Effect of Colchicine on Ganglioside Composition of Rat Primary Culture Neurons

Abstract

Developmental changes in gangliosides in the course of neurite outgrowth were examined in dissociated fetal rat cerebral neurons in culture. About a 2-fold increase in ganglioside levels was seen with the progression of neurite formation for up to 24 h in predominantly neuronal cultures. Ganglioside patterns appeared to be unchanged during the first 24 h, subsequently consisted of higher amounts of GD3 and b-series gangliosides (such as GD1b, GT1b, and GQ1b), and lower amounts of a-series gangliosides (GM1 and GD1a). Although the addition of colchicine to the cell growth medium inhibited neurite outgrowth in developing neurons, little if any differences in ganglioside patterns were found between control and colchicine-treated cells. Ganglioside levels decreased slightly in colchicine-treated cells in agreement with the decrease in cell attachment to culture dishes. Although colchicine treatment 8 h after plating caused complete retraction of formed neurites, the ganglioside level of the cells continued to increase during the following 16-hour incubation. Thus, the data suggest that ganglioside synthesis in differentiating neurons does not primarily accompany the expansion in cell surfaces due to neurite formation, and raises the possibility that a large proportion of gangliosides is retained in intracellular compartments.

Lack of Cell Density-Dependent Changes in Gangliosides of Rat Primary Culture Neurons

“…The immunocytochemical approach using antiganglioside antibodies revealed the intracellu lar distribution of most gangliosides 24 h after seeding and the subsequent expression of gangliosides on neu ronal membranes with a lag [11]. Inhibition of neurite outgrowth by colchicine during the first 24 h following plating caused neither the compositional change nor syn thetic inhibition of gangliosides [12], Recently, Lelonget al [30] have also reported that some of gangliosides are not exposed to the extracellular environment during divi sion and maturation phases in cultured chick neurons. In the ontogénie development a major ganglioside of fetal neurons, GD3, is reported to be strongly reactive to anti-GD3 antibody in proliferative neuronal cell precursors and to become unreactive in the subsequent stages [25][26][27].…”

“…Primary cultures of dissoci ated neurons were prepared from the embryos' cerebral hemi spheres as described elsewhere [11,12], Four groups containing 10s cells from the same batch were seeded on different numbers of 60-mm diameter collagen-coated culture dishes (Corning, 25010-COL1), which were coated with poly-D-lysine (10 pg/ml), to change cell density. The cells were grown in basal medium Eagle (BME) supplemented with 3% newborn calf serum (Gibco) and 0.6% glucose in a humidified atmosphere of 95% air/5% COi.…”

“…Total lipids were extracted from the collected cells with 20 vol umes of chloroform/methanol (2:1 and 1:2, V/V) as described previously [11,12]. Briefly, the ganglioside fraction was separated from the total lipid fraction by DEAE-Sephadex column chro matography (1x5 cm, A-25, Pharmacia-LKB) according to Ledeen ct al.…”

“…The specificity of the antibody to rat neuronal gan gliosides was examined by the combination of ganglioside mapping and enzyme-linked immunosorbent assay (ELISA). Ganglioside mapping was carried out on an HPLC apparatus equipped with a DEAE-Iatrobeads column (DEA6RSP-8010, 0.46 x 25 cm, Iatron, Tokyo) [12], and aliquots of fractions were analyzed by TLC and ELISA.…”

“…In contrast, the adrenergic cells showed virtually no density-dependent change in the ganglioside composition. Liepkalns et al [10] observed increases in ganglioside content on a pro tein content basis in human glioma (12)(13)(14)(15)(16)(17)(18)) and fetal neural cell line (CH) with increasing cell density. In creases in GM1 and di-and trisialogangliosides were ob served in glioma cells from sparse to confluent densities, but there were no apparent trends in the levels of GM 1 or disialogangliosides in fetal neural cells.…”