Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan.

Abstract

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°C; it was stable to heating to 60°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 laSS]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparan 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 heparan 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.An important determinant in the development of multicellular organisms is the extracellular matrix (ECM) upon which cells attach, migrate, and differentiate. It seems likely that a class of substances that affect neuronal development will be found to be associated with this extracellular matrix. The importance of the interaction between neuron and substratum has already been demonstrated in vitro (1-3). In vivo, axons appear to follow routes determined by the substratum (4). In some cases, this reflects the association of axons with already oriented cells, e.g., radial glia in the cerebellum (5) and the pioneering optic nerve fibers of Daphnia (6).The existence of factors necessary for the normal developmerit of certain neurons is established. Sympathetic and sensory neurons require nerve growth factor (NGF) for survival and development in vivo and in vitro (7). Certain other factors appear to be necessary for the survival and development of 574 cultured parasympathetic neurons (8, 9). Other factors, few of which have been significantly purified, have recently been reported to promote the development of sympathetic, parasympathetic, sensory, and spinal cord neurons in culture (10-24). If such factors are supplied by the cells that innervate or are innervated by certain types of neurons, it may explain why appropriate intercellular contacts must be made in order for many types of neurons to su...

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

“…Recently, several independent investigations have reported that heparan sulfate proteoglycans are concentrated in the basal lamina at the neuromuscular junction (2), are present in cholinergic synaptic vesicles (7, 10, 43), show an unusually high avidity for the synaptic form of acetylcholinesterase (47), and may function as substrate-attached nerve growth factors, even for neurons that do not respond to conventional nerve growth factor (27).…”

Response of purified chick motoneurons to myotube conditioned medium: Laminin is essential for the substratum-binding, neurite outgrowth-promoting activity

“…This band is converted, upon reduction, to two bands with Mrs of ~400,000 and ~ 200,000, appropriate for the heavy and light chains of LA. An earlier characterization of the motoneuron NOPA from MCM indicated that it was similar in its mode of action, size, density and enzymatic susceptibilities to a 'neurite outgrowth-promoting factor' from bovine corneal endothelial cell conditioned medium (BCE-CM) [3,9]. It has recently been reported that immunoprecipiration of LA from BCE-CM, as well as several other types of conditioned media, eliminates the ability of these conditioned media to promote neurite outgrowth by rat sympathetic neurons [10].…”

Is Endothelial Nitric Oxide Synthase a Moonlighting Protein Whose Day Job is Cholesterol Sulfate Synthesis? Implications for Cholesterol Transport, Diabetes and Cardiovascular Disease

“…Neurite outgrowth depends upon ATP produced by glycolysis, which is actively suppressed by nitric oxide [180]. On the other hand, heparan sulfate plays an essential role in neurite outgrowth; several different sulfated glycosaminoglycans will stimulate growth-factor induced neurite outgrowth [181], and heparan sulfate supplied to the medium stimulates neurite outgrowth even in the absence of growth factors [182]. Once again, there is a compelling reason to produce sulfate, and clear justification not to produce nitric oxide.…”