NS-1 (Nervous System Antigen-1), a Glial-Cell-Specific Antigenic Component of the Surface Membrane

A methylcholanthrene-induced glioblastoma of the C57BL/6 inbred mouse strain was used to raise antibodies in C57BL/6 and C57BL/10 inbred mice and in (C57BL/6 x DBA/2) and (C57BL/6 x Balb/c) F(1) hybrids. When examined by the cytotoxicity test, these antibodies define a cell-surface component (or components) found exclusively on brain tissue of all mouse strains studied and of several other mammalian species including man. The antigen, named NS-1 (nervous system antigen-1), is present on cells of three of the four mouse-glial-cell tumors tested, but not on the C1300 neuroblastoma, a tumor of neuronal origin. NS-1 occurs in higher concentration in regions of the nervous system richer in white than in gray matter, and in lower than normal concentrations in brains of myelindeficient neurological mutant mice. The concentration of NS-1 gradually increases postnatally and reaches the adult level between the third and fourth week. The existence of more than one allele or genetic locus controlling NS-1 activity is suggested by the occurrence of higher amounts of NS-1 in brains of the A and C57BL/6 than of the Balb/c and DBA/2 mouse strains. NS-1 is the first cellsurface component to be described that is not only unique to nervous tissue, but specific for glial cells.     

Functional Involvement of Sciatic Nerve-derived Versicanand Decorin-like Molecules and other Chondroitin Sulphate Proteoglycans in ECM-mediated Cell Adhesion and Neurite Outgrowth

We have previously described two proteoglycans from human sciatic nerve which are immunochemically related to the chondroitin sulphate proteoglycans versican and decorin. The chondroitin sulphate of the versican-like molecule and the core protein of the decorin-like molecule have been found previously to be up-regulated after lesioning the adult mouse sciatic nerve. To investigate if the versican- and decorin-like molecules are involved in cell-extracellular matrix interactions, we studied the effect of both molecules on cell adhesion. The versican- and decorin-like molecules, substrate-coated in a mixture with fibronectin, but not with laminin or collagen types I or IV, inhibited the adhesion of several cell lines, neonatal dorsal root ganglion neurons and Schwann cells. The inhibitory activity was concentration-dependent and mediated by the chondroitin sulphate. Furthermore, when different proteoglycans were incubated with fibronectin, only the versican- and decorin-like molecules and the chondroitin sulphate proteoglycan aggrecan, but not the heparan sulphate proteoglycan perlecan, were able to inhibit fibronectin-mediated cell adhesion. The versican- and decorin-like molecules, substrate-coated alone or in a mixture with fibronectin or laminin, were at most slightly inhibitory to neurite outgrowth from PC12 phaeochromocytoma cells and neonatal dorsal root ganglion neurons. In a solid-phase ligand-binding assay theversican- and decorin-like molecules interacted with fibronectin, but not with laminin or collagen types I and IV. Binding of the versican-like molecule to fibronectin and inhibition of cell adhesion by this molecule was mediated via the heparin and cell-binding domains of fibronectin. These observations suggest that binding of the two proteoglycans to fibronectin is involved in the modulation of adhesion of cells to fibronectin.     

The Neurotrophin Receptors TrkA and TrkB are Inhibitory for Neurite Outgrowth

To investigate the possibility that the neurotrophin tyrosine kinase receptors are also recognition molecules by virtue of their immunoglobulin-like domains, the ability of TrkA and TrkB to influence neurite outgrowth was tested in vitro. Cell monolayers of fibroblasts transfected to express either the TrkA or TrkB receptor reduced neurite outgrowth of phaeochromocytoma PC12 cells by 50–60% when compared to mock transfected fibroblasts or fibroblasts transfected with the epidermal growth factor receptor. Neurite outgrowth from cerebellar neurons was inhibited by 30–40% on these substrates. When a recombinantly expressed fragment of TrkA comprising the two immunoglobulin-like domains was coated as a substrate in combination with poly-L-lysine and laminin, neurite outgrowth was inhibited in a dose-dependent manner. This inhibition of neurite outgrowth was not mediated via an interaction with laminin as there is no specific binding of the TrkA fragment to laminin. The adhesion of cell bodies to this substrate was not affected by the immunoglobulin-like domains. These observations suggest that the mammalian neurotrophin receptors not only influence neurite outgrowth by neurotrophin triggered activation of the receptor, but also by cell surface recognition processes conveyed by the immunoglobulin-like domains.     

Behaviour of Small Inhibitory Interneurons in Early Postnatal Mouse Cerebellar Microexplant Cultures: A Video Time-lapse Analysis

The aim of this work was to investigate how the environment of the neuropil determines the positioning and differentiation of neurons that are postsynaptic to them. We investigated how stellate and basket cells, the small inhibitory interneurons of the cerebellar cortex, find their perpendicular orientation to the direction of fasciculated granule cell axons. Cultures of early postnatal mouse cerebellar microexplants showing this cellular behaviour in vitro were analysed by video time-lapse cinematography and evaluated by morphometry. The small interneurons were first detectable when they migrated, intermingled with granule cells, away from the explant along the radial fascicles of granule cell neurites. During migration some cells suddenly changed their orientation by extending neurites in perpendicular orientation to the radial fascicles. These cells were all GABA-immunoreactive and expressed the cytoskeletal markers tau in the thin axon-like process and MAP2 in the thicker dendrite-like arborizations at the opposite pole of the cell body. After having translocated in perpendicular orientation, these neurons were again able to turn back to move along the radial neurite bundles to another position. Furthermore, while in perpendicular orientation, the processes of these cells repelled each other upon contact of their growth cones, leading to equal spacing between the cell bodies with time in culture.     

Growth and degeneration of axons on astrocyte surfaces: Effects on extracellular matrix and on later axonal growth

Cultured astrocytes deposit an extracellular matrix which has been shown by immunocytochemistry to react with antibodies to tenascin, laminin, and fibronectin. Neuronal-glial interaction down-regulates these components of the matrix, causing a reduction in extracellular matrix localized to areas of contact with axons. Axons used for these experiments were from embryonic rat retinal explants. In some experiments explants were removed from the co-cultures and their axons allowed to degenerate. Degeneration of axons did not reverse the local reduction of extracellular matrix brought about by axon outgrowth. The period of axon outgrowth studied was 4-5 days; the period of degeneration was 2-3 days. Astrocytes alone, astrocytes with intact retinal explants, and astrocytes with 2-day degenerated retinal axons were tested for their ability to support neurite outgrowth from embryonic rat cortical neurons. Neurite outgrowth occurred on all astrocyte cultures. Cortical neurite lengths, measured 2 days after plating, were not significantly different between astrocytes alone and astrocytes with degenerated retinal axons. However, there was a tendency for neurites to be shorter on astrocytes with intact retinal axons present. Two conclusions may be drawn from these results. First, the state of differentiation of astrocytes, as marked by their assembly of extracellular matrix, is altered by contact with axons. Second, degeneration of axons alone, in the absence of other cell types, is not a sufficient signal to reestablish assembly of extracellular matrix. However, neither is it a sufficient signal to render astrocytes inhospitable to further axonal outgrowth or regeneration. © 1993 Wiley-Liss, Inc.     

Characterization of isolated mouse cerebellar cell populations in vitro

The expression of several cell surface components (Thy-1, H-2 and NS-4 antigens and tetanus toxin receptors) was studied by indirect immunofluorescence in situ using histological sections and in vitro using freshly dissociated and cultured cells from mouse cerebellum. Thy-1 alloantigen is expressed in adult cerebellum predominantly in neuron-rich regions, i.e. molecular, Purkinje cell, and granular layers, however, it is not detectable at postnatal day 8. In cerebellar cultures of 6-day-old mice Thy-1 is absent from more than 99% of all cells when these are maintained as monolayers in vitro for up to 3 days. After 4 days in vitro some GFA protein-positive astrocytes and some fibronectin-positive fibroblast-like cells start to express Thy-1 antigen. After 14 days in vitro not all fibroblast-like cells and astrocytes are Thy-1 antigen-positive. Neurons with small cell bodies and oligodendrocytes never express Thy-1 at any stage examined. H-2 is not expressed sufficiently to be detectable in histological sections in early postnatal or adult cerebellum. In cerebellar cultures of 6-day-old mice H-2 becomes detectable on some fibroblast-like cells and some astrocytes after 7 days in culture. In histological sections of adult and early postnatal cerebellum NS-4 antigen and tetanus toxin receptors are expressed predominantly in neuron-rich regions. In the developing cerebellum both are expressed at higher levels on more mature granule cells. In cerebellar cultures NS-4 antigen and tetanus toxin receptors are expressed on neurons. Occasionally some astroglia can also show detectable levels of expression. NS-4 antigen is also present on some 04 antigen-positive oligodendrocytes, while tetanus toxin receptors are never detectable on these cells.     

Glycomimetic improves recovery after femoral injury in a non-human primate

In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.     

Transplanted L1 expressing radial glia and astrocytes enhance recovery after spinal cord injury

A major obstacle for the transplantation of neural stem cells (NSCs) into the lesioned spinal cord is their predominant astrocytic differentiation after transplantation. We took advantage of this predominant astrocytic differentiation of NSCs and expressed the paradigmatic beneficial neural cell adhesion molecule L1 in radial glial cells and reactive and nonreactive astrocytes as novel cellular vehicles to express L1 under the control of the promoter for the human glial fibrillary acidic protein (GFAP-L1 NSCs). Behavioral analysis and electrophysiological H-reflex recordings revealed that mice transplanted with GFAP-L1 NSCs showed enhanced locomotor recovery in comparison to mice injected with wild type (WT) NSCs or control mice injected with phosphate-buffered saline (PBS). This functional recovery was further accelerated in mice transplanted with L1-expressing radial glial cells that had been immunoisolated from GFAP-L1 NSCs (GFAP-L1-i cells). Morphological analysis revealed that mice grafted with GFAP-L1 NSCs exhibited increased neuronal differentiation and migration of transplanted cells, as well as increased soma size and cholinergic synaptic coverage of host motoneurons and increased numbers of endogenous catecholaminergic nerve fibers caudal to the lesion site. These findings show that L1-expressing astrocytes and radial glial cells isolated from GFAP-L1 NSC cultures represent a novel strategy for improving functional recovery after spinal cord injury, encouraging the use of the human GFAP promoter to target beneficial transgene expression in transplanted stem cells.