Differential Glycosylation of Tractin and LeechCAM, Two Novel Ig Superfamily Members, Regulates Neurite Extension and Fascicle Formation

By immunoaffinity purification with the mAb Lan3-2, we have identified two novel Ig superfamily members, Tractin and LeechCAM. LeechCAM is an NCAM/FasII/ApCAM homologue, whereas Tractin is a cleaved protein with several unique features that include a PG/YG repeat domain that may be part of or interact with the extracellular matrix. Tractin and LeechCAM are widely expressed neural proteins that are differentially glycosylated in sets and subsets of peripheral sensory neurons that form specific fascicles in the central nervous system. In vivo antibody perturbation of the Lan3-2 glycoepitope demonstrates that it can selectively regulate extension of neurites and filopodia. Thus, these experiments provide evidence that differential glycosylation can confer functional diversity and specificity to widely expressed neural proteins

Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated

Local embryonic matrices determine region-specific phenotypes in neural crest cells

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions

Repair strategies for traumatic spinal cord injury, with special emphasis on novel biomaterial-based approaches

International audienc

Repair strategies for traumatic spinal cord injury, with special emphasis on novel biomaterial-based approachesSylvia Soares*, Ysander von Boxberg*, Fatiha Nothias

International audienc

Differential reaction of crossing and non-crossing rat retinal axons on cell membrane preparations from the chiasm midline: an in vitro study

In the rat, a small subpopulation of retinal ganglion cell axons forms a persistent projection to the ipsilateral half of the brain. These fibres originate almost exclusively from the ventrotemporal margin of the retina. In contrast to all other retinal axons they seem to be deflected from the midline of the optic chiasm and thereby led into the ipsilateral optic tract. In order to analyse the interactions between growing fibres and chiasm midline, we have developed the following in vitro model. Axons of the embryonic rat retina are grown on a carpet of tectal cell membranes used as a general growth-permissive substratum. At a certain distance from the explant (200-450 microns), the advancing fibres are confronted with two stripes of cell membranes prepared from the chiasm midline. Such chiasm membranes are shown to act as a barrier for the presumptive non-crossing axons, while they do not influence growth of fibres originating from any other regions of the retina, including the dorsotemporal part. The repulsion of non-crossing fibres by chiasm membranes is observed in vitro only when retinal explants from embryonic day (E) 17/18 and chiasm preparations from E14/15 are used. Fibres and tissue from different regions of the brain as well as from different developmental ages, and even from different species, can be combined in this assay system. In a first attempt to characterize the molecular basis of the repulsive effect of chiasm membranes on ventrotemporal fibres, similar assays were performed with membranes derived from other regions of the central nervous system midline, some of which are known to have repulsive properties against certain axon populations. Since these cell membranes did not act as a barrier for the ventrotemporal retinal axons, we suggest that the guidance cues at the chiasm are very specific. Our results are consistent with the hypothesis that certain cells at the chiasm midline (very likely radial glial cells) express 'repulsive or inhibitory' molecules, which act in a specific way on ipsilaterally projecting axons

Impact of different chitosan hydrogel formulations on macrophage polarization in vitro

International audienceInflammation is a normal event following a lesion, and is usually resolved after a certain time together with the reconstitution of the damaged tissue. In case of a central nervous system lesion, however, in particular a traumatic spinal cord injury (SCI), inflammation is somehow "uncontrolled", and will generally spread from the site of the initial impact towards originally unaffected tissue, creating important secondary damage. Today, development of novel therapeutic strategies for SCI should thus include a better control of the inflammatory reaction. We have recently shown that a fragmented physical hydrogel suspension (FPHS) of chitosan (4% DA, 2.5% Cp), implanted into a rat spinal cord hemisection, is a very efficient bio-scaffold material, allowing already by itself for massive axon regrowth through the lesion site, neovascularization, neural tissue reconstitution and reduction of cystic cavitation, long-term survival of (re-)myelinating Schwann cells, sand last not least, enhanced functional recovery [1]. This appears to be linked, at least in part, to a reduction -in comparison to a non-treated lesion- in classically activated, inflammatory ("M1") macrophages, and a prolonged presence of anti-inflammatory ("M2") macrophages that normally disappear from the lesion site after only about one week.To address the question whether the observed effect on macrophage polarization was due to the chitosan itself, or rather secondary to, for example, the invasion of diverse cell types attaching to the biomaterial serving as matrix replacement, the reduced astrocytic reaction, or better oxygen supply thanks to efficient revascularization, we introduced an in vitro assay in which different formulations of chitosan are used as substrate for culturing pre-polarized macrophages of the M1 and M2 subtypes. We show that indeed, chitosan directly influences the level of expression of typical M1 and M2 macrophage "marker molecules", dependent on the degree of acetylation (DA), and to a lesser extent also the concentration of chitosan within the hydrogel. The effect is only observed if macrophages are in direct contact with the hydrogel particles. This assay may thus be used to fine-tune the formulation of chitosan hydrogel intended for SCI treatment, and also serve as a rapid and easy means to test a newly generated biomaterial for its impact on macrophage polarization