Biomedical subjects
Publications and source records attributed to A D Lander.
Neural cells make and break many contacts during their lifetime. The processes of neuroblast migration, axon elongation and guidance, synaptogenesis, myelination and synaptic rearrangement all require the selective formation and elimination of cell-cell and cell-substratum associations.
The study of neurite growth in tissue culture has been a productive way to identify substances that may control the behavior of axons in vivo. Molecules that promote the outgrowth of neurites include nerve growth factor, laminin, fibronectin, and a protease inhibitor derived from glia. Evidence that these molecules may influence axon growth and guidance in vivo is discussed. The effects these molecules have at the cellular level are compared, in an attempt to identify common mechanisms of action. Several less well-characterized molecules that influence the behavior of neurites are also discussed.
Conditioned media (CMs) from many cell types contain a factor that can adsorb to a polycationic substratum and cause neurons plated on that substratum to extend neurites rapidly and profusely. The extracellular matrix glycoprotein laminin, when bound to a substratum, elicits a similar response by neurons. In this report, six CMs that contain a "neurite outgrowth-promoting factor" were studied. Immunoprecipitation with affinity-purified anti-laminin antibodies demonstrated that laminin is present in all of them, and when it was selectively removed, there was a corresponding loss of neurite outgrowth-promoting activity in each CM. Antibodies to purified laminin failed, however, to block the outgrowth-promoting activity of five of the CMs tested, even though these antibodies blocked the outgrowth-promoting activity of purified laminin in the same assay. This result could reflect differences in amino acid sequence or protein modification between CM-derived laminin and the purified laminin used in generating antibodies. Alternatively, it could reflect the fact that other molecules bind to laminin in CMs and could interfere with the binding of antibodies to sites on laminin that are important for biological activity.
When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated laminin-binding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.
We have identified a unique heparan sulfate (HeS) proteoglycan synthesized by the neuronal-like cell line PC12. The proteoglycan, purified with monoclonal antibodies from medium conditioned by PC12 cells, has an apparent molecular weight of 350,000, and it contains a Mr 80,000 core protein and HeS side chains of Mr 15,000 each. The purified molecule has the same apparent size and density as it has in conditioned medium. HeS proteoglycans that are indistinguishable antigenically but very difficult to solubilize are found on the external surface and in the interior of PC12 cells and neurons. Mild proteolysis converts the surface proteoglycan into a molecule closely resembling that found in the medium. The same surface antigens are also present on a subpopulation of T-cells and on a non-neuronal accessory cell found in dorsal root ganglion cultures. The PC12 cell line and the non-neuronal dorsal root ganglion cells secrete a factor into medium that, after adsorption to polylysine-coated surfaces, induces rapid neurite out-growth by primary sympathetic neurons. The monoclonal antibodies used to purify the neuronal HeS proteoglycan from PC12 cells are capable of depleting this conditioned medium of its neurite-promoting activity. These studies suggest that a HeS proteoglycan synthesized and secreted by neurons and certain accessory cells plays a role in regulating neurite outgrowth.
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Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMSF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMSF over half its surface, only the neurons on the treated half extended neurites. The active factor in CMSF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80 degrees C; it was stable to heating to 60 degrees C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with [35S]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparin sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparin sulfate proteoglycan and other molecules. CMSF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMSF.