogenese’) van myeline [1]. Lokale en tijdelijk aanwezige signaalmoleculen in de
extracellulaire omgeving dragen bij aan deze strak gereguleerde uitrijping van OPCs
[2]. Bij afbraak van myeline (‘demyelinisatie’) zal de machinerie voor myelinisatie
opnieuw in werking moeten worden gezet, omdat alleen OPCs en niet volwassen
OLGs in staat zijn om de ‘kale’ axonen opnieuw te omhullen met een functioneel
laagje myeline (‘remyelinisatie’) [3,4]. In het geval van de ziekte multiple sclerosis
(MS) is de (re)myelinisatie machinery echter ontregeld. Het verlies aan OLGs
en de aanwezigheid van latente OPCs die niet kunnen differentiëren, leiden tot
permanente demyelinisatie in MS en uiteindelijk als secundair effect tot het verlies
van axonen [3,5]. Het falen van remyelinisatie in MS is waarschijnlijk het gevolg
van veranderingen in de externe omgeving van de aangetaste gebieden (‘laesies’),
onder andere vanwege de blijvende aanwezigheid van normaliter tijdelijke
signaalmoleculen, zoals pro-inflammatoire cytokines, waaronder TNFα [6,7], en
extracellulaire matrix (ECM) moleculen, zoals fibronectine [8,9] (hoofdstuk 1). Om
het onderliggende mechanisme(n) van het falen van remyelinisatie op te helderen,
is uitgebreide kennis van de werking van de myelinisatie machinerie vereist. Om
onze kennis van OLGs en myelinisatie te kunnen verbeteren is het cruciaal om de
precieze rol van structurele myeline lipiden en eiwitten te ontrafelen, alsmede de
werking van de extracellulaire omgeving zoals de ECM en oplosbare signalen. Dit
was dan ook de focus van de studies zoals deze beschreven zijn in dit proefschrift.
Tijdens de differentiatie synthetiseren OLGs myeline eiwitten en lipiden in een
nauwgezette volgorde. Door de relatief hoge lipide-eiwit verhouding (70:30) is de
samenstelling van myeline membranen, in vergelijking tot andere membranen, uniek
[10,11]. Bijna een derde van de myeline lipide fractie bestaat uit de galactolipiden
galactosylceramide (GalC) en sulfatide. Daarnaast brengen OLGs myeline-
specifieke eiwitten tot expressie, waarvan proteolipid protein (PLP) en myeline
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Netherlandse Samenvatting
basic protein (MBP) de voornaamste zijn. OLGs zijn gepolariseerde cellen en maken
gebruik van verschillende transport routes om eiwitten en lipiden naar de juiste
membraandomeinen te vervoeren. Transport naar de groeiende myeline membranen
verloopt via een basolaterale route [10,12]. Daarnaast is ook de rangschikking
van myeline eiwitten en lipiden in het membraan cruciaal voor het vormen van
functioneel myeline en het bereiken van sprongsgewijze zenuwgeleiding. Zo zijn
de galactolipiden belangrijke componenten van microdomeinen, de zogenaamde
‘lipid rafts’, in het membraan, welke een belangrijke rol spelen in de ontwikkeling
van OLGs [13]. Bovendien is de aanwezigheid van specifieke myeline eiwitten in
deze membraan microdomeinen essentieel voor myeline vorming [10, hoofdstuk
3,4]. Al deze aspecten en de voortdurende reorganisatie van myeline suggereren
dat myeline membranen dynamisch zijn. Zo kan de rangschikking of plaatselijke
oppervlakte expressie van myeline lipiden de vloeibaarheid van de membranen
veranderen, wat vervolgens leidt tot veranderingen in de laterale organisatie en
mobiliteit van myeline eiwitten. Om meer gedetailleerd inzicht te krijgen in de
dynamiek van myeline eiwitten enerzijds in het plasma membraan van het cellichaam,
en anderzijds in het myeline membraan, kan het gebruik van niet-invasieve optische
microscopische technieken, zoals fluorescentie correlatie spectroscopie (FCS) en
rasterafbeelding correlatie spectroscopie (RISC) nuttig zijn [15-17]. In hoofdstuk
2 hebben we inzicht gegeven in hoe deze biofysische technieken op het gebied van
myeline kunnen worden toegepast. Verder is de beschikbaarheid van verschillende
modelsystemen beschreven, zoals OLG cellijnen en modelmembraan systemen,
bijvoorbeeld grote en zeer grote unilamellaire blaasjes (LUVs/GUVs [18,19]. Dit
soort modelmembranen kunnen worden samengesteld met een minimum aan
elementen zoals lipiden alleen of een geselecteerde set van lipiden en eiwitten,
rekening houdend met de juiste fysiologische verhoudingen. Op deze manier
kan antwoord worden gekregen op lipide-specifieke vragen. Zo kan bijvoorbeeld
het effect van de lengte van de vetzuurketens van galactolipiden op de vorming
van membraan microdomeinen worden onderzocht [20] of informatie worden
verkregen over specifieke interacties tussen myeline eiwitten en lipiden [21].
Het myeline eiwit PLP is een essentieel eiwit voor het in stand houden van de
integriteit van myeline, omdat het de toenadering van de buitenzijden van de lipide
bilaag, dus van de twee verschillende omwindingen, faciliteert [22,23]. PLP wordt
via vesiculair transport naar het myeline membraan getransporteerd, en zoals
weergeven in hoofdstuk 3, is dit transport in lijn met het gepolariseerd karakter
van OLGs gereguleerd; het myeline membraan is het doelwit van basolateraal
transport, terwijl het plasma membraan van het cellichaam het doelwit is van
apicale transport mechanismen[10,24,25]. We hebben aangetoond dat PLP,
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voorafgaand aan de inbouw in CHAPS-resistente membraan microdomeinen in
het myeline membraan, eerst wordt getransporteerd naar het plasma membraan
van het cellichaam via een syntaxin-3 (t-SNARE)-afhankelijke route, waarbij
het is ingebouwd in TX-100-resistente microdomeinen. De resultaten laten
verder een sulfatide-gemedieerde verschuiving van PLP van TX-100-resistente
membraan microdomeinen naar CHAPS-resistente membraan microdomeinen
op het celoppervlak zien. De apicale-basolaterale transcytotische route van
PLP naar het myeline membraan kan worden nagebootst in de gepolariseerde
levercellijn HepG2. Tevens hebben wij kunnen aantonen dat de conformatie van
de tweede extracellulaire lus van PLP verandert in aanwezigheid van sulfatide.
Na de rol van sulfatide in het transcytotische transport van PLP en de
verandering in de laterale organisatie van PLP in het membraan te hebben
vastgesteld, hebben we in hoofdstuk 4 naar een mogelijk verband gezocht tussen
deze laterale organisatie en het dynamisch gedrag van PLP. Een extractie studie
met CHAPS in een OLG cellijn, OLN-93, waarin een selectieve expressie van GalC
alleen en GalC en sulfatide mogelijk is, is gebleken dat na een transiente transfectie
met PLP, sulfatide de CHAPS onoplosbaarheid van PLP verhoogd. Tevens werd
een sulfatide-gemedieerde afname van de laterale beweeglijkheid van PLP
gedetecteerd. Een vergelijkbare sulfatide-geïnduceerde toename in de associatie
met CHAPS-resistente membraan microdomeinen, samen met een verminderde
beweeglijkheid van PLP in het membraan, werd ook waargenomen wanneer
de cellen werden gekweekt op laminine-2, een fysiologisch ECM substraat dat
myelinisatie stimuleert en tevens bindingsplaatsen voor sulfatide bevat [26–29]
. Daarentegen bleek PLP op fibronectine, een pathologisch ECM substraat dat de
vorming van myeline membranen remt en remyelinisatie in MS laesies [8,9,30]
schaadt, niet aanwezig te zijn in deze CHAPS-resistente membraan microdomeinen,
wat gepaard ging met een toename van de laterale beweeglijkheid van PLP.
In hoofdstuk 4 werd tevens de laterale organisatie en de mobiliteit van een
andere belangrijke myeline eiwit, 18,5 kDa MBP, in het membraan onderzocht.
De aanwezigheid van GalC, maar niet van sulfatide, verhoogde de aanwezigheid
van 18,5 kDa MBP in CHAPS-resistente membraan microdomeinen, wat
correleerde met een verhoging van de laterale beweeglijkheid van het eiwit.
In tegenstelling tot PLP bleek de laterale mobiliteit van 18,5 kDa MBP niet te
verschillen in cellen die gekweekt werden op laminine-2 en fibronectine. Inderdaad,
eerdere studies hebben aangetoond dat de laterale mobiliteit van MBP in
myeline waarschijnlijk gereguleerd wordt door oplosbare signalen [31]. Daarom
zullen tijdens pathologische condities oplosbare signalen in de extracellulaire
omgeving, en niet ECM eiwitten, de laterale organisatie van MBP beïnvloeden.
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Netherlandse Samenvatting
In MS laesies is de expressie van pro-inflammatoire cytokines, zoals TNFα,
verhoogd [32]. In deze context werd in hoofdstuk 5 het effect van TNFα op MBP
in myeliniserende kweken onderzocht. Verrassenderwijs bleek dat blootstelling
aan relatief lage hoeveelheden van TNFα de lengte van de myeline segmenten
kleiner maakte. In OLG monokweken kwam dit tot uiting in een duidelijke en
omkeerbare herverdeling in de lokalisatie van MBP, dat wil zeggen van myeline
membranen naar primaire uitlopers. Dit werd gemedieerd door TNF receptor 1
(TNFR1). De overleving van de OLG, de hoeveelheid aan MBP eiwit en RNA, en
de lokalisatie van MBP mRNA bleven onveranderd na behandeling met TNFα.
Onze resultaten laten verder zien dat de TNFα-gemedieerde herverdeling van
MBP gerelateerd was aan een dysorganisatie van het actine cytoskelet, wat
gepaard ging met een verschuiving van MBP van actine-afhankelijke naar actine-
onafhankelijke membraan microdomeinen. Dit verstoort waarschijnlijk de
barrière functie van MBP aangezien een soortgelijke herverdeling van PLP naar
primaire uitlopers werd waargenomen en een herlokalisatie van CNP, een ander
OLG specifiek eiwit, in de richting van myeline membranen. Deze bevindingen
suggereren dat de permanente aanwezigheid van TNFα het in stand houden
van myeline membranen verstoort, op een MBP- en actine-afhankelijke manier.
De MBP eiwit familie omvat verschillende isoformen [33] als gevolg van
alternatieve splicing van een MBP transcript dat is gegenereerd uit een uit 11 exonen
bestaand gen complex genaamd Golli (Gene in Oligodendrocyte Lineage) [34]. Zo
ontbreekt exon-II in de postnatale 14 en 18,5 kDa MBP isoformen. Deze isoformen
bevinden zich voornamelijk in het myeline membraan waar ze een rol spelen in de
‘verdichting’ van het myeline membraan aan de cytoplasmatische zijde en daarnaast
ook optreden als een moleculaire barrière voor de opname van eiwitten met grote
cytoplasmatische staarten in het myeline membraan [34,35]. Daarentegen zijn de
postnatale exon-II positieve 17 en 21,5 kDa MBP isoformen gelokaliseerd in de kern
en het cytoplasma [36,37]. De expressie van deze isoformen piekt tijdens de vroege
ontwikkeling van OLGs, maar de functie van deze isoformen is echter nog onduidelijk.
Daarom is in hoofdstuk 6 de functie van deze exon-II bevattende isoformen
onderzocht. Hiertoe is gebruik gemaakt van de galactolipide deficiënte OLN-93 cellen
die niet in staat zijn om postnatale MBP isoformen te produceren. Uit de resultaten
bleek dat OLN-93 cellen wel een MBP isoform van ongeveer 16 kDa tot expressie
brengen, welke wij geïdentificeerd hebben als een exon-II positieve embryonale
isoform van MBP (e-MBP). e-MBP verschijnt in een vergelijkbaar lokalisatie patroon
als postnatale exon-II positieve MBP isoformen; voornamelijk in de kern en het
cytoplasma. Als de proliferatie van de cellen werd geremd, werd e-MBP uitgesloten
van de kern, terwijl bij het herstel van proliferatie e-MBP weer terugkeert in de kern.
186
Chapter 7
Dit suggereert een actief pendelen tussen het cytoplasma en de kern als reactie op
proliferatie. Opvallend is dat e-MBP ook tot expressie komt in niet-OLG cellijnen,
zoals HepG2, HeLa en HEK293 cellen. Direct bewijs voor een rol van e-MBP in cel
proliferatie werd verkregen na downregulatie van MBP middels shRNA, waardoor de
proliferatie in alle geteste cellijnen minder werd. Bovendien lieten imaging studies
met levende cellen en FRAP (fluorescence recovery after photobleaching)-analyse
met fluorescent-gelabeld exon-II positief postnataal 21,5 kDa MBP zien, dat onder
prolifererende condities 21,5 kDa MBP hoofdzakelijk in de kern was gelokaliseerd,
terwijl het werd uitgesloten van de kern als de proliferatie werd geremd.
Leptomycin-B (LMB), een remmende stof voor het transport van eiwitten uit de kern,
verhinderde ook de export van MBP uit de kern. Kortom evenals e-MBP pendelt 21,5
kDa MBP actief tussen het cytoplasma en de kern als reactie op mitogene modulatie.
Waarschijnlijk zijn de exon-II bevattende MBP isoformen cruciale spelers in de
proliferatie van cellen tijdens de embryonale ontwikkeling en na geboorte voor OPCs.
Kortom, het werk zoals beschreven in dit proefschrift heeft nieuw inzicht
verschaft in mechanismen die verband houden met de aanmaak van myeline,
en laten een belangrijke rol voor de myeline eiwitten PLP en MBP en de typische
myeline galactolipiden, GalC en sulfatide zien. Verder is in dit proefschrift
bewijs geleverd dat gedetailleerde kennis van de associatie van myeline
eiwitten met membraan microdomeinen belangrijke nieuwe inzichten geeft in
de vorming van myeline membranen tijdens gezonde en ziekte-gerelateerde
omstandigheden. De verkregen kennis draagt bij aan het beter begrijpen
waarom de (her)aanmaak en het onderhoud van myeline faalt in MS, en
biedt daardoor perspectief voor het verder ontwikkelen van therapeutische
mogelijkheden voor een ziekte waarvan het ontstaan nog grotendeels onbekend is.
187
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189
Abbreviations
Abbreviations
Approx, approxemately
BC, bile canaliculus
BSA, bovine serum albümin
BrdU, 5-Bromo-2′-deoxy-uridine
cAMP, Cyclic adenosine monophosphate
CDK, cyclin-dependent kinase
CGT, ceramide glucosyltransferase
CHAPS, 3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate
CNP, 2’, 3’-cyclic-nucleotide 3’-
phosphodiesterase
CNS, central nervous system;
CRM1, chromosome region maintenance1;
CN, cytoplasm nucleus;
CSPGs, chondroitin sulfate proteoglycans
CST, cerebroside sulfotransferase
Ctrl, control
DIV, days in vitro
FCS, fetal calf serum;
FCS, fluorescence correlation spectroscopy;
FCCS, fluorescence cross-correlation
spectroscopy;
FGF, fibroblast growth factor;
Fn, fibronectin
FRAP, fluorescence recovery after
photobleaching;
FRET, Förster resonance energy transfer
e-MBP, embryonic myelin basic protein;
EM, electron microscopy
ECM, extracellular matrix
GalC, galactosylceramide
GD3, ganglioside 3
GFP, green fluorescent protein;
Golli, gene in the oligodendrocyte lineage;
GUVs, giant unilamellar vesicles;
GSL, glycosphingolipids
HD, high density;
IFNγ, interferon gama
IGF-1, insulin like growth factor 1
IL1β, interleukin 1 beta
IPL, intraperiod line
LD, low density;
Ld, liquid disordered
LDH, lactate dehydrogenase
LMB, leptomycin B;
Ln2, laminin-2
Lo, liquid ordered
LUVs, large unilamellar vesicles
mAb, monoclonal antibody;
MAG, myelin associated glycoprotein
MBP, myelin basic protein;
MDCK, Madin-Darby canine kidney
MOG, myelin oligodendrocyte glycoprotein;
MS, multiple sclerosis
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
NC, nucleus cytoplasm;
NES, nuclear export signal;
NF155, neurofascin155
NGS, normal goat serum
N&B, number and brightness analyses
OLGs, oligodendrocytes
OPC, oligodendrocyte progenitor cell;
pAb, polyclonal antibody;
PCH, photon counting histogram
PBS, phosphate-buffered saline;
PDGF, platelet-derived growth factor;
PIE-FI, pulsed interleaved excitation
fluctuation imaging;
PFA, paraformaldehyde;
PNS, post-nuclear supernatant
PSF, point spread function
RER, rough endoplasmic reticulum
RICS, raster image correlation spectroscopy
PI, phosphatidylinositol
PIP2, phosphatidylinositol 4,5-Biphosphate
PLL, poly-L-lysine;
PLP, proteolipid protein
PMD, Pelizaeus-Merzbacher disease
PS, phosphatidylserine
RFP, red fluorescent protein;
RT, room temperature;
SD, standard deviation
SEM, standard error of the mean
SF, serum-free
s-FCS, scanning fluorescence correlation
spectroscopy
SPLSs, supported bilayers
sv-FCS, spot variation fluorescence correlation
spectroscopy
S3, syntaxin 3
qPCR, Real-Time quantitative polymerase
chain reaction
TLC, thin layer chromatography
TNF1, TNF receptor 1
TNFα, tumor necrosis factor α
2C1D, 2 components 1 dimension;
2C2D, 2 components 2 dimensions
190
Acknowledgement
Acknowledgement
It was 5 years ago I took my two luggage and tried to find a city where I had never
been in my life. I believe that my first train journey from Schiphol to Groningen was
the longest journey ever during my stay in Netherlands. What I was seeing through the
windows of the train were the feelings such as curiosity, excitement and fear about my
new life in Groningen. I did not know a single person or a place. 5 years later; now, it is
time leave Groningen, the city which now means a lot to me…
I had many people supported me during this journey. First, I would like to take
this opportunity to express my sincere gratitude and deep regards to my supervisors
Nicoletta, Wia and my promoter Dick; without their support, the completion of this
degree would not have been possible. Nicoletta; thank you for introducing me to the
fascinating biophysics world from which I learned a lot and also thanks for supporting
me from a distance. Wia; many thanks for your great support along my projects,
and for sharing your expertise in myelin biology with me. Dick; thank you for your
continuous guidance, support and encouragement.
I thank to all (former) MCB members, DCB members and BCN members, Mirjana,
Anita, Sven, Inge, Jan Willem, Ena, Peng, Inge (MS group), Leon, Edwin, Peter,
Frederike, Lucja, Faya, Marjolein, Chantal, Judith, Katica, Roberta, Magda, Julia, Judith,
Erik, Michel, Nai-Hua, Ruby, Jeroen, Yvon, Tini, Jan Wijbenga, Wya, Greetje, Diana and
Janine. I also would like to thank Karin, Jenny and Ina for helping me to perform some
experiments and for our nice lab chats. Special thanks to Gerry for her great support
from day 1 until my last day. Another special person is Klaas Sjollema who spent
almost all his time to help me with all kind of settings at the microscopy floor without
any complains. Because of his generous time and effort I have greatly improved
my microscopy knowledge. I am also thankful to Ben Giepmans for his interest and
support to my projects.
Many, many thanks to my dear colleague and close friend Erdinç, who made this
opportunity real by encouraging me to step into this life-time experience. During
my Ph.D. period, I had so many wonderful friends with whom I shared my life with.
I want to thank Ana, for being a real sister and sharing unforgettable memories with
me (Groningen curtain princess); also to Jing for being an awesome friend, colleague
and sister, and for supporting me during my difficult times; to Charlotte for being
a very sincere friend and sending me positive energy all the time; to Christiaan
191
Acknowledgement
(annoying orange) for sometimes being the only person who understands me, sharing
his experiences with me and of course sharing Sheff’s pictures :) ; to Herschel for his
supportive friendship, funny stories, mid-night lab chats and 6th floor memories;
to Bispo for introducing so many colors to my life; to Zia for being patient all the
time to chat with depressed Hande with a big smile; to Josephine for very fruitful
conversations and nice coffee breaks; to Peter for being a very friendly officemate; to
Arend for saving me from a killer (!) bee and to Fung for making me laugh all the time.
My special friend Jethro, thanks a lot for everything! I also express my warm thanks to
my friends Mehran and Milind for being loyal friends.
In addition, I would like to thank Don Lamb, Jelle Hendrix, Waldemar Schrimpf
and all the other lab members for our fruitful collaboration. I also would like to thank
Graham Smith, Prof. George Harauz and Prof. Joan Boggs for our collaboration.
Special thanks to Jimmy (Cumhur) for being with me all the time and sharing the
different flavors of life during my Ph.D. period.
Ve Groningenli Turk ahalisi, sizlerin yeri ayrı dili de ayrı olsun dedim :) Groningen
dönüşü ağlamama sebep olan İlke, Bora ve Kübraya buradan selamlar sevgiler. Sizin
yüzünden Groningenden hiç ayrılamayacaktım az kalsın! Nedenleri niyeleri de bizde
saklı kalsın:) . Doktoram esnasında tanıstığım hayatıma anlam katan; biricik adaşım
Hande, Şebnem, Naima, Orcun, Seniz, Ozan, Piray, Ebru, Berfu, Devrim, Volkan, Turan
ve Serra’ya da tesekkur ederim. Doktoram süresince telefonlarımla bunalttığım,
bıkmadan usanmadan beni dinleyen ve yanımda olan annem Yegane, babam Ünsal ve
halam Zerrin’e de çok teşekkür ediyorum.
My final deepest thanks and indebtedness to Can, for not only being with me
during the tears of joy but also during the tears of sadness…
I will miss you Groningen, thanks for being a wonderful city!
Hande Ozgen, 2014
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