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A characteristic feature of the vertebrate nervous system is the ensheathment of axons by myelin, a multilamellar membrane specialization produced by polarized glial cells. Although the main protein and lipid components of the myelin sheath are well characterized, relatively little is known about the mechanisms of their intracellular distribution to the respective sites of assembly within the myelin sheath. To analyze whether peripheral myelin protein trafficking is mediated by glycosphingolipid/cholesterol-enriched membranes (GEMs), we studied the association of established myelin proteins, peripheral myelin protein 22 (PMP22), protein zero (P0), plasmolipin, and myelin basic protein (MBP), with these membrane microdomains. To examine the association of the selected peripheral myelin proteins with detergent-insoluble GEMs, purified myelin from sciatic nerve of adult rat was extracted with Triton X-100 at 4 degrees C and 37 degrees C and, in additional experiments, was pretreated with the cholesterol chelator methyl-beta-cyclodextrin. The material was then centrifuged to equilibrium in sucrose gradients, and fractions were analyzed by Western blotting. Here we demonstrate for the first time that PMP22, P0, and plasmolipin prepared from purified peripheral myelin are associated with GEMs. To characterize whether the association of these proteins is a specialized feature of myelinating Schwann cells, we studied the distribution of PMP22, P0, and plasmolipin in transiently transfected HeLa cells. These experiments confirm the specific association of these proteins with GEMs in both neural and nonneural cell types., (Copyright 2002 Wiley-Liss, Inc.)

Neurofibromas are benign tumors arising from the peripheral nerve sheath and are a typical finding in neurofibromatosis type 1 (NF1). Schwann cells are the predominant cell type in neurofibromas and thus are supposed to play a major role in the pathogenesis of these tumors. It is not known, however, if NF1 mutations in Schwann cells result in an altered phenotype that subsequently leads to tumor formation. To characterize the biological properties of neurofibroma-derived Schwann cells we developed cell culture techniques that enabled us to isolate Schwann cells from neurofibromas and grow them in vitro for several weeks without significant fibroblast contamination. Neurofibroma-derived Schwann cells were characterized by altered morphology, heterogeneous growth behavior, and increased expression of the P0 antigen while several other features of normal human Schwann cells were retained. We conclude that neurofibroma-derived Schwann cells exhibit a distinct phenotype in vitro but that the observed abnormalities by themselves are insufficient to explain neurofibroma formation. Application of our improved culture conditions makes neurofibroma-derived Schwann cells readily available for further studies to define their role in tumorigenesis in neurofibromatosis type 1., (Copyright 2000 Wiley-Liss, Inc.)

Tenascin-R (TN-R), an extracellular matrix constituent of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions underlying axon growth inhibition/guidance, myelination and neural cell migration during development and regeneration. Although most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide first evidence that TN-R derived from whole rat brain or cultured oligodendrocytes expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e., C-4S and C-6S, that are recognized by CS-56, a CS/dermatan sulfate-specific monoclonal antibody. Based on different in vitro approaches utilizing substrate-bound glycoprotein, we found that TN-R-linked CS GAGs (1) promote oligodendrocyte migration from white matter microexplants and increase the motility of oligodendrocyte lineage cells; (2) similar to soluble CS GAGs, induce the formation of glial scar-like structures by cultured cerebral astrocytes; and (3) contribute to the antiadhesive properties of TN-R for neuronal cell adhesion in an F3/F11-independent manner, but not to neurite outgrowth inhibition, by mechanism(s) sensitive to chondroitinase or CS-56 treatments. Furthermore, after transection of the postcommissural fornix in adult rat, CS-bearing TN-R was found to be stably upregulated at the lesion site. Our findings suggest the functional impact of TN-R-linked CS on neural cell adhesion and migration during brain morphogenesis and the contribution of TN-R to astroglial scar formation (CS-dependent) and axon growth inhibition (CS-independent), i.e., suppression of axon regeneration after CNS injury., (Copyright 2000 Wiley-Liss, Inc.)

The peripheral myelin protein PMP22 gene has been described as a growth arrest-specific gene gas3 and has been identified as disease gene of various demyelinating neuropathies. The gene consists of two highly conserved alternative noncoding 5'-exons la (CD25) and 1b (SR13), respectively. Differential expression patterns of these transcripts in vivo and in vitro suggest a very complex mode of PMP22 gene regulation, which cannot be explained merely by transcriptional control. In fact, the PMP22 gene is regulated on different post-transcriptional levels. While reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no alterations in stability for both PMP22 transcripts in randomly growing Schwann cell cultures of rat sciatic nerve for at least 8 hours, in serum-induced synchronized cultures of resting cells we observed a specific cell cycle-regulated degradation of both transcripts. We further prepared diverse PMP22/CAT fusion genes to study the influence of the alternative 5'UTRs on PMP22 translation. Transient transfection of NIH3T3-fibroblasts and rat Schwann cells demonstrated that the alternative 5'UTRs (CD25 and SR13) and the 3'UTR exert differential regulatory influences on the translation efficiency.

Molecular genetic studies have shown that the peripheral myelin protein 22 (PMP22) is a key gene in hereditary peripheral neuropathies and appears to be essential for the formation and maintenance of myelin in the PNS. Based on the amino acid sequence the predicted structure of PMP22 protein contains two major distinct hydrophilic regions and four transmembrane domains. To analyze the cellular localization and membrane topology of PMP22 we inserted an octapeptide tag-sequence at the amino or at the carboxyl terminus of the PMP22 open reading frame and generated different chimeric constructs which were expressed in HeLa cells. The expression of the tagged PMP22 protein and its orientation with respect to the plasma membrane were analyzed using antibodies raised against specific PMP22 epitopes and the tag sequence. Combined indirect, double-immunofluorescence labeling and confocal microscopy showed that PMP22 is synthesized in the rough endoplasmic reticulum of transfected cells and passes through the Golgi apparatus to the cell surface. We determined the transmembrane organization of PMP22 providing the first experimental evidence that confirms the cytoplasmic disposition of its N and C termini and the extracellular localization of the two hydrophilic domains containing amino acids 28-40 and 118-131. This study provides the basis for further analysis aimed to identify functional domains of wild-type PMP22 and the cellular sorting of mutant forms of PMP22.

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot-Marie-Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over- or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT-PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation.

A nearly pure neuronal culture from embryonic rat brain (hippocampus) has been established in order to observe individual neurons during their in vitro development. This neuronal culture has been used as a test system for a neurotrophic factor (NTF) which is released into a serum-free defined medium, presumably by astrocytes, the dominant cell type in a primary glial culture from rat brain. NTF is essential for the development of the hippocampal neurons in cell culture. The number of neurons responding to NTF by neurite extension is highly dependent on the concentration of the factor in the culture medium. The amount of factor in serum-free glial conditioned medium can be estimated in a rapid (20-hour) bioassay.