Evaluation of chondroitin sulfate bioactivity in hippocampal neurones and the astrocyte cell line U373: influence of position of sulfate groups and charge density

Abstract

Chondroitin sulfates are linear polysaccharides of alternating glucuronic acid and N-acetylgalactosamine, sulfated in varying positions. They form the extracellular framework providing the information for the structural establishment of tissues in multicellular organisms. Growth cones of neurones modulate their outgrowth according to signals received from proteoglycans. The exact molecular structures behind these functions are not fully understood, but structural details of the carbohydrate backbone are crucial. In this report we have employed quantitative cytometry on hippocampal neurite outgrowth in the presence of chondroitin sulfate added in solution to determine the influence of the position and density of the sulfate groups of the N-acetyl-D-galactosamine-residues of chondroitin sulfates. It is of profound interest whether externally added chondroitin sulfates can compete with core protein bound chondroitin sulfate to modulate the effects of tissue-synthesized matrix. In series of microscopic images 3 parameters of neuritic outgrowth activity, neurite length, number of neurites and fasciculation (thickness of neurites) are analyzed at concentrations occurring in intact tissues. Fasciculation increased and number of neurites decreased with high di-sulfation. No significant differences on process length reduction were found between the isotypes. Specificity of effects found is emphasized, as no influence on cell proliferation with U373 human astrocyte cell line is detectable, while neurones clearly are inhibited. The IC30 and IC50 values of chondroitin sulfates isoforms are presented for neurones. The data indicate that the soluble fragments from chondroitin sulfate are actively modulating cell development. Besides dosage, sulfation density and position are relevant for effects of chondroitin sulfate in neuronal regenerative activity.