Adrian Spillmann

Identification and characterization of a bovine neurite growth inhibitor (bNI-220).

The poor axonal regeneration that follows lesions of the central nervous system (CNS) is crucially influenced by the local CNS tissue environment through which neurites have to grow. In addition to an inhibitory role of the glial scar, inhibitory substrate effects of CNS myelin and oligodendrocytes have been demonstrated. Several proteins including NI-35/250, myelin-associated glycoprotein, tenascin-R, and NG-2 have been described to have neurite outgrowth inhibitory or repulsive properties in vitro. Antibodies raised against NI-35/250 (monoclonal antibody IN-1) were shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long distance fiber regeneration in the lesioned adult mammalian CNS in vivo. We report here the purification of a myelin protein to apparent homogeneity from bovine spinal cord which exerts a potent neurite outgrowth inhibitory effect on PC12 cells and chick dorsal root ganglion cells, induces collapse of growth cones of chick dorsal root ganglion cells, and also inhibits the spreading of 3T3 fibroblasts. These activities could be neutralized by the monoclonal antibody IN-1. The purification procedure includes detergent solubilization, anion exchange chromatography, gel filtration, and elution from high resolution SDS-polyacrylamide gel electrophoresis. The active protein has a molecular mass of 220 kDa and an isoelectric point between 5.9 and 6.2. Its inhibitory activity is sensitive to protease treatment and resists harsh treatments like 9m urea or short heating. Glycosylation is, if present at all, not detectable. Microsequencing resulted in six peptides and strongly suggests that this proteins is novel.

Purification of a neurite growth inhibitor (NI-250) from bovine CNS myelin

In the central nervous system (CNS) the ability of nerve fibres to regenerate upon injury is very limited. In contrast, peripheral nerve fibres have been observed to regrow over long distances. Transplantation experiments with CNS and PNS nerve explants have shown that the local microenvironment is crucial for regenerating nerve fibers: CNS neurons can extend their lesioned neurites into the peripheral nerve environment, but cease growth at the transition between PNS and CNS (Aguayo et al 1990; David and Aguayo 1981; Ramon y Cajal 1928; Tello 1911). To test if a possible lack of trophic factors in the adult CNS might be responsible for this observation, perinatal rat sensory, sympathetic, or retinal neurons were cocultured with explants of adult rat sciatic (PNS) or optic nerves (CNS) in the presence of nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) (Schwab & Thoenen 1985). The pattern of fiber growth corresponded exactly to that found in vivo: many axons grew into the sciatic nerve explants, but none grew into the optic nerves. These results suggested that there is a neurite growth inhibitory activity present in adult CNS tissue that cannot be overcome by the stimulatory effects of neurotrophic factors (Schwab & Thoenen, 1985).