Effects of prior sterol depletion on neurite outgrowth in neuroblastoma cells

The effect of decreasing cellular sterol content on neurite outgrowth in C1300 (Neuro 2A) neuroblastoma cells in serum-free medium has been studied. Sterol-depleted, undifferentiated neuroblastoma cells were obtained by growing cells for 24 h in medium containing lipoprotein-poor serum and 25-hydroxycholesterol (25-OHC). Under these conditions the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase and the incorporation of [14C] acetate into sterols were almost completely suppressed, and the sterol/phospholipid ratio of the cells declined to 60% of that in cultures grown without 25-OHC. The sterol-depleted cells were viable and exhibited rats of DNA, RNA, protein and fatty acid synthesis comparable to those measured in control cultures. Sterol depletion had no detectable effect on the number of cells that were able to undergo morphological differentiation within 3 h after removal of serum from the medium. However, by 24 h most of the sterol-depleted cells had retracted their neurites. The observation that addition of low-density lipoprotein was able to restore neurite outgrowth in cultures treated with 25-OHC indicates that the inability of sterol-depleted cells to maintain their neurites is related specifically to the decline in the sterol content rather than to a general cytotoxic effect of 25-OHC. Our findings suggest that incorporation of cholesterol into the cell membrane is important for long-term maintenance and elongation of neuroblastoma neurites, but that the initial morphological change (e.e., within 3 h after removal of serum) is apparently a separate and distinct event, not dependent on the availability of cholesterol.