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The Role of Nerve Growth Factor in Neuronal Survival, Maintenance, and Regrowth

E.M. Shooter

Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305

Nerve growth factor (NGF) plays a major role in regulating neuronal cell death in sensory, sympathetic, and CNS cholinergic neurons. The underlying physiological process that determines how many neurons survive or die is a retrograde flow of NGF from the target to the cell body (Hendry et al. 1974). The neurons that survive are the ones that establish a significant level of this retrograde flow of NGF. The targets of the NGF-responsive neurons synthesize NGF, and the level of NGF or NGF mRNA correlates well with the density of innervation, as it should if NGF regulates neuronal cell death (Thoenen et al. 1987).

In one well-defined sensory target, the whisker pad of the mouse, NGF synthesis begins when the nerve fiber from the neurons in the trigeminal ganglia first contact the target (Davies et al. 1987). Although this suggests that NGF gene transcription in the target could be controlled by the nerve, this has turned out not to be so. NGF synthesis begins in targets deprived of their sensory input at exactly the same time as in targets that receive innervation (Rohrer et al. 1988). Some clues as to the possible regulation of NGF synthesis are mentioned later. What is clear from these data, and from the finding that NGF neither enhances nor anti-NGF antibody inhibits neurite outgrowth from early sensory or sympathetic neurons, is that NGF plays no direct role in initial axon growth or guidance.

A continual flow of NGF is required throughout the lifetime of those neurons that succeed in starting this flow if they are to maintain their fully differentiated state. This is most readily seen in neonatal animals where interruption of the retrograde flow by a variety of mechanisms leads to degeneration of the neurons and loss of neurotransmitter synthetic enzyme activity (Thoenen and Barde 1980). The molecular mechanisms invoked by the retrograde flow are not yet understood in detail, although some of the

pieces of the puzzle have been identified. NGF synthesized by, apparently, all different types of cells in the target (Bandtlow et al. 1987) is released and interacts with NGF receptors on the neuronal membranes at the nerve terminal. Again, the nature of the signal transduction mechanism generated by NGF binding to its receptor is not known, nor whether the retrograde flow of a second messenger instead of, or as well as, NGF is important.

Internalization of NGF at the nerve terminal occurs on the high-affinity receptors (Hosang and Shooter 1987), and the NGF-containing vesicles formed in the process attach to and move rapidly along microtubules to the neuronal cell body (Vale 1987). Once there, it is quite rapidly degraded in a lysosomal process that can be inhibited without affecting the biological activity of NGF (Layer and Shooter 1983).

The binding of NGF to its receptors elicits a number of short-term changes, such as membrane ruffling and growth cone motility, suggesting that part of the transduction signal readily interacts with the cell's cytoskeleton (Greene and Shooter 1980). Longer term changes generally involve gene transcription and include, for example, transient expression of genes coding for proteins that bind to DNA, such as c-fos, c-myc, a transactivating factor, and a homolog of a glucocorticoid receptor (Millbrandt 1987, 1988) as well as more stable expression of genes coding for the neurotransmitter synthetic enzymes, the cytoskeleton and its associated proteins (Drubin et al. 1985; Aletta et al. 1988), the NGFR itself, and at least two proteins of the S-100 family (Masiakowski and Shooter 1988).

One of the two forms of the NGFR has recently been cloned from the rat and human, and they show remarkable conservation of structure (Radeke et al. 1987; Johnson et al. 1986). This single peptide chain binds NGF with an affinity that is 100-fold less than the other form of the NGFR (the high-affinity NGFR), and its structure reveals no particular clues about the signal transduction mechanism. It is fairly small as receptors go (42,000 mol.wt. for the peptide chain and another approximately 40,000 mol.wt. as carbohydrate), but the extracellular domain contains repeats of short cysteinerich regions characteristic of other peptide or protein-binding receptors. The intracellular domain is too short to encompass an endogenous tyrosine kinase activity found in the mitogenic growth factor receptors. The fact that the cDNA for this receptor hydridizes to only one mRNA, even in cells that display both forms, together with a lot of other indirect evidence, suggests that the low-affinity receptor is the NGF binding subunit and that its interaction with another cytosolic or membrane-bound protein generates the highaffinity form.

Since the retrograde flow of NGF from the target is so crucial to the maintenance of the differentiated state of the neuron, some alternative source of NGF must become available after peripheral sciatic nerve injury, otherwise the proximal stump and cell body (Heumann et al. 1987a) would degenerate. The nonneuronal cells of the sciatic nerve provide this source. Very rapidly

after nerve injury, these cells, particularly around the site of the injury, synthesize and secrete NGF and act as a substitute target. On a longer time scale, all the nonneuronal cells in the distal injured nerve synthesize NGF mRNA and NGF (Heumann et al. 1987b) in response to interleukin-1 released by the invading, activated macrophage (Lindholm et al. 1987). NGF now shows its well-known property of invoking neurite regrowth. As regeneration of the nerve fibers proceeds, the macrophage slowly leaves the nerve, and NGF production in the nerve slowly declines. With the reestablishment of connections with the target, the original source of NGF for retrograde flow is required, and from here on, the only NGF found in the regenerated nerve is that undergoing retrograde flow.

Besides responding to injury by producing NGF, the Schwann cells distal to the injury also up-regulate the gene for the NGFR and begin to express NGFR on their surface (Taniuchi et al. 1986; Heumann et al. 1987b). The regulation of the NGFR gene is not controlled by macrophage but rather by contact (or lack of contact) between the axon and Schwann cell membranes. Thus, regeneration of nerve fibers by bringing axonal membranes back into contact with Schwann cell membranes down-regulates the NGFR expression. Although the NGFR are all of the low-affinity form, the coexpression of NGF and its receptor provides the possibility of forming a bed of NGF on the surface of the Schwann cells over which the growth cones can travel. Moreover, these receptors appear to be functional because their occupation leads to the induction of the genes for c-fos and one of the cell adhesion molecules (Thoenen et al. 1987).

The work briefly outlined above has defined the roles of NGF in neuronal survival, maintenance, and regeneration in the periphery. The discovery of one group of neurons in the central nervous system that are also NGF-responsive raises the interesting question as to whether NGF plays the same roles in the brain. This group, the cholinergic neurons in the basal forebrain nuclei, express NGF receptors and retrogradely transport NGF from their targets in the hippocampus and cortex (Schwab et al. 1979). Moreover, the density of the cholinergic innervation in these two regions and also within the hippocampus correlates well with the levels of NGF and NGF mRNA (Korsching et al. 1985; Shelton and Reichardt 1986) as it does for targets in the periphery.

Infusion of NGF into newborn rats increases neurotransmitter synthesis in this instance by increasing choline acetyltransferase activity in the hippocampus, medial septum, and cortex (Gnahn et al. 1983) as well as in the striatum. (Mobley et al. 1986). Lesioning of the septo-hippocampal fibers results not only in ingrowth of sympathetic fibers into the hippocampus, an effect that can be partially inhibited by anti-NGF antibodies, but also in the loss of about 50 percent of the cholinergic cell bodies, which can be largely prevented by NGF (Hefti 1986; Williams et al. 1986).

Interestingly, the specific learning task (ability to find a platform under water) that these lesioned rats lose is recovered after prolonged infusion of

NGF (Will and Hefti 1985). Moreover, in some aged rats with both degeneration of these same cholinergic neurons and a decline in the ability to perform the underwater task, infusion of NGF can, again, rescue both the neurons and the learning abilities (Fischer et al. 1987). All these experiments suggest that NGF does play similar roles in the central and the peripheral nervous systems. However, there are enough differences in, for example, the time course of the developmental expression of NGF, NGF mRNA, and NGFR to indicate that there may also be functions for NGF that are unique to the central nervous system (Thoenen et al. 1987). The interplay of NGF with brain systems known to be involved in learning and memory makes an especially attractive theme for further research sponsored by the National Institute of Mental Health.

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