Binding, internalization, and retrograde transport of 125I-nerve growth factor in cultured rat sympathetic neurons

Abstract

Sympathetic neurons internalize nerve growth factor (NGF) and transport it retrogradely to their cell bodies where it appears to serve a trophic function in maintaining neuronal survival. We have characterized the binding, internalization, and retrograde transport of 125I-NGF by cultured rat sympathetic neurons. After 3 to 4 weeks in culture, sympathetic neurons possessed approximately 2x107 specific, cell surface NGF binding sites per neuron with an apparent affinity constant of 2 to 5x10-9 M. The density of binding sites on the plasma membrane of the neurites was approximately twice that on the plasma membrane of the cell bodies. Because of the extensive network of neuronal processes, the neurites probably account for more than 99.5% of the total binding in mature cultures. Using electron microscope autoradiography, we localized the distribution of 125I-NGF in the cell body following a 1-hr exposure to 125I-NGF. The majority of silver grains were associated with lysosomal organelles, including secondary lysosomes, residual bodies, and multivesicular bodies (MVB). The MVB were the most heavily labeled, with a labeling density (L.D.) of 21, while the lysosomes had a L.D. of 3.1. To study the retrograde transport of 125I-NGF, neurons were grown in compartmentalized culture dishes and their distal processes were exposed to 125I-NGF. Radioactive material was transported to the cell bodies at the rate of approximately 3 mm/hr. The transport mechanism was sensitive to colchicine and was saturable with respect to NGF. After 8 hr of transport, when the radioactivity in the cell bodies had reached a steady state, the label again was localized primarily to the MVB (L.D. = 16.8) and the lysosomes (L.D. = 3.8). The nuclei were not labeled significantly and had an overall L.D. of 0.47. We saw no evidence for the accumulation of NGF by the nulclear membrane, the nucleolus, or chromatin.