The major myelin-resident protein PLP is transported to myelin membranes via a transcytotic mechanism: involvement of sulfatide.

Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cell's 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 PLP's 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.

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.

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.

Fibronectin impedes "myelin" sheet-directed flow in oligodendrocytes: a role for a beta 1 integrin-mediated PKC signaling pathway in vesicular trafficking.

Differentiation of oligodendrocytes results in the formation of the myelin sheath, a dramatic morphological alteration that accompanies cell specialization. Here, we demonstrate that changes in the extracellular microenvironment may regulate these morphological changes by altering intracellular vesicular trafficking of myelin sheet-directed proteins. The data reveal that fibronectin, in contrast to laminin-2, decreased membrane-directed transport of endogenous NCAM 140 and the model viral protein VSV G, both proteins normally residing in the myelin membrane. The underlying mechanism relies on an integrin-mediated activation of PKC, which causes stable phosphorylation of MARCKS. As a result, dynamic reorganization of the cortical actin cytoskeleton necessary for the targeting of vesicular trafficking to the myelin sheet is precluded, a prerequisite for morphological differentiation. These data are discussed in the context of the demyelinating disease multiple sclerosis, i.e., that leakage of fibronectin across the blood-brain barrier may impede myelination by interference with intracellular myelin sheet-directed membrane transport.

The function of neurofascin155 in oligodendrocytes is regulated by metalloprotease-mediated cleavage and ectodomain shedding.

Formation of the paranodal axo-glial junction requires the oligodendrocyte-specific 155-kDa isoform of neurofascin (NF155). Here, we report the presence of two peptides in cultured oligodendrocytes, which are recognized by distinct NF155-specific antibodies and correspond to a membrane anchor of 30 kDa and a 125 kDa peptide, which is shed from the cells, indicating that it consists of the NF155 ectodomain. Transfection of OLN-93 cells with NF155 verified that both peptides originate from NF155 cleavage, and we present evidence that metalloproteases mediate NF155 processing. Interestingly, metalloprotease activity is required for NF155 transport into oligodendrocyte processes supporting the functional significance of NF155 cleavage. To further characterize NF155 cleavage and function, we transfected MDCK cells with NF155. Although ectodomain shedding was observed in polarized and non-polarized MDCK cells, surface localization of NF155 was restricted to the lateral membrane of polarized cells consistent with a role in cell-cell adhesion. Aggregation assays performed with OLN-93 cells confirmed that NF155 accelerates cell-cell adhesion in a metalloprotease-dependent manner. The physiological relevance of NF155 processing is corroborated by the presence of NF155 cleavage products in heavy myelin, suggesting a role of NF155 ectodomain shedding for the generation and/or stabilization of the nodal/paranodal architecture.

Leucocyte depletion in a drowning victim during rewarming with extracorporeal circulation may limit pulmonary oedema.

INTRODUCTION: We report two drowning victims with hypothermic circulatory arrest who were resuscitated with the use of extracorporeal circulation (ECC). The first patient developed severe post-bypass pulmonary oedema and inspired us to use a leucocyte-depletion filter in the second patient to attenuate leucocyte-mediated pulmonary reperfusion injury. METHODS: In the first patient, a standard extracorporeal circuit was used. In the second patient, systemic leucocyte depletion was applied using leucocyte-depletion filters (Pall RS 1, Pall, Portsmouth, UK), in the venous side of the extracorporeal circuit. Circulating leucocyte counts were measured and arterial blood gas analysis and chest X-rays were performed. RESULTS: Both patients showed a decrease of the circulating leucocyte counts during rewarming and had nearly similar leucocyte counts on arrival at the intensive care unit (ICU). The first patient developed severe pulmonary oedema, with poor arterial blood gases, whereas the second patient, who had leucocyte-depletion by filtration, did not develop severe pulmonary oedema, and had good arterial blood gases. CONCLUSION: Profound leucocyte-depletion by means of filtration may have contributed to limit leucocyte-mediated pulmonary reperfusion injury.

Operational storm surge forecasting in the Netherlands: developments in the last decade.

The accurate forecasting of storm surges is an important issue in the Netherlands. With the emergence of the first numerical hydrodynamic models for surge forecasting at the beginning of the 1980s, new demands and possibilities were raised. This article describes the main phases of the development and the present operational set-up of the Dutch continental shelf model, which is the main hydrodynamic model for storm surges in the Netherlands. It includes a brief discussion of applied data-assimilation techniques, such as Kalman filtering, the model calibration process and some thoughts on quality assurance in an operational environment. After further describing some select recent investigations, the paper concludes with some remarks on future developments in a European context.

Alteration of the extracellular matrix interferes with raft association of neurofascin in oligodendrocytes. Potential significance for multiple sclerosis?

Remyelination, as potential treatment for demyelinating diseases like multiple sclerosis (MS), requires the formation of new axoglial interactions by differentiating oligodendrocyte progenitor cells. Since the oligodendrocyte-specific isoform of neurofascin, NF155 (neurofascin isoform of 155 kDa), may be important for establishing axoglial interactions, we analyzed whether its expression is changed in chronic relapsing experimental allergic encephalomyelinitis (EAE). Although overall expression of NF155 was not changed, immunoreactivity of NF155 was dramatically increased in EAE lesion sites indicating an enhanced accessibility of NF155 epitopes. As this may be due to infiltrating plasma components, for example, fibronectin, we analyzed whether fibronectin affects the intracellular distribution and membrane association of NF155 in primary oligodendrocytes. In oligodendrocytes cultivated on polylysine, NF155 was recruited to membrane microdomains (rafts) during development and became enriched in secondary and tertiary processes. Fibronectin perturbed localization and raft association of NF155 and inhibited the morphological differentiation of oligodendrocytes. Consistent with the in vitro data, raft association of NF155 was reduced in spinal cord of EAE rats. The results suggest that the association of NF155 to microdomains in the oligodendrocyte membrane is required for its participation in intermolecular interactions, which are important for myelination and/or myelin integrity.

Insulin-like growth factor-binding protein 6 inhibits survival and differentiation of rat oligodendrocyte precursor cells.

Insulin-like growth factor 1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and promotes myelination. We demonstrate that IGF-binding protein 6 (IGFBP-6) is expressed and localized to the Golgi complex in rat oligodendrocyte precursor (O2A) cells. IGFBP-6 mRNA showed a developmentally regulated expression pattern, displaying a transient decrease during early development, and enhanced levels upon cell maturation. IGFBP-6 mRNA expression could be reduced by addition of basic fibroblast growth factor and progesterone while estrogen increased IGFBP-6 mRNA. IGF-1, platelet-derived growth factor, and insulin had no effect. When added exogenously, IGFBP-6 reduced O2A cell survival in the absence of IGF-1 and inhibited IGF-1-stimulated survival in a partially IGF-1-dependent and partially IGF-1-independent fashion. In addition, IGFBP-6 reduced the IGF-stimulated expression of two myelin proteins, CNPase and MAG. Taken together, the data show that IGFBP-6 is a new negative effector of oligodendrocyte survival and differentiation.

Insulin-like growth factor binding proteins-1 and -2 differentially inhibit rat oligodendrocyte precursor cell survival and differentiation in vitro.

Insulin-like growth factor-1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and induces myelination. Its actions are modulated by IGF binding proteins (IGFBPs) that are present in the extracellular fluids or on the cell surface. Additionally, IGFBPs are also known to exert actions that are independent of IGF-1. We studied whether IGF-binding proteins (IGFBPs)-1 and -2 modulate rat oligodendrocyte precursor (O2A) cell survival and differentiation in vitro both in the absence and presence of exogenously added IGF-1. The data reveal that IGFBP-1 and -2 reduced O2A cell survival in the absence and presence of exogenously added IGF-1. The effects of IGFBP-1 on cell survival in the presence of exogenously added IGF-1 were IGF-1-dependent, whereas IGFBP-2 displayed both IGF-1-dependent and IGF-1-independent effects. Furthermore, IGFBP-1 and -2 inhibited O2A cell differentiation in the presence of IGF-1 as reflected by decreased expression levels of two myelin proteins, CNPase (2',3'-cyclic nucleotide 3'-phosphohydrolase) and MAG (myelin associated glycoprotein). Analysis of medium samples revealed that O2A cells do not secrete proteases that degrade these IGFBPs. Taken together the data show that IGFBP-1 and -2 are negative effectors of oligodendrocyte survival and differentiation. Accordingly, the role of IGFBPs should be explicitly taken into account when investigating IGF-1 effects on oligodendrocytes, especially in the context of therapeutic purposes.

Survival benefit of cardiopulmonary bypass support in bilateral lung transplantation for emphysema patients.

BACKGROUND: This study is designed to examine a possible association of cardiopulmonary bypass (CPB) support and outcome of lung transplantation in a well-balanced group of emphysema patients. METHODS: We performed a retrospective analysis of 62 consecutive primary bilateral lung transplantations for emphysema. Risk factors for their possible association with patient survival were analyzed by multivariate logistic regression. RESULTS: The use of CPB support was associated with improved survival (odds ratio=0.25; P=0.038). The actuarial survival at 1 year was 97% for patients treated with CPB and 77% for patients treated without CPB support. In 28 patients (45%), 2 human leukocyte antigen (HLA)-DR mismatches between donor and recipient occurred, whereas 34 patients had 0 or 1 HLA-DR mismatches. The use of CPB support in the group with two HLA-DR mismatches was associated with improved survival (odds ratio=0.06; P=0.020). This association was not present in the group with 0 or 1 HLA-DR mismatches. CONCLUSIONS: These results demonstrate a significant survival benefit of CPB support during bilateral lung transplantation in emphysema patients. The difference in survival benefit of CPB support between the patients with 0 or 1 HLA-DR mismatches and the patients with 2 HLA-DR mismatches indicates that the immunosuppressive effect of CPB support might be responsible for this survival benefit. The underlying immunological mechanism might be important in the future treatment of organ transplantation.