Specific and potent mitogenic effect of axolemmal fraction on schwann cells from rat sciatic nerves in serum-containing and defined media

Using cultures of Schwann cells from neonatal rat sciatic nerves, we examined the mitogenic activity of an axolemmal fraction from adult rat CNS. Axolemmal fraction proved a potent mitogen, stimulating [³H]thymidine incorporation into Schwann cell DNA 13.5 fold over control values when axolemmal fraction equivalent to 16 μg of protein per culture microwell or more was added. Half maximal stimulation was obtained with addition of axolemmal fraction equivalent to 4 μg of protein. The concentration-dependence and magnitude of the mitogenic response of the cultured cells were nearly identical whether they were maintained in vitro for 1 day or for 2 weeks prior to addition of the axolemmal fraction. A study of the time-course of the effect of axolemmal fraction on Schwann cell mitosis showed that maximal [³H]thymidine incorporation took place during the fifth day after addition of axolemmal fraction. Axolemmal fraction also produced stimulation of [³H]thymidine incorporation into Schwann cells, seeded and cultured in a serum-free defined medium. Though the concentration-dependence of the mitogenic effect in the absence of serum was similar to that in a serum-containing medium, maximal stimulation in the defined medium was only 2.8-fold. The mitogenic activity of axolemmal fraction was rapidly and almost totally inactivated by sonication or homogenization, and was partially lost after exposure to heat. The mitogenic activities of plasma membrane fragments from rat skeletal muscle or rat erythrocytes, andof mitochondrial fragments (the major contaminant of the axolemmal fraction) were one-tenth that of axolemmal fraction or less. In contrast to glial growth factor prepared from bovine pituitaries (GGF-BP), which stimulates proliferation of both fibroblasts and Schwann cells, axolemmal fraction induced proliferation of Schwann cells but not of endoneurial fibroblasts; cultures treated with axolemmal fraction demonstrated a 3-fold increase in Schwann cell population in 10 days without detectable increase in number of fibroblasts. Also in contrast to GGF-BP, the mitogenic effect of which is considerably enhanced by simultaneous addition of cholera toxin to the medium, cholera toxin had no effect on the Schwann cell proliferative response to axolemmal fraction.     

Adhesion of axolemmal fragments to Schwann cells: a signal- and target-specific process closely linked to axolemmal induction of Schwann cell mitosis

Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125I-axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more.     

Regulation of glucose transport in cultured Schwann cells

Glucose is the major source of metabolic energy in the peripheral nerve. Energy derived from glucose is mostly utilized for axonal repolarization. One route by which glucose may reach the axon is by crossing the Schwann cells that initially surround the axons. Considering the ability of neurons to control many glial cell functions, we postulated that Schwann cell glucose transporters might be transiently regulated by axonal contact. Glucose transport was studied in a cultured, differentiated rat Schwann cell line stably expressing SV40 T antigen regulated by a synthetic mouse metallothionein promoter. 3[H]-2-deoxy-D-glucose uptake was measured in cultured cells in basal and in various experimental conditions. Glucose transporter gene expression was determined after RNA isolation from cultured cells through Northern and RNAse protection assay. In vitro, Schwann cells were found to express high-affinity, insulin-insensitive, facilitative glucose transporters and predominantly GLUT1 mRNA. Schwann cell 2-deoxyglucose uptake was increased by axolemmal membranes or forskolin but unchanged by elevated glucose levels. Regulation of Schwann cell glucose transporters by axolemma and their resistance to glucose-induced down-regulation suggest extrinsic rather than intrinsic regulation that might enhance Schwann cell vulnerability to glucotoxicity.     

Tissue culture studies of Schwann cell proliferation and differentiation

Neonatal rat sciatic nerve Schwann cells in monolayer culture are stimulated to proliferate and to express a lipid and a protein characteristic of myelin by agents which raise intracellular cyclic adenosine 3',5'-monophosphate. Both glial growth factor and axolemmal fragments increase the rate of mitosis of cultured rat and human Schwann cells. Rat Schwann cell mitosis is enhanced by a soluble factor produced by concanavalin A-stimulated blood mononuclear cells and inhibited by lead salts. Schwann-like cells cultured from human dermal and plexiform neurofibromas resemble normal human Schwann cells in phenotype and response to mitogens.     

Evidence that the axolemmal mitogen for cultured Schwann cells is a positively charged, heparan sulfate proteoglycan-bound, heparin-displaceable molecule

Treatment of axolemma with pH 9 buffer results in a pellet enriched two-fold in the mitogen for cultured Schwann cells. Heparitinase treatment releases 8% of the mitogen into solution, while heparin selectively solubilizes the mitogen, resulting in an extract which has a specific mitogenic activity approximately 2.5 times greater than the mitogenicity of the starting axolemma membrane. These data support a model in which the axolemmal mitogen is a positively charged molecule associated with negatively charged sulfated proteoglycans.     

Axolemmal differentiation in myelinated fibers of rat peripheral nerves

In developing rat peripheral fibers, nodal specialization appears early, prior to myelin compaction, and is first detected as a junction between the axon and the overhanging Schwann cell process characterized by a uniformly wide (approximately 18 nm) intercellular gap containing a patchy dense substance and a cytoplasmic undercoating subjacent to the axolemma. The gap width is rather consistent but the axolemmal undercoating is more variable and lower in density than that found at more mature nodes of Ranvier, and it is also highly variable in length, ranging from 0.5 to 3 micron. The outermost Schwann cell layer is usually prominent with a large volume of cytoplasm and many organelles. In freeze-fracture replicas, modal specializations are characterized by accumulations of large (approximately 10 nm) particles in the axolemma, especially the E face, but immature nodes generally have a lower particle concentration than mature nodes. No node-like particle aggregates have been found in axons not intimately associated with Schwann cells. Mature paranodal axon-Schwann cell junctions are usually formed first by the loops closest to the node and are characterized by a 2-3 nm gap between the apposed membranes, periodic intercellular densities (transverse bands) in the gap and cisternae flattened against the junctional Schwann cell membrane. The loops further removed from the node display a wider gap containing irregularly spaced or diffuse intercellular densities, or none. Mature junctions appear relatively late in the rat, and it is not unusual to find developing nodes with several Schwann cell loops present that do not indent the axolemma significantly and are not associated with the paracrystalline pattern characteristic of the mature junctional axolemma. In such instances, the nodal particle aggregates do not have sharply circumscribed boundaries. The majority of the developing nodes are asymmetric with one paranodal segment more mature than the other.     

Tissue culture studies of neurofibromatosis: effects of axolemmal fragments and cyclic adenosine 3',5'-monophosphate analogues on proliferation of Schwann-like and fibroblast-like neurofibroma cells

Six dermal neurofibromas obtained from 5 patients with neurofibromatosis were dissociated and the cells were plated on polylysine-coated glass. Two principal cell types were observed in the cultures: elongated and bipolar Schwann-like cells (SLCs), and polymorphic flattened fibroblast-like cells (FLCs). Indirect immunofluorescence demonstrated that SLCs expressed surface laminin but not surface fibronectin; FLCs expressed surface fibronectin but were only weakly positive for surface laminin. Tritiated thymidine autoradiography demonstrated that cultured SLCs proliferated slowly (labeling index, 0.7 to 4.0%), whereas FLCs divided more rapidly (labeling index, 7.5 to 26.4%). Axolemmal fragments prepared from human or rat central nervous system specimens adhered to SLCs derived from each of the 6 neurofibromas, but not to FLCs. Axolemmal fragments induced a marked proliferative response of SLCs from 2 of the 6 neurofibromas but had no effect on proliferation of SLCs from the other 4 neurofibromas or FLCs from any of the 6 neurofibromas. In one patient from whom 2 neurofibromas were obtained, SLCs from one neurofibroma responded to axolemmal fragments, while SLCs from the other did not. Treatment of the cultures with 0.1 mM cyclic adenosine 3'5'-monophosphate (cAMP) analogue, 8-bromo cAMP, caused marked inhibition of proliferation of both SLCs and FLCs derived from all 6 neurofibromas. The same concentration of another cAMP analogue, dibutyryl cAMP, inhibited proliferation of SLCs but not of FLCs.     

Inhibition of reactive gliosis in vivo by exogenous axolemma and myelin fractions

Exogenous myelin- or axolemma-enriched fractions were assessed for the ability to inhibit biochemical and morphological expressions of reactive gliosis in rat optic nerve. Elvax pellets containing exogenous myelin, axolemma, whole-brain homogenate, liver, or red cell extracts or no homogenate were inserted into a dural slit in distal regions of crushed optic nerve. Biochemical and morphological expressions of reactive gliosis were assessed at 7 or 14 days postoperatively. Post-traumatic elevations in lactic dehydrogenase activity normally seen at 7 days postoperatively were prevented by placement of Elvax pellets containing myelin or axolemmal fractions into the optic nerve. Morphological analyses indicated an inhibition of post-traumatic elevations in glial cell numbers, surface area, and nuclear size at the 14-day time point. Exposure of the axolemmal fraction to heat or trypsin inactivated its ability to modulate reactive gliotic changes. Myelin fractions were trypsin-sensitive, but not heat-sensitive. In contrast, Elvax pellets containing whole-brain tissue homogenates or liver and red cell membranes had no significant effects on post-traumatic glial changes relative to preparations in which homogenate-free pellets were used.     

Temporary adhesions between axons and myelin-forming processes

Following irradiation, the dorsal funiculus of the lumbosacral spinal cord in the rat undergoes the following sequence of events: (a) a marked reduction of the normal glial population, (b) an absence of oligodendrocyte myelin formation, (c) the invasion and proliferation of Schwann cells, and (d) the myelination of axons within the cord by Schwann cells. The present study demonstrates that, during the latter process, junctional complexes develop between these intraspinal Schwann cells and the axolemma. These complexes are present at sites of probable initial contact between the two membranes. As the Schwann cell process begins to wrap the axons, these junctional complexes are located between the inner spiraling process of the Schwann cell and the axon. With the advancement of myelin formation to the stage of 8 to 9 compact spirals, these contacts are rarely observed. Spinal cords from normal 8-day-old rats were examined in order to determine if such contacts occur during myelination by oligodendrocytes. Although they are more difficult to detect in the normal animal due to the abundance of glial processes, similar junctional complexes occur between oligodendrocyte processes and axons. These observations suggest that these complexes may serve to stabilize and to guide the myelin-forming process around the perimeter of the axon. Additionally, these junctions may play an active role in the advancement of the inner spiraling process by forming temporary adhesions between the axolemma and the adjacent myelin-forming process. Coated vesicles are commonly observed fused with the axolemma of axons which are in the early stages of myelination. These coated vesicles may be involved in the insertion or the deletion of junctional membrane.     

Localization of sodium/potassium adenosine triphosphatase in multiple cell types of the murine nervous system with antibodies raised against the enzyme from kidney

This report describes the development and characterization of a battery of highly specific antibodies to sodium/potassium (Na+ + K+)-ATPase and their use in localizing this enzyme in nervous tissue. The immunolabeling characteristics of polyclonal antibodies and monoclonal antibodies (Schenk, D. B., and H. L. Leffert (1983) Proc. Natl. Acad. Sci. U. S. A. 80: 5281-5285) raised against rat renal (Na+ + K+)-ATPase were compared. The interspecies cross-reactivity of the polyclonal anti-rat antibodies was examined by determining their binding to purified rat, eel, or dog enzyme. The immunostaining characteristics of the IgG fraction of the polyclonal antibody preparations, their affinity-purified derivatives, and the monoclonal antibodies were compared. The results obtained with each of these were similar, providing information about where focal concentrations of the enzyme exist within nervous tissue. The IgG fraction of the polyclonal antibody preparations provided the most sensitive probe, facilitating localization of the (Na+ + K+)-ATPase in the tissue sections from various regions of the nervous system. (Na+ + K+)-ATPase-like immunoreactivity was observed along the plasmalemma of alpha-motor neurons and at the nodal axolemma of myelinated axons from the central or peripheral nervous system. It was determined that the absence of labeling for the enzyme along the paranodal or internodal regions of the axolemma was not an artifact due to a limited accessibility of antibody to these regions. Some central nervous system glial cells demonstrated abundant amounts of plasmalemmal and intracellular (Na+ + K+)-ATPase-like immunoreactivity. These cells were identified as astroglia by positive labeling of cells in serial sections for glial fibrillary acid protein immunoreactivity in the soma and radial processes in optic nerve, or velous processes in the cerebellum. Astrocyte processes overlying the nodal axolemma also stained positively for the enzyme. (Na+ + K+)-ATPase-like immunoreactivity was not observed in association with oligodendroglia cell bodies or their processes forming myelin sheaths. In contrast, the plasmalemma of myelinating Schwann cells showed greatest immunoreactivity in the region of the node of Ranvier. Although a focal concentration of immunoreactivity was observed along node- and paranode-associated regions of Schwann cells, a lower level of uniform staining was noted along the entire Schwann cell surface membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Astroglial proliferation and phenotype are modulated by neuronal plasma membrane

The rate of proliferation of rat astroglia cultured in a serum-free medium, estimated by tritiated thymidine radioautography, was diminished by more than 50% by addition of rat central nervous system axolemmal fragments to the culture medium. Addition of the axolemmal fragments also induced a phenotypic alteration of the cultured astroglia, from cells of irregular shape containing a fine meshwork of intracytoplasmic glial fibrils to star-shaped cells with thicker, cable-like glial fibrils.     

Evidence for the colocalization of the axonal mitogen for Schwann cells and oligodendrocytes

Axons that normally will encounter either CNS or PNS glia have been shown to contain a powerful mitogen for both Schwann cells and oligodendrocytes. The normally nonmyelinated, nonglial ensheathed cerebellar granule cells have been shown to possess a proliferative signal for Schwann cells, suggesting that a glial mitogen is common to all axons. To determine if a glial mitogen capable of stimulating both Schwann cells and oligodendrocytes is colocalized on all types of axons we have (1) cocultured granule cells with oligodendrocytes, (2) incubated oligodendrocytes with granule cell membranes, and (3) evaluated the ability of heparin extracts of granule cell membranes, splenic nerve microsomes, and axolemma-enriched fractions isolated from rat and bovine CNS to stimulate mitosis of cultured oligodendrocytes. Neither the intact granule cells nor the granule cell membrane fraction stimulated cultured oligodendrocytes to divide. However, heparin extracts of the granule cell membranes were significantly mitogenic to the cultured oligodendrocytes. Heparin extracts of splenic nerve microsomes were more mitogenic than the comparable extract obtained from bovine CNS axolemma-enriched fractions. These results suggest that the neuronal mitogen for oligodendroglia is colocalized with the neuronal mitogen for Schwann cells.     

A quantitative morphometric analysis of rat spinal cord remyelination following transplantation of allogenic Schwann cells

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords.     

Isolated growth cones stimulate proliferation of cultured Schwann cells.

A growth cone-enriched fraction was prepared from 3-4 day rat cerebra. Examination of the growth cone fraction by electron microscopy revealed numerous structures circular in appearance that contain a number of features common to neuronal growth cones in vivo. The isolated growth cones stimulated a dose-dependent incorporation of [3H]-thymidine into cultured Schwann cells in a manner similar to that observed with an axolemma-enriched fraction prepared from adult rat brainstem. The mitogenic activities of both the growth cone fraction and axolemma-enriched fraction were decreased 50% and 20%, respectively, by treatment with heparitinase I. The mitogen for Schwann cells present in the isolated growth cones appears to be similar to that found in axolemma-enriched fractions prepared from adult rats.     

Gangliosides modulate Schwann cell proliferation and morphology

We examined the effect of gangliosides on Schwann cell cultures isolated from neonatal rat sciatic nerves. Addition of gangliosides (GM1, GM3, and ganglioside mixture) at concentrations between 0.25 and 2 mg/ml significantly diminished both the baseline rate of proliferation of the Schwann cells and their response to two types of mitogens, the axolemmal fragments and derivatives of adenosine 3'-5'-monophosphate (cAMP). Gangliosides, the sialic acid residue of which had been removed, were highly toxic to the Schwann cells, which went to indicate that sialic acid is necessary to produce the inhibitory effects. Gangliosides also produced prominent changes in the morphological appearance of the Schwann cells. Most of the Schwann cells treated with gangliosides had an elongated shape with long processes and an alignment of end-to-end or side-by-side cell adhesion. These effects of gangliosides apparently were not mediated by cAMP, since intracellular cyclic adenosine monophosphate (cAMP) of Schwann cells at a basal- and forskolin-stimulated level was not altered by the exogenous gangliosides. These findings indicate that the direct effect of gangliosides on Schwann cells should also be considered as a background mechanism of ganglioside-induced facilitation of neuronal regeneration.     

Schwann cell proliferation in the postnatal mouse: Timing and topography

We examined the intensity and timing of Schwann cell proliferation in the developing mouse using autoradiographic techniques. Schwann cell labeling indices were highest in the first few postnatal days and decreased gradually thereafter. Highest labeling indices and onset of myelination occurred later in the $\text{C57B16}\text{C3H}$ than the $\text{Balb}\text{c}$ mouse, suggesting strain differences in Schwann cell behavior. The timing of Schwann cell proliferation was similar from L5 roots to the posterior tibial nerves, with no evidence of a proximaldistal gradient during this period. Cessation of proliferation was not abrupt, but occurred throughout the first 10 postnatal days. These observations can be explained by the presence of a fixed-size proliferating Schwann cell pool, in which half of all daughter cells cease division with each cell cycle and differentiate into their adult roles.     

Postnatal differentiation of rat optic nerve fibers: Electron microscopic observations on the development of nodes of Ranvier and axoglial relations

The postnatal differentiation of rat optic nerve fibres was examined by transmission electron microscopy. The results show that many early developing axons contain clusters of vesiculotubular profiles prior to Myelination. At places vesicular elements appear to fuse with the axolemma, and, in addition, some axons exhibit deep axolemmal invaginations and axoplasmic lamellated bodies. It is suggested that these feature might reflect axolemmal remodeling, possibly involving axoglial signalling and/or functional differentiation of the axolemma. The size distribution of unmyelinated optic nerve axons changes little during development. Ensheathment of larger axons commences 6 days postnatally. The subsequent formation of compact sheaths are a few microns long and separated by long bare axon segments. In optic nerves from 10–12-day-old rat pups, a few sheaths consisting of about five layers border primitive asymmetric nodes with a patchy axolemmal undercoating. Extensions from one of the terminating sheaths are often associated with undercoated patches of axolemma. Relatively differentiated nodes of Ranvier first appear 14–16 days after birth. The continued nodal maturation involves establishment of a regular nodal geometry, increasing distinctness of the axolemmal undercoating, and formation of perinodal astrocytic processes embedded in an extracellular node gap substance. The results are compared with available data on the conduction properties of rat optic nerve fibres during development.     

Cultured rat Schwann cells express low affinity receptors for nerve growth factor

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.     

Experimental lead neuropathy: inorganic lead inhibits proliferation but not differentiation of Schwann cells

Schwann cells were prepared from the sciatic nerves of newborn rats and cultured in a monolayer. Addition of lead acetate at concentrations between 0.4 and 10.0 micrograms/ml, levels comparable to those occurring in neural tissues and physiological fluids of lead-intoxicated rats, diminished both the baseline rate of proliferation of the Schwann cells and their response to the mitogens, axolemmal fragments, glial growth factor, and the adenosine 3':5'-cyclic monophosphate (cAMP) analogues 8-bromo-cAMP and dibutyryl-cAMP. This demonstrates a direct toxic effect of inorganic lead on Schwann cells. Lead acetate in this concentration range did not, however, inhibit the cAMP analogue-induced appearance of the "myelin marker" lipid galactocerebroside on the surfaces of the cultured Schwann cells.