Once the elongating fibers have established the appropriate synaptic contacts with the target cells, the continued survival of the innervating cells in the ganglion appears to depend on the availability of NGF. Studies conducted by Hendry at the Australian National University and by K. Stockel and H. Thoenen at the Basel Institute for Immunology have demonstrated that NGF is taken up at the terminal nerve endings of the sympathetic fibers and transported back to the neuronal cell body along the axon. This retrograde axonal transport of NGF is absolutely essential for the survival of the innervating neurons. When it is experimentally prevented (either by severing the projecting axons, by treating them with the drug vinblastine, which blocks axonal transport, or by administering 6-hydroxydopamine, which destroys the nerve endings), the innervating sympathetic neurons in the ganglion die off. The lethal effects of blocking the axonal transport of NGF can be completely overcome, however, by supplying the cell bodies with externally administered NGF. In this case the external NGF makes up for the NGF that would normally be transported back inside the axon to the cell body from the innervated cells.
Work in several laboratories has shown that the retrograde axonal transport of NGF follows its interaction with specific receptor sites on the nerve terminals of the newly established fibers. Receptors are proteins that are usually located on the external surface of the cell membrane; they provide specific recognition sites for messenger substances such as hormones, neurotransmitters and growth factors. The existence of such specific receptors on the neuronal surface makes it possible for NGF to exert its effects at exceedingly low concentrations (about 2.8 micrograms per liter). The binding of NGF to its receptors triggers a chain of biochemical events that leads ultimately to the outgrowth of the nerve fiber.
Immature sympathetic neurons respond to NGF with a burst of metabolic activity that provides the material necessary for the growth of the nerve fiber and the manufacture of molecules of neurotransmitter. The cells make more proteins and lipids, take up amino acids from the surrounding medium and burn glucose and other energy-rich compounds at a faster rate. These effects can be seen as a general response to the primitive signal conveyed by NGF through its specific receptors on the cell surface.
Within a short time after the binding of NGF to its receptors the protein constituents of the cytoplasm of the immature sympathetic neuron are profoundly rearranged. In particular the filamentous structures called microtubules and microfilaments come to fill all the available space between the cell nucleus and the cell membrane. These filaments play a key role in the growth of the nerve fiber by providing a structural framework and the propulsive force for its elongation.
How does NGF control the assembly of these filamentous proteins? One possibility is that it acts directly to enhance the polymerization of tubulin and actin, the monomeric proteins that give rise respectively to microtubules and micro filaments. Working at the Laboratory of Cell Biology in Rome, we tested the hypothesis by measuring the rate of assembly of tubulin and actin in the test tube in both the presence and the absence of NGF. In the absence of NGF dilute solutions of the proteins were unable to polymerize into filaments (or did so at a very low rate) because the thermodynamic tendency of the monomers to stay far apart in solution was greater than their tendency to aggregate. The addition of NGF to the solution, however, induced a rapid and massive polymerization reaction. Subsequent investigation revealed that NGF joins together the minimum number of monomers needed to initiate polymerization, after which the reaction proceeds spontaneously to completion. On the basis of these test-tube findings we have hypothesized that the massive formation of microtubules and microfilaments in the developing sympathetic neuron is triggered by NGF, and that this effect may lead directly to the growth and elongation of the nerve fiber.