Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord.

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2-8 mm from a lysolecithin-induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2-3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 microns/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 microns/hr. Migration was much slower in white matter tracts (95 microns/hr +/- 54 in the shiverer and only 38 microns/hr +/- 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin-induced lesion since 1) their number increased progressively with time in the lesion, and 2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, P0, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non-immortalized rat Schwann cells.

Establishment and characterization of a mouse Schwann cell line which produces myelin in vivo.

A Schwann cell line (MSC 80) was established from purified mouse Schwann cell cultures using large doses of serum. MSC 80 cell line is an aneuploid cell line which has a doubling time of 17 hr and has been maintained through more than 110 passages. Most of MSC 80 cells are of bipolar or stellate (3-5 processes) shape. A few others are irregular in shape, gigantic, and multinucleated. All MSC 80 cells express antigens of myelin-forming Schwann cells such as S-100, 224/58, laminin, and other glycoproteins of the extracellular matrix. However, they also express the non-myelin-forming Schwann cell antigen GFAP. By time-lapse cinematography, MSC 80 cells exhibit the Schwann cell characteristic rhythmical undulations. When induced to form aggregates in agar, they form intercellular junctions and basement membrane-like structures. In addition, after transplantation in or at a distance from a lysolecithin induced lesion, MSC 80 cells form myelin around the host demyelinated axons. MSC 80 cells thus express, when isolated in vitro, some of the normal myelin-forming Schwann cell phenotype. In addition, they present the major advantage of forming myelin when associated with axons in vivo.

Endogenous and exogenous alpha-fetoproteins as differential markers of cultured neonatal mouse Schwann cells and fibroblasts.

alpha-Fetoprotein (AFP) and AFP-gene transcripts were demonstrated in vitro in Schwann (S) cell and fibroblast (F) cell cultures of neonatal mouse origin. All S and F cells of primary cultures and of established cell lines expressed the AFP gene. AFP mRNA was detected by an in situ hybridization technique using a 35S-AFP-cDNA probe. AFP was localized by immunocytoperoxidase labelling using purified anti-AFP antibodies. The amounts of stained endogenous AFP, estimated semiquantitatively, were about 3-fold higher in S cells than in F cells. After incubating the cultures with exogenous mouse AFP, both S and F cells showed significant ability to take up the protein; the amount of internalized protein was found to be higher in F cells than in S cells. Moreover, the uptake of AFP fluorescein conjugates (FITC-AFP), estimated quantitatively by fluorometry, also gave higher values for F cells. The cytoplasm of F cells exhibited a characteristic fluorescence pattern, strongly illuminated and dispersed grains; the cytoplasm of S cells was regularly labelled. If exogenous FITC-AFP uptake could be used to distinguish labelled F cells from S cells (with application for identification and selection of F cells), the immunocytochemically stained endogenous AFP could allow S cells to be distinguished from F cells (using the dilutions of antibodies still staining the S cells but which lead to the absence of F-cell labelling). The two procedures, which can be used independently or together, may constitute differential markers for S cell and F cultures in, i.e., nerve regeneration of neurofibroma studies using the model of mixed S and F culture also containing other types of cells.

Adult sphingomyelinase deficiency: report of 2 patients who initially presented with psychiatric disorders.

We studied 2 unrelated adult patients under neuroleptic treatment who met all phenotypic and biochemical criteria for Niemann-Pick disease type B. In addition, they had chronic psychiatric disorders and low blood levels of HDL cholesterol. The marked and persistent deficiency of acid sphingomyelinase and the disturbance of sphingomyelin metabolism in skin fibroblast subcultures ruled out a pure drug-induced lipidosis. The association of Niemann-Pick disease type B with psychiatric disorders and with low levels of HDL cholesterol could be a chance association of 2 diseases, a new phenotype of Niemann-Pick type B, or the revelation by the neuroleptic treatment of a subclinical inborn sphingomyelinase deficiency.

Calmodulin antagonist W-7 inhibits lysosomal sphingomyelinase activity in C6 glioma cells.

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) is known to be a potent calmodulin antagonist and inhibitor of calmodulin-dependent protein kinases. W-7 and 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) are inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. In C6 glioma cells, W-7 and not H-7 inhibited dose-dependently acid sphingomyelinase, a result indicating the modulation of this lysosomal enzyme by a calmodulin-dependent system. Other lysosomal enzymes, such as beta-glucosidase, alpha-galactosidase, and arylsulfatase A, were unaffected by W-7 and H-7, a finding indicating a selective effect of W-7 on sphingomyelinase.

Effects of vigabatrin and of GABA on myelinated rat cerebellar cultures: preliminary data.

1. The aim of this study was to evaluate the effect of high concentrations of vigabatrin (gamma-vinyl GABA) and of GABA on myelin of the central nervous system cultures. 2. Explants of rat cerebellum were cultured for 14-19 days in vitro on collagen-coated coverslips in Leighton tubes. They were exposed for up to 14 days to 500 nmol ml-1 vigabatrin or to 1000 nmol ml-1 GABA. 3. Qualitative and quantitative blind examination of living cultures and of Sudan black B-stained slides showed mild toxicity of both drugs for myelinated fibres. No clear-cut differences could be demonstrated between the two compounds, although vigabatrin seemed slightly more toxic than GABA at these doses. 4. In electron microscopy, no patent intramyelinic oedema nor primary demyelination were seen. On the contrary, some degenerating myelinated fibres and astrocytic gliosis were seen in both experimental conditions. The changes involved axons as well as myelin sheaths. 5. The toxicity of GABA and vigabatrin was surprisingly mild in this very sensitive model.

Acidic sphingomyelinase: relationship with antidepressant-induced desensitization of beta-adrenoceptors.

Previous results indicate a dose-dependent decrease of lysosomal sphingomyelinase activity induced by tricyclic antidepressants in cell cultures. A possible association of this effect with the antidepressant-induced down-regulation of beta-adrenoceptors was postulated. We report here the determination of beta-adrenoceptor binding sites and lysosomal sphingomyelinase activity in the cerebral cortex of rats treated chronically with desipramine or with the potential antidepressant drug midalcipran (which is devoid of effect on beta-adrenoceptors). The effect of midalcipran on lysosomal sphingomyelinase activity was also determined on C6 glioma cells. In C6 glioma cells, midalcipran did not decrease sphingomyelinase activity, at variance with the enzymatic inhibition induced by desipramine (DMI). In the rat cerebral cortex, neither DMI nor midalcipran modified sphingomyelinase activity. In agreement with previously reported effects, DMI induced beta-adrenoceptor desensitization in the rat cerebral cortex, while midalcipran remained ineffective. Our results indicate that in the rat cerebral cortex, the activity of lysosomal sphingomyelinase is not modulated by chronic treatment with antidepressant drugs, whatever their effect on beta-adrenoceptor sites. Our results suggest that sphingomyelinase activity is not associated with the desensitization of beta-adrenoceptors, taken as an index of the therapeutic action of antidepressants. The results indicate that care should be taken when extrapolating to in vivo situations the conclusions derived from cell culture conditions.

[Demyelination in in vitro culture in multiple sclerosis: facts and controversies]. / Démyélinisation en culture in vitro au cours de la sclérose en plaques: faits et controverses.

The myelinotoxicity of sera from multiple sclerosis (MS) patients was determined by the assessment of visible myelin damage in guinea-pig spinal cord-spinal ganglia and in rat cerebella cultivated on collagen-coated coverslips in Leighton tubes. No change was seen in 22 cases. These data show that serum myelinotoxicity is low in MS - as compared with that of Experimental Allergic Encephalomyelitis. It appears a little specific phenomenon, the mechanism of which remains unclear. It can be easily assessed only in very sensitive culture techniques and is best measured by biochemical methods. This does not preclude the pathophysiological significance of the serum myelinotoxicity in MS. Supernatants of cerebro-spinal fluid (CSF) cell cultures in 10 MS patients caused no demyelination. Pooled concentrated supernatants of CSF cell cultures from 13 to 20 MS patients--containing from 7.3 to 11.7 micrograms IgG/ml gave no patent in vitro demyelination in 3 different experiments. The released products of CSF cell cultures in MS are not very toxic for myelin. However these experiments have to be repeated with more sensitive culture techniques, biochemical assay of myelinotoxicity and more concentrated CSF cultures supernatants.

The M2 autoantigen of central nervous system myelin, a glycoprotein present in oligodendrocyte membrane.

Autoantibodies with in-vitro demyelinating capacity induced in Hartley and strain 13 guinea pigs with homologous central nervous system (CNS) tissue were used to characterize the target autoantigen M2. Using the Dot Immunobinding technique, M2 was found to be a component of CNS myelin different from basic protein (BP) and from cerebroside. The expression of M2 on oligodendrocytes, cells known to produce CNS myelin, also confirmed that M2 was a component of CNS myelin. Furthermore, the autoradiography of immunoprecipitates formed with radiolabelled guinea pig myelin and analysed in sodium dodecyl sulphate gels showed that M2 was specific to CNS myelin and absent in peripheral nervous system (PNS) myelin. On electrophoresis M2 appeared as two CNS myelin protein bands at the 27 and 54 KD molecular weight levels, distinct from the major protein bands of proteolipid and BP. M2 bands were of glycoprotein nature, as was demonstrated by affinity chromatography of CNS myelin on wheat germ agglutinin (WGA)-Sepharose. A monoclonal antibody induced by BP-free CNS glycoproteins recognized the same bands as anti-M2 serum in guinea pig CNS myelin. This would imply that both M2 bands share common determinants. M2 bands similar to the above in guinea pig were also shown in rat, rabbit and bovine CNS myelin with guinea pig antibodies. The same type of anti-M2 antibodies were induced in rabbit immunized with homologous CNS tissue. Although only a minor component of myelin, M2 is strongly immunogenic compared to BP. M2 antigen could thus be the target of chronic demyelinating processes such as experimental allergic encephalomyelitis.

Schwann cell marker defined by a monoclonal antibody (224-58) with species cross-reactivity. I. Cellular localization.

We have demonstrated by indirect immunofluorescence the cellular localization of a monoclonal antibody (mAb 224-58), produced after immunization of a mouse with human central nervous system (CNS) myelin. Serologically, mAb 224-58 was found to be specific for 3'-sulfomonogalactosylglycolipids, namely 3'-sulfogalactosylceramide (SGC) and 3'-sulfogalactosyl 1-O-alkyl ether 2-O-acylglycerol (seminolipid). This mAb did not bind to SGC-containing tissues such as kidney, liver, spleen, or brain, nor to muscle. However mAb 224-58 did stain positively mouse, rat, and human peripheral nerve sections. In these latter sections, mAb 224-58 was bound to Schwann cell bodies and processes. The specificity of mAb 224-58 for Schwann cells was ascertained on teased rat sciatic nerves and rat Schwann cell cultures. Cells positive for mAb 224-58 were also positive for laminin, and negative for Thy 1-1 antigens both in teased fibers and Schwann cell cultures. In addition, in teased nerve preparations, mAb 224-58-positive cells were also galactosylceramide (GalC)- and SGC-positive. Isolated Schwann cells also expressed 224-58 antigen, even after prolonged time in culture. On testis sections, which contain both SGC and seminolipid, the SGC-positive cells, i.e., the spermatogonia, were always 224-58-negative. But the other germinal cells were 224-58-positive. This suggests that although 224-58 does not discriminate between SGC and seminolipid in serological tests, these lipids in their naturally occurring membrane acquire a spatial configuration that renders them distinguishable to their respective antibody.

[A new promotor of nerve growth: naftidrofuryl]. / Un nouveau promoteur de croissance nerveuse: le naftidrofuryl.

20 day-old rat thoracic dorsal root ganglia were grown for 48 hrs. in Iscove's medium supplemented with 8% fetal calf serum and 600 mg/100 ml glucose. Naftidrofuryl was added at 10(-6), 10(-7), 10(-8) and 10(-9) M concentrations to the culture medium. The 10(-7) and 10(-8) M concentrations induced a statistically significant increase of the number (30 to 40%; p = .0054 and .0016, respectively) and length (20 to 30%; p = .0012 and .001, respectively) of neurites of the outgrowth measured after Bodian's protargol impregnation. The width of the cell spread in the outgrowth was also enlarged at the 10(-7) and 10(-8) M concentrations (18 to 26%; p = .0012; p less than .0001, respectively).

Desipramine elicits the expression of opiate receptors and sulfogalactosylceramide synthesis in rat C6 glioma cells.

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.

Effect of tricyclic antidepressants on sphingomyelinase and other sphingolipid hydrolases in C6 cultured glioma cells.

Cationic amphiphilic drugs, which include tricyclic antidepressants, have been shown to give rise to lipidoses under experimental conditions, with a general increase of lipids especially phospholipids. We report here an early and important decrease in sphingomyelinase activity in C6 glioma cells cultured in the presence of imipramine or desipramine at final concentrations of 0.01 and 0.05 mM. The effect was both dose-dependent and time-dependent and was observed before any lipid accumulation. Cerebroside beta-glucosidase and cerebroside beta-galactosidase had normal activities under the same experimental conditions and thus there was no general effect on membrane-bound sphingolipid hydrolases. A decrease of sphingomyelinase activity has been previously reported for two amphiphilic compounds, perhexiline maleate and AY 9944. These results suggest a potential function of sphingomyelinase in the mode of action of these drugs.

Tricyclic antidepressant desipramine induces stereospecific opiate binding and lipid modifications in rat glioma C6 cells.

Incubation for 48 hours of C6 glioma cell cultures with 10(-4)M tricyclic antidepressant desipramine gave rise to a quantitative increase of total lipids and to qualitative modifications of glycosphinegolipids involving detection by thin-layer chromatography of spots migrating according to cerebroside and sulfatide and presence of an abnormal ganglioside pattern. These lipid modifications were associated with the appearance of stereospecific binding of opiates (dihydromorphine) with a dissociation constant of 30-60 nM. These results favor an important role of lipids in opioid receptor function.

Peripheral nerve cells in culture rich in Schwann cells incorporate and metabolize trans-unsaturated fatty acid (elaidic acid) as well as physiological dis isomer (oleic acid).

A culture of peripheral nerve cells enriched in Schwann cells was obtained from sciatic nerve in normal and demyelinating trembler mutant. These cells incorporated and metabolized a non-physiological trans fatty acid (elaidic aid) as well as the physiological cis isomer (oleic acid). Both acids were incorporated similarly in all lipids studied (phosphatidylcholine was a very potent acceptor) only cholesterol-esters' formation was slightly reduced from elaidic acid. Both acids were partially degradates into sub-units, in turn used for synthesis of new fatty acids. However elaidic acid was less degraded by the cells thus providing more C14:1, C16:1 fatty acids and less cholesterol. The sub-units were also used to provide very long chains, saturated and mono-unsaturated; only synthesis of nervonic acid was at variance when using oleic and elaidic acids. The presence of elaidic acid diminished the elongation-desaturation of essential fatty acids. No major differences were found between control and trembler cells, however cholesterol-esters' synthesis was slightly enhances in the mutant cells, when using both acids.

Tricyclic antidepressants induce sphingomyelinase deficiency in fibroblast and neuroblastoma cell cultures.

Tricyclic antidepressants (imipramine and desipramine) gave rise to an important decrease of sphingomyelinase activity in murine neuroblastoma and human fibroblast cell cultures. It occurred within 1 to 2 hours at a final concentration of 1 or 2 X 10(-5) M in cell culture medium. Other lysosomal enzymes such as acid lipase, arylsulfatases A and B and hexosaminidases were not modified. Low level of sphingomyelinase activity may be related to the amphiphilic characteristics of the drugs: iminodibenzyle which has the same tricyclic core but is devoid of the side chain necessary for amphiphilic properties had no effect. As iminodibenzyle has no therapeutic action, amphiphilic may be requisite to antidepressant properties of tricyclic drugs.