On the cause of multiple sclerosis:Molecular mechanisms regulating myelin biogenesis

Hoe myeline wordt gemaakt en hoe die kennis van nut kan zijn om een effectieve therapie voor MS te ontwikkelen. Myeline, de vette isolatielaag rondom zenuwcellen, raakt bij multiple sclerose (MS)-patiënten beschadigd door onder andere ontstekingsreacties. Die beschadigingen worden op den duur niet meer hersteld. Om te begrijpen waarom dat niet meer gebeurt, is het belangrijk te weten hoe in normale situaties de myelinemembranen worden gevormd. Met die kennis verwachten we gereedschap in handen te krijgen om de aanmaak van nieuw myeline in de aangetaste gebieden (‘laesies’) weer in gang te zetten en zo de beschermende myeline-laag weer op te bouwen. In dit onderzoek hebben we o.a. inzicht gekregen in de wijze waarop de twee belangrijkste myeline eiwitten, MBP en PLP, beïnvloed kunnen worden in de aanmaak van myeline. Zo hebben we bijvoorbeeld waargenomen dat wanneer de aanmaak van syntaxine 4, een bekend ankereiwit in transportprocessen, in voorlopercellen van oligodendrocyten wordt geblokkeerd, er geen MBP eiwit meer wordt ingebouwd in het myeline. Ook hebben we vastgesteld dat de transportroute van PLP verandert, wanneer in rijpere oligodendrocyten MAL eiwit – een transport-regulerend eiwit – wordt gemaakt. Maar ook factoren van buiten de cel hebben invloed op de aanmaak van myeline. Zoals de ontstekingsmediator TNFα, aanwezig in MS laesies, die ervoor zorgt dat het geraamte van de cel (cytoskelet) in volwassen oligodendrocyten dusdanig wordt aangetast, dat het MBP eiwit niet meer volledig in myeline terechtkomt. Dit onderzoek geeft nieuw en gedetailleerd inzicht in de moleculaire mechanismen die de lokalisatie van de belangrijkste myeline componenten reguleren, en daarmee de myeline aanmaak. Deze resultaten kunnen in de toekomst helpen om nieuwe behandelingen voor MS te ontwikkelen

Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma

Transcriptional Expression of Myelin Basic Protein in Oligodendrocytes Depends on Functional Syntaxin 4:a Potential Correlation with Autocrine Signaling

Myelination of axons by oligodendrocytes is essential for saltatory nerve conduction. To form myelin membranes, a coordinated synthesis and subsequent polarized transport of myelin components are necessary. Here, we show that as part of the mechanism to establish membrane polarity, oligodendrocytes exploit a polarized distribution of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery components syntaxins 3 and 4, localizing to the cell body and the myelin membrane, respectively. Our data further reveal that the expression of myelin basic protein (MBP), a myelin-specific protein that is synthesized "on site" after transport of its mRNA, depends on the correct functioning of the SNARE machinery, which is not required for mRNA granule assembly and transport per se. Thus, downregulation and overexpression of syntaxin 4 but not syntaxin 3 in oligodendrocyte progenitor cells but not immature oligodendrocytes impeded MBP mRNA transcription, thereby preventing MBP protein synthesis. The expression and localization of another myelin-specific protein, proteolipid protein (PLP), was unaltered. Strikingly, conditioned medium obtained from developing oligodendrocytes was able to rescue the block of MBP mRNA transcription in syntaxin 4-downregulated cells. These findings indicate that the initiation of the biosynthesis of MBP mRNA relies on a syntaxin 4-dependent mechanism, which likely involves activation of an autocrine signaling pathway

MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains

In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known regulator of polarized trafficking in epithelial cells, and given its presence in OLGs it was therefore of interest to investigate whether MAL played a similar role in PLP transport in OLGs, taking into account its timely expression in these cells. Our data revealed that premature expression of mCherry-MAL in oligodendrocyte progenitor cells interfered with terminal OLG differentiation, although myelin membrane formation per se was not impaired. In fact, also PLP transport to myelin membranes via the cell body plasma membrane was unaffected. However, the typical shift of PLP from TX-100-insoluble membrane domains to CHAPS-resistant, but TX-100-soluble membrane domains, seen in the absence of MAL expression, is substantially reduced upon expression of the MAL protein. Interestingly, not only in vitro, but also in developing brain a strongly diminished shift from TX-100 resistant to TX-100 soluble domains was observed. Consistently, the MAL-expression mediated annihilation of the typical membrane microdomain shift of PLP is also reflected by a loss of the characteristic surface expression profile of conformation-sensitive anti-PLP antibodies. Hence, these findings suggest that MAL is not involved in vesicular PLP trafficking to either the plasma membrane and/or the myelin membrane as such. Rather, we propose that MAL may regulate PLP's distribution into distinct membrane micro-domains that allow for lateral diffusion of PLP, directly from the plasma membrane to the myelin membrane once the myelin sheath has been assembled

Sulfatide-mediated control of extracellular matrix-dependent oligodendrocyte maturation

In the central nervous system, the extracellular matrix (ECM) compound laminin-2, present on developing axons, is essential in regulating oligodendrocyte (OLG) maturation. For example, laminin-2 is involved in mediating interactions between integrins and growth factors, initially localizing in separate membrane microdomains. The galactosphingolipid sulfatide is an important constituent of these microdomains and may serve as a receptor for laminin-2. Here, we investigated whether sulfatide interferes with ECM-integrin interactions and, in this manner, modulates OLG maturation. Our data reveal that disruption of laminin-2-sulfatide interactions impeded OLG differentiation and myelin-like membrane formation. On laminin-2, but not on (re)myelination-inhibiting fibronectin, sulfatide laterally associated with integrin alpha 6 in membrane microdomains. Sulfatide was partly excluded from membrane microdomains on fibronectin, thereby likely precluding laminin-2-mediated myelination. Anti-sulfatide antibodies disrupted integrin alpha 6-PDGF alpha R interactions on laminin-2 and induced demyelination in myelinated spheroid cultures, but intriguingly stimulated myelin-like membrane formation on fibronectin. Taken together, these findings highlight the importance of laminin-sulfatide interactions in the formation of functional membrane microdomains essential for myelination. Thus, laminin-sulfatide interactions might control the asynchronous localized differentiation of OLGs, thereby allowing myelination to be triggered by axonal demand. Given the accumulation of fibronectin in multiple sclerosis lesions, the findings also provide a molecular rationale for the potential of anti-sulfatide antibodies to trigger quiescent endogenous OLG progenitor cells in axon remyelination. GLIA 2014;62:927-94

Localization and membrane microdomain association of PLP in OLN-PLP-GS and OLN-PLP-GS-MAL cells.

<p>(A) Localization of PLP in OLN-cells in the presence (OLN-PLP-GS-MAL) and absence of MAL (OLN-PLP-GS). MAL is expressed after PLP-eGFP. ET3 and O10 recognize distinct extracellular PLP epitopes. Representative pictures of at least three independent experiments are shown. The PLP-eGFP expression in the cytoplasm marks the morphology of the cells, including the processes. Scale bars are 10 μm. Note that upon expression of MAL the ET3 PLP surface epitope is exposed along the entire membrane (arrows), while being restricted to the cell body plasma membrane in the absence of MAL (arrowheads). (B,C) OLN-PLP-GS (‘without MAL’), OLN-PLP-GS-MAL (‘MAL after PLP’) and OLN-GS-MAL-PLP (‘MAL before PLP’) cells were extracted with TX-100 (1%) or CHAPS (20 mM) at 4°C and subjected to OptiPrep density centrifugation. PLP-eGFP (anti-GFP) and caveolin were visualized by Western blot. Representative blots of two to four independent experiments are shown in B, quantitative analyses in C. The protein percentage of each fraction was calculated by dividing the protein percentage in that fraction by total protein expression. Bar graphs of the pooled fraction percentage of (membrane microdomain, ‘raft’) fractions 3–5 and (‘non-raft’) fractions 6 and 7 are shown. Statistical differences with OLN-PLP-GS cells as assessed with a student’s <i>t</i>-test are shown (* p<0.05). Note that upon premature expression of MAL, i.e., prior to transient expression of PLP (OLN-GS-MAL-PLP) the characteristic distribution of PLP in CHAPS-insoluble (membrane microdomain) fractions is reduced, while the lateral distribution of the membrane microdomain marker caveolin is not altered.</p

Localization of PLP in HepG2-PLP and HepG2-PLP-MAL cells.

<p>(A) Localization of PLP-eGFP in HepG2-cells in the presence (HepG2-PLP-MAL) and absence of MAL (HepG2-PLP). MAL is expressed after PLP-eGFP. MRP2 is an apical marker, ET3 recognizes an extracellular PLP epitope. Representative pictures of at least three independent experiments are shown. Scale bars are 10 μm. Note that upon expression of MAL the expression of PLP-eGFP at the apical surface seems to be reduced; more intracellular structures appear, while still present at the basolateral membrane surface. (B) Quantitative analysis of the number of PLP-positive of total MRP2-positive bile canaliculi (BCs, apical surface) in HepG2-PLP and HepG2-PLP-MAL cells. Each bar represents the mean + SD of three independent experiments. In each experiment, the % of PLP-positive of total MRP2-positive BCs was set to 100%. In HepG2-PLP cells 41.0 ± 9.4% of the MRP2-postitive BCs were PLP-positive. Statistical differences with HepG2-PLP cells as assessed with a one sample <i>t</i>-test are shown (* p< 0.05). Note that the number of PLP-positive BCs is reduced upon expression of MAL. (C) Western blot analysis of the expression levels of PLP (anti-GFP) and MAL (anti-MAL antibody, 6D9) in HepG2-PLP and HepG2-PLP-MAL cells. Note that PLP expression is similar in the presence or absence of MAL.</p

Schematic overview of the trafficking of newly synthesized PLP to myelin membranes in the presence or absence of MAL.

<p>In the absence of MAL (upper part), newly synthesized PLP is transported to the myelin membrane via an indirect pathway, involving (i) vesicular transport to the apical-like cell body plasma membrane prior to basolateral-like myelin membranes and (ii) temporal accumulation in a late endosomal compartment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155317#pone.0155317.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155317#pone.0155317.ref020" target="_blank">20</a>]. Along this route, PLP partitions in distinct membrane domains, i.e., TX100-resistant or CHAPS-resistant, and expose different conformational epitopes during respectively biosynthetic (red, ET3) and transcytotic (blue, O10) transport. In the presence of MAL (lower part), the distinct partitioning of PLP in distinct membrane microdomains is lost and both conformational epitopes are exposed along the entire membrane. These MAL-mediated changes allow for lateral diffusion of PLP from the cell body plasma membrane to the myelin membrane (green, ET3/O10).</p