Jenny C. de Jonge

Fibronectin aggregation in multiple sclerosis lesions impairs remyelination

Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.

Differential and cell development-dependent localization of myelin mRNAs in oligodendrocytes

In oligodendrocytes (OLG), the mRNAs for the various myelin proteins localize to different intracellular sites. Whereas the confinement of myelin basic protein (MBP) mRNA to the processes of the cell has been well established, we demonstrate that most other myelin mRNA species are mainly present in the perinuclear region. Using in situ hybridization of cultured rat OLG we found that mRNAs are localized to at least three different locations: 1) to the perinuclear region [myelin‐associated glycoprotein (MAG) mRNA]; 2) mainly to the processes (the mRNA for the 14‐kDa isoform of MBP); and 3) to both the perinuclear region and the primary processes [2′,3′‐cyclic nucleotide phosphodiesterase (CNPase) and proteolipid protein (PLP) mRNAs]. Thus, depending on their primary structure, the mRNA species in OLG either remain near the nucleus or localize to primary or secondary processes before their translation. The myelin mRNA localization correlates well with that of the proteins encoded in them, as demonstrated by immunocytochemistry. Since different isoforms of MBP have different locations in transfected HeLa cells (Staugaitis et al.: J Cell Biol 110:1719–1727, 1990), we also have investigated the localization of the various mRNAs in OLG, using exon 2‐minus and exon 2‐specific probes for in situ hybridization. The exon 2‐minus MBP mRNAs are transported far into the processes, whereas exon 2‐specific mRNA was only detected in the cell body. This suggests that sorting and trafficking of MBP mRNA are regulated by the presence or absence of the exon 2 sequence. Furthermore, during maturation of OLG, exon 2‐plus mRNAs disappear, whereas exon 2‐minus mRNAs increase. The developmentally regulated expression of exon 2‐plus transcripts suggests a role of their protein products in differentiation rather than in myelination.

Lovastatin induces the formation of abnormal myelin-like membrane sheets in primary oligodendrocytes.

Statins, well‐known inhibitors of cholesterol synthesis and protein isoprenylation, have been proposed as therapeutic drugs for multiple sclerosis (MS). As lovastatin and simvastatin, which are currently tested for their use in MS, can cross the blood‐brain barrier, they may affect cellular processes in the central nervous system. This is especially relevant with respect to remyelination as a proposed additional treatment for MS, because cholesterol is a major component of myelin. Here, we show that primary oligodendrocytes, treated with lovastatin, form extensive membrane sheets, which contain galactosphingolipids. However, these membrane sheets are devoid of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). Reduced MBP protein expression was confirmed by SDS‐PAGE and Western blotting, and in situ hybridization experiments revealed that lovastatin blocks MBP mRNA transport into oligodendrocyte processes. In contrast, PLP expression was only mildly affected by lovastatin. However, lovastatin treatment resulted in intracellular accumulation of PLP and prevented its translocation to the cell surface. Interestingly, another inhibitor of cholesterol synthesis (ro48‐8071), which does not interfere with isoprenylation, had a similar effect on the localization of PLP, but it did not affect MBP expression and localization. These results suggest that lovastatin affects PLP transport predominantly by the inhibition of cholesterol synthesis, whereas reduced MBP expression is caused by impaired isoprenylation. Based on these results we recommend to carefully monitor the effect of statins on myelination prior to their use in demyelinating diseases.

Perturbation of myelination by activation of distinct signaling pathways: an in vitro study in a myelinating culture derived from fetal rat brain.

An in vitro myelinating mouse‐derived model system has been adapted and optimized for fetal rat brain. In these mixed brain cell (MBC) cultures, myelinogenesis was studied by examining the effect of signaling pathways that are involved in the timing of oligodendrocyte differentiation. When PMA, a protein kinase C (PKC) activator, was kept present during development, the early myelin protein, CNP, was expressed in oligodendrocytes as promptly as in control MBC cultures. In contrast, continuous activation of signaling pathways triggered by FGF‐2 caused a delay in the expression of CNP. The expression of the late myelin proteins MBP and PLP in oligodendrocytes was impeded by both PMA‐ and FGF‐2‐treatment, and, as a consequence, also myelin formation. Surprisingly, the continuous presence of PDGF during development also prevented myelin formation, even though all myelin‐specific proteins were significantly expressed. Taken together, the data indicate that this in vitro myelinating culture system represents an excellent system to study signaling events necessary for the onset of myelination. Moreover, the present results demonstrate that oligodendrocyte differentiation in the presence of neurons and astrocytes can be manipulated both by extracellular and intracellular signaling factors. Importantly, differentiation per se is not necessarily culminating into myelination. J. Neurosci. Res. 59:74–85, 2000

Galectin-4, a novel neuronal regulator of myelination

Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin‐specific proteins and lipids. Galectin‐4, a tandem‐repeat‐type lectin with affinity to sulfatide and nonsialylated termini of N‐glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin‐4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin‐4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin‐4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin‐4 or anti‐galectin‐4 antibodies were added to co‐cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin‐4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin‐4. Interestingly, in co‐cultures, a reduced release of endogenous galectin‐4 correlated with the onset of myelination. Moreover, galectin‐4‐reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin‐4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination.

The lateral membrane organization and dynamics of myelin proteins PLP and MBP are dictated by distinct galactolipids and the extracellular matrix.

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLPs diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.

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 α6 in membrane microdomains. Sulfatide was partly excluded from membrane microdomains on fibronectin, thereby likely precluding laminin‐2‐mediated myelination. Anti‐sulfatide antibodies disrupted integrin α6‐PDGFαR 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;62:927–942

Sorting signals and regulation of cognate basolateral trafficking in myelin biogenesis.

A detailed understanding of trafficking pathways in mature oligodendrocytes is essential for addressing issues aimed at controlling (re)myelination by modulating myelin‐directed transport. Previously, we have shown that viral marker proteins HA and VSV G, on reaching the apical and basolateral surfaces of polarized epithelial cells, respectively, are primarily transported to the plasma membrane and myelin sheet, respectively, in oligodendrocytes (OLGs). In the present study, we demonstrated that in OLGs basolateral sorting signals similar to those in epithelial cells may target proteins to the myelin sheet, emphasizing the basolateral‐ and apical‐like nature of the myelin sheet and plasma membrane, respectively. Thus, substitution of essential amino acids reverses the direction of targeting of these proteins, whereas elimination of apical targeting of HA coincides with its dissipation from detergent‐resistant microdomains. Furthermore, protein kinase C activation negatively regulated transport of the OLG resident transmembrane protein PLP to the myelin sheet, like that of VSV G as shown previously, but did not affect the localization of the membrane‐associated myelin‐specific proteins MBP and CNP. These data imply that several distinctly regulated pathways operate in myelin sheet directed–transport that at least partly rely on a cognate basolateral sorting signal.

The Major Myelin-Resident Protein PLP Is Transported to Myelin Membranes via a Transcytotic Mechanism: Involvement of Sulfatide

ABSTRACT Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cells plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLPs reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A: Implications for Multiple Sclerosis

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro, and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factor cAMP response element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination. SIGNIFICANCE STATEMENT As an environmental factor, aggregates of the extracellular matrix protein fibronectin perturb the maturation of oligodendrocyte progenitor cells (OPCs), thereby impeding remyelination, in the demyelinating disease multiple sclerosis (MS). Here we demonstrate that exogenous addition of ganglioside GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation, both in vitro and in vivo, by activating a PKA-dependent signaling pathway. We propose that targeted delivery of GD1a to MS lesions may act as a potential novel molecular tool to boost maturation of resident OPCs to overcome remyelination failure and halt disease progression.