The Extracellular Matrix Molecule Tenascin: Expression in the Developing Chick Retinotectal System and Substrate Properties for Retinal Ganglion Cell Neurites In vitro

To investigate the molecular mechanisms involved in the outgrowth of retinal ganglion cell axons in the tectum, the expression of the extracellular matrix molecule tenascin was analysed in the tectum and retina of chickens by immunocytochemistry and in situ hybridization. Tissue was analysed between embryonic days 4 and 12, just before and during the period when retinal ganglion cell axons innervate their target region, the optic tectum. In the tectum, tenascin immunoreactivity becomes detectable at the anterior pole at embryonic day 4, 2 days before retinal ganglion cell axons arrive, and spreads caudally with increasing age. At early stages, tenascin is predominantly accumulated in the stratum opticum, the zone of ingrowing retinal ganglion cell axons, and along their prospective pathway. In the stratum opticum, the molecule is associated with radial glial fibres, glial endfeet and retinal ganglion cell axons located in the immediate neighbourhood of radial glial fibres. At all ages investigated, tenascin mRNA is mainly restricted to cells located in the periventricular region, suggesting that the molecule is synthesized by radial glial cells. In the retina, tenascin is expressed by amacrine, displaced amacrine and horizontal cells but not by retinal ganglion cells. To investigate whether the accumulation of tenascin in the developing and prospective pathway of retinal ganglion cell axons may affect their rate of growth we assayed the substrate properties of tenascin for retinal ganglion cell neurites in vitro. When retinal ganglion cell suspensions from 6–day-old chick embryos were maintained on homogeneous mouse or chick tenascin/ polyornithine substrates, neurite length was significantly increased when compared to polyornithine substrates at coating concentrations of 10 or 20 μg/ml. Higher coating concentrations (35 or 70 μg/ml) resulted in neurite lengths comparable to control values. Together, these observations suggest that tenascin in the developing and prospective stratum opticum might serve as a preformed pathway to support growth of retinal ganglion cell axons in the tectum.     

Up-regulation of a Chondroitin Sulphate Epitope during Regeneration of Mouse Sciatic Nerve: Evidence that the Immunoreactive Molecules are Related to the Chondroitin Sulphate Proteoglycans Decorin and Versican

After transection of adult mouse sciatic nerve, the expression of a chondroitin sulphate epitope recognized by the monoclonal antibody 473-HD (mAb 473-HD) was found to be up-regulated. The epitope was localized immunocytochemically mainly in Schwann cell basal laminae and, more weakly, also in the endoneurium. In cultures of mouse dorsal root ganglion cells, Schwann cells expressed high levels but fibroblasts only low levels of the epitope. To identify the molecule(s) carrying this chondroitin sulphate epitope, human sciatic nerves were extracted with phosphate-buffered saline and shown to contain two chondroitin sulphate proteoglycans of apparent molecular weights of 130 and 900 kDa. The 900 kDa and, more weakly, the 130 kDa proteoglycan were reactive with mAb 473-HD, which was found to recognize chondroitin-6-sulphate as epitope. Following chondroitinase ABC treatment of the 130 kDa proteoglycan, a core protein of ∼45 kDa was seen and shown to react with polyclonal antibodies against the chondroitin-dermatan sulphate proteoglycan decorin from human fibroblasts. Chondroitinase ABC treatment of the 900 kDa proteoglycan yielded a core protein with a molecular weight of ∼400 kDa that was recognized by polyclonal antibodies against recombinantly expressed fusion proteins from human versican. After transection of adult mouse sciatic nerves, the distal nerve stumps showed up-regulation of the chondroitin-6-sulphate epitope of the 900 kDa proteoglycan, whereas the core protein of this proteoglycan did not show any detectable change in the level of expression. In contrast, the core protein of the 130 kDa proteoglycan was up-regulated in expression. These observations suggest that versican- and decorinlike molecules may contribute to successful regeneration in the peripheral nervous system of mammals.     

Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in binding to some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipids of the PNS.     

Functional Topography of the Myelin-associated Glycoprotein. I. Mapping of Domains by Electron Microscopy

The functional topography of the myelin-associated glycoprotein (MAG) was investigated by electron microscopic analysis of rotary-shadowed molecules of a MAG fragment (MAG 90) comprising the five immunoglobulin-like domains of the extracellular part of the molecule. MAG 90 molecules appeared as rod-like structures (18.5±1.2 nm long and 4.0±0.8 nm wide) with a globular domain at one end. Antibodies directed against the amino- and carboxy-terminus of MAG 90 interacted with the non-globular terminal region, indicating that the molecule is bent in the globular region with the amino- and carboxy-terminal arms in close apposition to each other. An antibody which interferes with the binding of MAG to neurons interacted predominantly with the globular domain of MAG 90. The fibril-forming collagen types I, III and V bound mainly to the non-globular terminal region of MAG 90, whereas the majority of heparin molecules interacted with the globular region of the molecule. The L2/HNK-1 carbohydrate structure was localized at the non-globular region in the protein fragment comprising the fourth and fifth immunoglobulin-like domains.     

Multiply myelinated axons in the optic nerve of mice deficient for the myelin-associated glycoprotein

We recently reported that some retinal ganglion cell axons in mice deficient for the myelin-associated glycoprotein are concentrically surrounded by more than one myelin sheath. In the present study, we demonstrate that myelin sheaths displaced from the axon reveal a normal ultrastructure of compact myelin, with the only exception that multiple myelination of axons frequently correlates with the presence of unfused regions of major dense lines. Supernumerary sheaths terminated on other sheaths or on astrocyte cell surfaces in a pattern closely resembling the morphology of a true paranode. The thickness of compact myelin of multiply myelinated axons was significantly increased when compared with axons of similar caliber surrounded by a single myelin sheath. Our observations demonstrate that maintenance of compact myelin and paranodal regions is not dependent on direct axonal contact and that the presence of more than one concentric myelin sheath around an axon results in dysregulation of the axon-to-fiber ratio. © 1995 Wiley-Liss, Inc.     

L1 Antibodies Block Lymph Node Fibroblastic Reticular Matrix Remodeling In Vivo

L1 is an immunoglobulin superfamily adhesion molecule highly expressed on neurons and involved in cell motility, neurite outgrowth, axon fasciculation, myelination, and synaptic plasticity. L1 is also expressed by nonneural cells, but its function outside of the nervous system has not been studied extensively. We find that administration of an L1 monoclonal antibody in vivo disrupts the normal remodeling of lymph node reticular matrix during an immune response. Ultrastructural examination reveals that reticular fibroblasts in mice treated with L1 monoclonal antibodies fail to spread and envelop collagen fibers with their cellular processes. The induced defect in the remodeling of the fibroblastic reticular system results in the loss of normal nodal architecture, collapsed cortical sinusoids, and macrophage accumulation in malformed sinuses. Surprisingly, such profound architectural abnormalities have no detectable effects on the primary immune response to protein antigens.     

Tenascin-C is involved in motor axon outgrowth in the trunk of developing zebrafish.

Motor axons in the trunk of the developing zebrafish exit from the ventral spinal cord in one ventral root per hemisegment and grow on a common path toward the region of the horizontal myoseptum, where they select their specific pathways. Tenascin-C, a component of the extracellular matrix, is concentrated in this choice region. Adaxial cells and other myotomal cells express tenascin-C mRNA, suggesting that these cells are the source of tenascin-C protein. Overexpressing an axon repellent fragment containing the cysteine-rich region and the epidermal growth factor-like repeats of tenascin-C led to retarded growth of ventral motor nerves between their spinal exit point and the horizontal myoseptum. Injection of a protein fragment containing the same part of tenascin-C also induced slower growth of motor nerves. Conversely, knock down of tenascin-C protein resulted in abnormal lateral branching of ventral motor nerves. In the zebrafish unplugged mutant, in which axons display pathfinding defects in the region of the horizontal myoseptum, tenascin-C immunoreactivity was not detectable in this region, indicating an abnormal extracellular matrix in unplugged. We conclude that tenascin-C is part of a specialized extracellular matrix in the region of the horizontal myoseptum that influences the growth of motor axons.     

Maintenance of immunocytologically identified Purkinje cells from mouse cerebellum in monolayer culture

Purkinje cells were identified in monolayer cultures obtained from trypsin-dissociated cerebella of embryonic and early postnatal mice by the Purkinje cell-specific monoclonal antibodies PC1, PC2, PC3 and UCHT1. These cells also expressed the neuronal marker L1 antigen but not the glial markers, glial fibrillary acidic protein or 04 antigen. They also expressed tetanus toxin receptors, PC4, M1 and Thy-1 antigens. Survival of Purkinje cells was best: (a) when cerebella were taken from mice not older than one day of age: (b) when cells were seeded at higher plating densities; and (c) cultured in chemically defined medium which facilitates the survival of neurons. No Purkinje cells could be detected in cultures from mice older than 6 days. PC1 antigen expression developed in vitro on the same time scale as in vivo, i.e. it was first detectable at the equivalent of postnatal days 3–4. At this stage cell bodies had a size of 13–14 μm in diameter and few processes. Dendrite-like arborizations, with more than one primary dendrite, extension of usually only one thin and long (0.5–1.6 mm) axon-like process and collaterals directed preferentially towards other Purkinje cells, developed with time in culture until the final form was reached by the equivalent of approximately day 16. Cell body size was 18–19 μm in diameter at this stage. Cell shapes were reminiscent of those described in certain cerebellar mouse mutants and in experimentally produced agranular cerebella. Many ultrastructural features of these cells correlated with those described for the in vivo counterpart. However, there was a lack of spiny branchlets and abnormally long persisting somatic spines. Synaptic contacts of the ‘en passant’ type could be seen at the Purkinje cell soma. Gray type I synapses were seen on Purkinje cell dendrites and spines.     

Time of transplantation and cell preparation determine neural stem cell survival in a mouse model of Huntington’s disease

Cell replacement therapies for neurodegenerative diseases, using multipotent neural stem cells (NSCs), require above all, a good survival of the graft. In this study, we unilaterally injected quinolinic acid (QA) into the striatum of adult mice and transplanted syngeneic NSCs of enhanced green fluorescent protein-transgenic mice into the lesioned striatum. The injection of QA leads to an excitotoxic lesion with selective cell death of the medium sized spiny neurons, the same cells that are affected in Huntington’s disease. In order to investigate the best timing of transplantation for the survival of donor cells, we transplanted the stem cells at 2, 7 and 14 days after injury. In addition, the influence of graft preparation prior to transplantation, i.e., intact neurospheres versus dissociated cell suspension on graft survival was investigated. By far the best survival was found with the combination of early transplantation (i.e., 2 days after QA-lesion) with the use of neurospheres instead of dissociated cell suspension. This might be due to the different states of host’s astrocytic and microglia activation which we found to be moderate at 2, but pronounced at 7 and 14 days after QA-lesion. We also investigated brain derived neurotrophic factor (BDNF)-expression in the striatum after QA-lesion and found no significant change in BDNF protein-level. We conclude that already the method of graft preparation of NSCs for transplantation, as well as the timing of the transplantation procedure strongly affects the survival of the donor cells when grafted into the QA-lesioned striatum of adult mice.