Mitogenicity of Brain Axolemma Membranes and Soluble Factors for Dorsal Root Ganglion Schwann Cells

Abstract

Previous studies in this laboratory have shown that membranes derived from dorsal root ganglia (DRG) neurites are mitogenic for cultured Schwann cells derived from the same source [Salzer et al (1980): J Cell Biol 84:767–778]. Improved procedures are described for preparing Schwann cells derived from dorsal root ganglia that are highly responsive to various mitogens. Under these conditions, the cells respond not only to the neurite mitogen but also to pituitary extracts, dibutyryl cyclic AMP, and cholera toxin that have been shown previously to be good mitogens for Schwannn cells derived from sciatic nerve [Raff et al (1978): Cell 15:813–822], thus reconciling discrepancies in the response of these different Schwann cell preparations to mitogens. Searching for a source of membranes more suitable for biochemical characterization of the neurite mitogen, we found that bovine brain axolemma, prepared by the method of DeVries et al [(1977): Brain Res 147:339–352] is highly mitogenic for Schwann cells. The milotic index of Schwann cells was increased by the addition of axolemma from 0.5%–2% to 30%–50% during 24‐h incubation with [³H]thymidine. Half maximal effect was obtained at about 0.4 μg axolemma protein per microwell containing 2–4 × 10 ³ cells. The axolemma mitogen appears to be an integral membrane protein that remains bound to the membrane under various ionic conditions but can be extracted in a partially active form with deoxycholate. Like the DRG neurite mitogen, the mitogenic activity of axolemma was abolished by trypsin treatment. Unlike the neurite preparation, however, the mitogenic activity of axolemma was only partially inactivated by heat treatment (60%–70% inactivation). A significant difference between the mitogenic activity of axolemma membranes and neurite membranes is the fact that axolemma membranes fail to stimulate Schwann cell proliferation in a defined, serum‐free medium (N‐2), whereas neurites show significant mitogenic activity in this medium. These findings indicate a possible difference between DRG neurites and brain axolemma either in the mitogen itself or surface components responsible for recognition between the membranes and the cells.