Increased expression of distinct galectins in multiple sclerosis lesions.

AIMS: Multiple sclerosis (MS) is a chronic progressive degenerative disorder of the central nervous system, characterized by inflammation, demyelination, ultimate failure of remyelination and axonal loss. Current research identifies galectins, adhesion/growth-regulatory effectors binding ß-galactosides, peptide motifs and lipids, as important immunomodulators in diverse inflammatory diseases. However, little is known about their expression, cellular localization and role in human central nervous system tissue. To identify a potential role of galectins in MS, their expression and localization in control white matter (CWM) and demyelinated MS lesions were examined. METHODS: qPCR, Western blot and immunohistochemical analyses were performed on human post mortem CWM and MS lesions at different stages. Cultured astrocytes, derived from healthy subjects and MS patients, were analysed similarly. RESULTS: Among 11 different galectins tested, galectins-1, -3, -8 and -9 were present at detectable levels in CWM, and, interestingly, significantly enhanced in active MS lesions. On the cellular level, galectins localized to microglia/macrophages, astrocytes and endothelial cells. Intriguingly, galectin-9 displayed a distinctly different intracellular localization in microglia/macrophages when comparing active and inactive MS lesions, being restricted to the nuclei in active lesions, and primarily localizing in the cytoplasm in inactive lesions. Furthermore, enhanced levels of galectin-1, detected as dimers in Western blot analysis, were released by cultured astrocytes from MS patients. CONCLUSIONS: This study provides a detailed analysis of galectins in MS lesions and assigns distinct galectins to different aspects of the disease. Thus, besides being known as modulators of inflammatory processes, our findings suggest additional association of distinct galectins with MS pathology.

Gene delivery by cationic lipids: in and out of an endosome.

Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required.

On the mechanism of cationic amphiphile-mediated transfection. To fuse or not to fuse: is that the question?

Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.

Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery.

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion.

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.

Cationic lipid-mediated transfection in vitro and in vivo (review).

Recent rapid developments in genomics will likely lead to a rapid expansion in identifying defective genes causing a variety of diseases, implying a vast increase in the number of therapeutic targets. Treatment of such diseases may include strategies ranging from gene delivery and replacement to antisense approaches. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene- or oligonucleotide-carrier systems, which are necessary for protective purposes (against nucleases) and transport (to target tissue and cells in vivo). Further, they should also display the propensity to efficiently translocate the oligonucleotides and gene constructs into cells, via passage across several membrane barriers. The emphasis in this review will be on the use of cationic lipids for that purpose. Crucial to successful application of this sophisticated technology in vivo will be a need for a better understanding of fundamental and structural parameters that govern transfection efficiency, including the issues of cationic lipid/DNA complex assembly (with or without helper lipid), stability towards biological fluids, complex-target membrane interaction and translocation, and gene-integration into the nucleus. Biophysical and biochemical characterization of so-called lipoplexes, and their interaction with cells in vitro, are considered instrumental in reaching such insight. Here, most recent advances in cationic lipid-mediated gene delivery are discussed from such a perspective.

Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor.

Oligonucleotides (ODNs) can be employed as effective gene-specific regulators. However, before ODNs can reach their targets, several physical barriers have to be overcome, as although ODNs may pass cell membranes, most become sequestered in endocytic compartments. Accordingly, sophisticated strategies are required for efficient delivery. Here we have employed a pyridinium-based synthetic amphiphile, called SAINT-2, which carries ODNs into cells in a highly efficient, essentially non-toxic and serum-insensitive manner. Intracellular delivery was examined by monitoring the trafficking of fluorescent ODNs and lipid, and by measuring the effect of specific antisense ODNs on target mRNA and protein levels of the receptor for the neuropeptide corticotropin-releasing factor (CRF-R), expressed in Chinese hamster ovary cells. ODN delivery is independent of lipoplex size, and fluorescently tagged ODNs readily acquire access to the nucleus, whereas the carrier itself remains sequestered in the endosomal-lysosomal pathway. While the release is independent of the presence of serum, it is not observed when serum proteins gain access within the lipoplex, and which likely stabilizes the lipoplex membrane. We propose that the amphiphile-dependent aggregate structure governs complex dissociation, and hence, the biological efficiency of ODNS: We demonstrate an essentially non-toxic and effective antisense-specific down-regulation of the CRF-R, both at the mRNA and protein level.

Membrane domains and polarized trafficking of sphingolipids.

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.

Lipoplex formation under equilibrium conditions reveals a three-step mechanism.

Cellular transfection can be accomplished by the use of synthetic amphiphiles as gene carrier system. To understand the mechanism and hence to improve the efficiency of transfection, insight into the assembly and properties of the amphiphile/gene complex is crucial. Here, we have studied the interaction between a plasmid and cationic amphiphiles, using a monolayer technique, and have examined complex assembly by atomic force microscopy. The data reveal a three-step mechanism for complex formation. In a first step, the plasmids, interacting with the monolayer, display a strong tendency of orientational ordering. Subsequently, individual plasmids enwrap themselves with amphiphile molecules in a multilamellar fashion. The size of the complex formed is determined by the supercoiled size of the plasmid, and calculations reveal that the plasmid can be surrounded by 3 to 5 bilayers of the amphiphile. The eventual size of the transfecting complex is finally governed by fusion events between individually wrapped amphiphile/DNA complexes. In bulk phase, where complex assembly is triggered by mixing amphiphilic vesicles and plasmids, a similar wrapping process is observed. However, in this case, imperfections in this process may give rise to a partial exposure of plasmids, i.e., part of the plasmid is not covered with a layer of amphiphile. We suggest that these exposed sites may act as nucleation sites for massive lipoplex clustering, which in turn may affect transfection efficiency.

The subapical compartment and its role in intracellular trafficking and cell polarity.

In polarized epithelial cells and hepatocytes, apical and basolateral plasma membrane surfaces are maintained, each displaying a distinct molecular composition. In recent years, it has become apparent that a subapical compartment, referred to as SAC, plays a prominent if not crucial role in the domain-specific sorting and targeting of proteins and lipids that are in dynamic transit between these plasma membrane domains. Although the molecular identity of the traffic-regulating devices is still obscure, the organization of SAC in distinct subcompartments and/or subdomains may well be instrumental to such functions. In this review, we will focus on the potential subcompartmentalization of the SAC in terms of regulation of membrane traffic, on how SAC relates to the endosomal system, and on how this compartment may operate in the context of other intracellular sorting organelles such as the Golgi complex, in generating and maintaining cell polarity.

Lipid trafficking and sorting: how cholesterol is filling gaps.

Recent research has highlighted a role for cholesterol homeostasis in the regulation of trafficking and sorting of sphingolipids. This sorting may dictate the nature of the acyl chain species of phospholipids in the plasma membrane which, in turn, may govern the selective partitioning of these lipids into lateral domains. Recently, several proteins have been identified that play a role in the flow and sorting of all major lipid classes.

PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways.

In this paper we address the linking of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF-2) to intracellular signaling molecules in oligodendrocyte progenitors. It is demonstrated that both growth factors activate downstream targets similar to those shown for protein kinase C (PKC) activation. Yet, neither the arrest of terminal oligodendrocyte differentiation nor the proliferation induced by PDGF or FGF-2 can be antagonized by inhibition of PKC. Rather, p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and pp70 S6 kinase were found to be necessary for the mitogenic activity of PDGF and FGF-2. Paradoxically, these kinases were also necessary for the onset of oligodendrocyte differentiation in control cells. In addition, cAMP-dependent kinase A (PKA) activation inhibited the mitogenic response of oligodendrocyte progenitors to FGF-2. Taken together, the molecular mechanism that controls oligodendrocyte lineage progression is operated by at least two signal pathways, which interfere either with proliferation and/or differentiation of oligodendrocyte progenitors.

Polarized sphingolipid transport from the subapical compartment changes during cell polarity development.

The subapical compartment (SAC) plays an important role in the polarized transport of proteins and lipids. In hepatoma-derived HepG2 cells, fluorescent analogues of sphingomyelin and glucosylceramide are sorted in the SAC. Here, evidence is provided that shows that polarity development is regulated by a transient activation of endogenous protein kinase A and involves a transient activation of a specific membrane transport pathway, marked by the trafficking of the labeled sphingomyelin, from the SAC to the apical membrane. This protein kinase A-regulated pathway differs from the apical recycling pathway, which also traverses SAC. After reaching optimal polarity, the direction of the apically activated pathway switches to one in the basolateral direction, without affecting the apical recycling pathway.

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide.

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.

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.

On the biogenesis of the myelin sheath: cognate polarized trafficking pathways in oligodendrocytes.

Oligodendrocytes, the myelinating cells of the central nervous system, are capable of transporting vast quantities of proteins and of lipids, in particular galactosphingolipids, to the myelin sheath. The sheath is continuous with the plasma membrane of the oligodendrocyte, but the composition of both membrane domains differs substantially. Given its high glycosphingolipid and cholesterol content the myelin sheath bears similarity to the lipid composition of the apical domain of a polarized cell. The question thus arises whether myelin components, like typical apical membrane proteins are transported by an apical-like trafficking mechanism to the sheath, involving a 'raft'-mediated mechanism. Indeed, the evidence indicates the presence of cognate apical and basolateral pathways in oligodendrocytes. However, all major myelin proteins do not participate in this pathway, and remarkably apical-like trafficking seems to be restricted to the oligodendrocyte cell body. In this review, we summarize the evidence on the existence of different trafficking pathways in the oligodendrocyte, and discuss possible mechanisms separating the oligodendrocyte's membrane domains.

Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability.

BACKGROUND: Cationic liposomes belong to the family of non-viral vectors for gene delivery. Despite several drawbacks, such as low efficiency compared to viruses and inactivation by serum, cationic liposomes remain a promising tool for gene therapy. Therefore further investigation of the mechanism of transfection and improvement of formulations are warranted. METHOD: In a comparative study, we investigated the effect of serum on the ability of SAINT, a novel synthetic amphiphile, and Lipofectin to mediate transfection in vitro, employing a variety of cell lines. RESULTS: In all cell types, SAINT-mediated transfection was not significantly affected by the presence of serum, in contrast to Lipofectin-mediated transfection. Intriguingly, the extent of complex association was enhanced in the presence of serum, while cell association of the Lipofectin complex was approximately two-fold higher than that of SAINT. These data imply that transfection efficiency and the amount of cell-associated complex are not related. However, when the helper lipid dioleoylphosphatidylethanolamine (DOPE) was substituted for cholesterol, SAINT-mediated transfection was reduced in the presence of serum. This indicates that lipoplex composition rather than the cationic lipid per se codetermines the effect of serum. Also, the presence of serum decreased cytotoxicity, while no correlation could be demonstrated between toxicity and transfection efficiency. The binding of serum proteins to either complex was identical, both in terms of protein identity and relative amounts. CONCLUSION: We propose that serum, in conjunction with cell-specific factors and lipoplex composition, determines complex (in)stability, which is crucial for effective gene delivery and expression.

On the mechanism of intracellular membrane fusion: in search of the genuine fusion factor.

Intracellular membrane fusion events require a general protein machinery that functions in vesicular traffic and in assembly and maintenance of organelles. An array of cytosolic and integral membrane proteins are currently identified, and in conjunction with ongoing detailed structural studies, rapid progress is made in understanding basic features of the overall mechanism of the fusion machinery, but above all a proper appreciation of its enormous complexity. Thus a highly sophisticated level of regulation of the different steps involved in tethering, docking and merging itself is apparent. Apart from the relevance of protein-protein interactions, also a role of distinct lipids is gradually emerging, particularly in fusion. However, although various suggestions have been made recently, largely based upon in vitro studies, the identity of the actual fusion factor(s) remains to be determined.