Purification and expansion of human Schwann cells in vitro.

The ability to culture cells from the human nervous system provides new insight into the pathophysiology of neurological diseases and could be crucial to the development of gene replacement therapies and neural transplantation. We report that the proliferation of human Schwann cells isolated from paediatric and adult nerves is sustained in vitro by recombinant glial growth factor. Agents that increase intracellular cyclic cAMP were also mitogenic towards Schwann cells but suppress growth of contaminating fibroblasts. As the lifespan of highly enriched cultures can be extended for up to twelve population doublings, large numbers of cells can be generated from nerve biopsies.

Restricted replication of lentiviruses. Visna viruses induce a unique interferon during interaction between lymphocytes and infected macrophages.

Lentivirus infections are characterized by a persistent, restricted type of virus replication in tissues. Using sheep and goat lentiviruses, whose target cells in vivo are macrophages, we explored virus-host cell interactions to determine whether an interferon (IFN) is produced during virus replication in vivo which causes restricted replication. We show that the lentiviruses were incapable of inducing IFN directly in any infected cell, including macrophages and lymphocytes. However, after infection with these viruses, sheep and goat macrophages acquired a factor that triggered IFN production by T lymphocytes. Only sheep/goat lentiviruses were capable of inducing the factor and, although these viruses replicated productively in various cell cultures of the natural host animal, only infected macrophages developed the IFN-inducing factor. The factor was produced continuously and was strictly cell associated, requiring direct contact with lymphocytes. The lymphocytes responded with a single, sudden release of IFN beginning 7 h after cocultivation and reaching peak values at 48 h, after which they ceased production and became refractory. IFN production was not immunologically specific and did not require histocompatibility between donors of the two cell types. The IFN is a nonglycosylated protein of molecular weight 54,000-64,000, and is stable to heat and acid treatments. These findings identify a unique IFN and a new method for virus induction of IFN. The novel two-stage process of induction provides a mechanism for local amplification and continuity of production of IFN in vivo. This is compatible with infection in the animal whose lentivirus-induced pathologic lesions consist of accumulations of lymphocytes and infected macrophages in target tissues.

Homophilic adhesion of the myelin P0 protein requires glycosylation of both molecules in the homophilic pair.

The myelin P0 protein is glycosylated at a single site, asparagine 93, within its only immunoglobulin (Ig)-like domain. We have previously shown that P0 behaves like a homophilic adhesion molecule (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.). 344:871-872). To determine if the sugar residues of this molecule contribute to its adhesiveness, the glycosylation site was eliminated by replacing asparagine 93 with an alanine, through site-directed mutagenesis of the P0 cDNA. The mutated P0 cDNA was transfected into CHO cells and surface expression of the mutated P0 was assessed by immunofluorescence, limited trypsinization and an ELISA. A cell line was chosen which expressed approximately equivalent amounts of the unglcosylated P0 (UNGP0) at the cell surface as did a cell line expressing the fully glycosylated P0 (GPo); the adhesive properties of these two cell lines were compared. It was found that when a single cell suspension of the UNGPo cells were incubated, by 60 min, unlike the GP0 cells, they had not formed large aggregates; they were indistinguishable from the control transfected cells. This suggests that the UNGP0 protein does not behave like an adhesion molecule. To establish if only one molecule in the P0:P0 homophilic pair must be glycosylated for adhesion to occur, the ability of UNGP0 cells to adhere to GP0 cells was assessed both qualitatively and quantitatively. The results of both types of assay imply that, indeed, both P0 molecules in the homophilic pair must be glycosylated for adhesion to take place.

Topography of cerebroside sulfotransferase in Golgi-enriched vesicles from rat brain.

Cerebroside sulfotransferase (CST) catalyzes the final step in the synthesis of sulfatide (sulfogalactocerebroside) by transferring the sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to galactocerebroside. Orientation of CST was studied in vesicles enriched in this enzyme obtained from 21-d-old rat brain. Several lines of evidence indicate that CST is located on the luminal side of these vesicles. (a) Sulfation of endogenous galactocerebroside occurred in vesicles only in the presence of a detergent to render the membranes permeable to exogenous PAPS. (b) There is a pool of latent enzyme within the vesicle, which is released by Triton X-100. (c) CST is not destroyed by trypsin unless the vesicle membranes are first made permeable by Triton X-100. (d) Glycolipid substrate, when covalently attached to agarose beads, was not sulfated unless the enzyme was solubilized. These results are similar to those obtained with thiamine pyrophosphatase, which is known to be located within the lumen of the vesicles. This study establishes that an enzyme synthesizing a complex glycolipid is localized within Golgi-enriched vesicles. Since the product of the CST reaction must also be localized to the luminal side of the vesicles, it is most likely that sulfatide is located at the intraperiod line (outer layer) of myelin. The orientation of CST within the vesicle provides a mechanism for the asymmetrical assembly of glycolipids in bilayers.

Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney.

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.

Myelinogenesis in optic nerve. A morphological, autoradiographic, and biochemical analysis.

Morphological, autoradiographic, and biochemical methods were used to study the time of appearance, distribution, and nature of sulfated constituents in the developing rat optic nerve. Electron microscope studies showed that myelination begins (6 days postnatal) shortly after the appearance of oligodendroglia (5 days postnatal). Over the ensuing 3 wk, myelination increased rapidly. During the 1st postnatal wk, mucopolysaccharides and glycoproteins were labeled with 35S and autoradiographs showed grains over arachnoidal cells, astroglia, and the glia limitans. These results indicated that astroglia synthesize sulfated mucopolysaccharides of the glia limitans. After the onset of myelination, however, the major portion of [35S]sulfate was incorporated into sulfatide. Autoradiographs showed a shift of radioactive grains from astroglia and arachnoidal cells to myelin, indicating that actively myelinating oligodendroglia incorporate [35S]sulfate into myelin sulfatide; there was a concomitant increase in the activity of cerebroside sulfotransferase. In addition, the increasing amounts of proteolipid protein and myelin basic protein corresponded with the morphological appearance of myelin. These results point to a strict correlation between the structural and biochemical changes occurring during myelination. This system provides a useful model for studies designed to evaluate the effects of various perturbations on the process of myelination.

Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter.

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor.

Some neurodegenerative disorders may be caused by abnormal synthesis or utilization of trophic molecules required to support neuronal survival. A test of this hypothesis requires that trophic agents specific for the affected neurons be identified. Cholinergic neurons in the corpus striatum of neonatal rats were found to respond to intracerebroventricular administration of nerve growth factor with prominent, dose-dependent, selective increases in choline acetyltransferase activity. Cholinergic neurons in the basal forebrain also respond to nerve growth factor in this way. These actions of nerve growth factor may indicate its involvement in the normal function of forebrain cholinergic neurons as well as in neurodegenerative disorders involving such cells.

The role of complex carbohydrates in adhesion of the myelin protein, P0.

The most abundant protein of peripheral nerve myelin, a glycoprotein termed P0, is believed to be involved in the compaction of the myelin sheath and is postulated to be the closest relative to the ancestral gene for the immunoglobulin superfamily. Recently, P0 has indeed been shown to behave like a homophilic adhesion molecule via interactions of its extracellular domains. Here we demonstrate the importance of the oligosaccharide moieties of P0 in its functioning as a homophilic adhesion molecule. Expression of the complex form of P0 glycoprotein in transfected Chinese hamster ovary cells greatly increased the adhesiveness of those cells, whereas expression of the high-mannose form of P0 glycoprotein did not. This is the first step in the dissection of P0-P0 interaction at the molecular level.

SV40 large T antigen with c-Jun down-regulates myelin P0 gene expression: a mechanism for papovaviral T antigen-mediated demyelination.

Expression of myelin proteins has been shown to be altered in transgenic mice that express papovaviral large tumor (T) antigens. This paper analyzes the effect on P0 gene expression in secondary Schwann cells transfected with the SV40 T antigen gene and in Schwann cells immortalized by T antigen. In secondary Schwann cells, both T antigen and c-Jun are required for significant inhibition of the P0 promoter; expression of only one of the proteins is insufficient for repression of the P0 gene. T antigen, c-Jun (p39), and c-Jun-related protein (p47) form an immunoprecipitable complex in SV40 immortalized Schwann cell lines, and T antigen and c-Jun bind independently and as a complex to the P0 promoter. Our data suggest that the probable molecular mechanism underlying the hypomyelination observed in transgenic animals expressing T antigen may be due to the repression of the P0 gene by T antigen and c-Jun.

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.

Schwann cell-conditioned medium promotes neuroblastoma survival and differentiation.

Neuroblastomas are histopathologically heterogeneous, ranging from immature malignant tumors to benign ganglioneuromas. The amount of Schwann cell stroma greatly increases with neuroblastoma differentiation, and these Schwann cells appear to be normal cells that infiltrate the tumor. To determine whether Schwann cells influence neuroblast differentiation, four human neuroblastoma cell lines were cultured in the presence or absence of human Schwann cell-conditioned medium for 7 days. Neuroblastoma cell survival, as determined by a colorimetric assay, more than doubled in three of the four neuroblastoma cell lines in the Schwann cell-conditioned medium. There was a corresponding reduction in apoptosis as measured by a nick-end labeling assay, with little change in mitotic rate. Schwann cell-conditioned medium induced extensive neurite outgrowth in all of the neuroblastoma cell lines, and these processes contained mature neurofilament in three of the cell lines. These results indicate that Schwann cells produce soluble substances capable of supporting survival and differentiation in neuroblastoma cell lines. This may represent a biological mechanism responsible for neuronal differentiation in stroma-rich neuroblastomas.

Fetal metachromatic leukodystrophy: pathology, biochemistry and a study of in vitro enzyme replacement in CNS tissue.

Brain tissue from a fetus with the diagnosis of metachromatic leukodystrophy (MLD) became available at autopsy. Pathologic studies of the CNS showed inclusion bodies within oligodendroglia. The morphology of myelin was normal. Cells and myelin were isolated from the cerebrum; there was an increased level of sulfatide present in both fractions. In vitro studies of enzyme replacement in cultured MLD brain cells indicated that it may be possible to correct the abnormal sulfatide accumulation.

Spin-label studies of erythrocytes in myotonic dystrophy: no increase in membrane fluidity.

The basic defect in myotonic dystrophy is thought to involve muscle cell membranes. Butterfield and associates have recently presented electron spin resonance data that suggest increased fluidity of erythrocyte membranes in patients with myotonic dystrophy. We studied erythrocytes from 11 patients with myotonic dystrophy and 14 age-matched controls, using spin-labeled fatty acid and ester probes. Despite attempts to reproduce the previously reported experimental conditions exactly, we found no significant differences in the electron spin resonance spectra of erythrocytes from normal and myotonic dystrophy subjects. These findings do not provide evidence of increased erythrocyte membrane fluidity in myotonic dystrophy; they fail to support the concept of an intrinsic defect of the lipid membrane in this disorder.

Signals for proinflammatory cytokine secretion by human Schwann cells.

Wallerian degeneration is a post-traumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infiltrating leukocytes. Our studies indicate that Schwann cells could initiate the process of Wallerian degeneration by releasing proinflammatory cytokines involved in leukocyte recruitment and differentiation including IL-1beta, MCP-1, IL-8 and IL-6. A comparison of the secretory pattern between nerve explants and cultured Schwann cells showed that each cytokine was differentially regulated by growth factor deprivation or axonal membrane fragments. Since Wallerian-like degeneration occurs in a wide variety of peripheral neuropathies, Schwann cell-mediated cytokine production may play an important role in many disease processes.

Production of Schwann cell lines using a regulated oncogene.

The process of myelination in the central and peripheral nervous systems has been well characterized morphologically by a variety of techniques. It is evident from these studies that, in the peripheral nervous system myelin formation is a multistep process. Clearly, a 1:1 relationship must be established with the axon, which is followed by formation of the basal lamina and eventually myelin. Because immortalized Schwann cell lines obtained using SV40 T antigen under the control of an inducible promoter have many properties of untransfected Schwann cells in culture, including their ability to form myelin in vitro, these cells will enable us to dissect more easily the process of myelination. Having successfully immortalized rat Schwann cells without affecting their ability to differentiate fully, we are applying this approach to generate analogous cell lines from the peripheral nerves of other species such as mouse and human. Unlike rat Schwann cells, there are no known mitogens for human and mouse Schwann cells, making it impossible to expand these cell populations. The ability to produce large numbers of human Schwann cells from nerve biopsy and to analyze their biochemical properties would be of enormous value in identifying the cellular abnormalities that result in demyelinating disease. Likewise, there are several mutant mouse strains with defects in myelin formation, and cell lines from these animals would facilitate our understanding of the process leading to dysmyelination.

Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons.

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.

Sulfatide synthesis in mice with defective synthesis of 3'-phosphoadenosine 5'-phosphosulfate.

The regulation of sulfation of galactocerebroside was examined in several tissues from brachymorphic mice, including the central nervous system and kidney. These animals have an inherited defect in the enzymes which synthesize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) resulting in decreased sulfation of glycosaminoglycans. In contrast, sulfation of galactocerebroside to form sulfatide (sulfogalactocerebroside) was normal in brachymorphic mice even in tissues with decreased ability to synthesize PAPS. This suggests that, unlike the regulation of glycosaminoglycan sulfation, the formation of sulfatide is not stringently regulated by tissue levels of PAPS.

Ocular clinicopathologic correlation of Hallervorden-Spatz syndrome with acanthocytosis and pigmentary retinopathy.

We studied the eyes of a 10-year-old girl with retinal degeneration, acanthocytosis, and normal betalipoprotein levels. The ophthalmoscopic pattern was characterized initially by a flecked retina and later by bone-spicule formation and "bull's-eye" annular maculopathy. On ultrastructural study, the retinal pigment epithelium varied in size and contained large, round single-membrane-bound aggregates composed of complex melanolipofuscin granules. Cells that had migrated into the outer retinal layers contained similar melanolipofuscin aggregates; these cells were identified as macrophages and correlated with the flecks and macular annulus seen on ophthalmoscopy. The cells around the retinal blood vessels contained normal melanin pigment, were identified as retinal pigment epithelial cells, and correlated with the bone spicule pigmentation found on ophthalmoscopic examination.

Progressive spinal muscular atrophies.

Spinal muscular atrophy is the most common autosomal-recessive genetic disorder lethal to infants. It was first described in the 1890s. Since then our understanding of the disorder has progressed significantly. Progression of the disease is due to loss of anterior horn cells, thought to be caused by apoptosis. Diagnosis is based on the course of the illness, as well as certain changes seen on nerve and muscle biopsy and electrodiagnostic studies. More recently, our understanding of the genetics of this disorder has provided a noninvasive approach to diagnosis. This method of testing has its downside, but the quest for a more sensitive analysis is still underway. Even though our knowledge of this disease has come a long way since its first recognition, the therapies available to these children are still only supportive. Again, researchers eagerly look for new therapeutic interventions to allow for improved quality of life and an extended life span.