Block of slow axonal transport and axonal growth by brefeldin A in compartmented cultures of rat sympathetic neurons.

Disruption of the Golgi by brefeldin A (BFA) has been reported to block fast axonal transport and axonal growth. We used compartmented cultures of rat sympathetic neurons to investigate its effects on slow axonal transport. BFA (1 micro g/ml) applied to cell bodies/proximal axons for 6-20 h disrupted the Golgi, reversibly blocked axonal growth, and reversibly blocked anterograde transport of all proteins, including tubulin. The retrograde transport of nerve growth factor (NGF) was also blocked. The phosphorylation of Erk1 and Erk2 in response to NGF was unaffected after 6 h of treatment with BFA, suggesting that the block of axonal transport was specific and direct. Consistent with its principal site of action at the Golgi, no effects were observed when BFA was applied only to the distal axons. Block of fast anterograde and retrograde axonal transport is consistent with the role of the Golgi in supplying transport vesicles. Block of slow axonal transport was surprising, and further results indicated that transport of tubulin en route along the axon was arrested by application of BFA to the cell bodies, suggesting that a continuous supply of anterograde transport vesicles from the Golgi is required to maintain slow axonal transport of cytoskeletal proteins.

Compartmentalization of choline and acetylcholine metabolism in cultured sympathetic neurons.

To determine the relative contribution of cell bodies and distal axons to the production of acetylcholine, we used retinoic acid to induce a cholinergic phenotype in compartmented cultures of rat sympathetic neurons. When [3H]choline was given to cell bodies/proximal axons for 24 h, 98% of the radiolabel was recovered as choline, phosphocholine, CDP-choline and phosphatidylcholine, whereas only 1 to 2% of the radiolabel was incorporated into acetylcholine. Choline taken up by cell bodies and transported to axons is poorly utilized for acetylcholine biosynthesis. In contrast, when distal axons were supplied with [3H]choline, 11% of the radiolabel was recovered in acetylcholine after 24 h, the remainder being incorporated into precursors/metabolites of phosphatidylcholine. The lack of acetylcholine synthesis in cell bodies/proximal axons could not be ascribed to an absence of choline acetyltransferase activity in this region of the neurons, since the specific activity of this enzyme was similar in cell bodies/proximal axons and distal axons. The rate of choline uptake by distal axons (15.3 4.4 nmol/5 min/mg protein) was approximately 10-fold greater than by cell bodies/proximal axons (1.6 0.8 nmol/5 min/mg protein). Moreover, choline uptake into distal axons was inhibited by 74.5% by hemicholinium-3, and by 80.1% by removal of Na(+) from the medium. In contrast, choline uptake by cell bodies/proximal axons was not significantly inhibited by hemicholinium-3 or Na(+) removal. These results suggest that the majority of axonal acetylcholine is synthesized in distal axons/axon terminals from choline taken up by a high-affinity choline transporter in distal axons.

Receptor binding, internalization, and retrograde transport of neurotrophic factors.

This review deals with the receptor interactions of neurotrophic factors, focusing on the neurotrophins of the nerve growth factor (NGF) family, the glial cell derived neurotrophic factor (GDNF) family, and the ciliary neurotrophic factor (CNTF) family. The finding that two proteins, p75NTR and Trk, act as receptors for NGF in neurons generated the discovery of other neurotrophic factors/receptor families and has enhanced our understanding of the development, survival, regeneration, and degeneration of the nervous system. The kinetics of binding, the structure of the ligand-receptor complex, and the mechanism of retrograde transport of the neurotrophins are discussed in detail and compared to information available on the GDNF and CNTF families. Each neurotrophic factor family, i.e., NGF, GDNF, and CNTF, has a set of receptors with specificity for individual members of the family and a common receptor without member specificity that, in some families, generates the cellular signal and retrograde transport.

Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons.

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of TrkA.

Uptake of lipoproteins for axonal growth of sympathetic neurons.

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.

The synthesis and transport of lipids for axonal growth and nerve regeneration.

Neurons are unique polarized cells in which the growing axon is often located up to a meter or more from the cell body. Consequently, the intracellular movement of membrane lipids and proteins between cell bodies and axons poses a special challenge. The mechanisms of lipid transport within neurons are, for the most part, unknown although lipid transport via vesicles and via cholesterol- and sphingolipid-rich 'rafts' are considered likely mechanisms. Very active anterograde and retrograde transport of lipid-containing vesicles occurs between the cell body and distal axons. However, it is becoming clear that the axon need not obtain all of its membrane constituents from the cell body. For example, the synthesis of phosphatidylcholine, the major membrane phospholipid, occurs in axons, and its synthesis at this location is required for axonal elongation. In contrast, cholesterol synthesis appears to occur only in cell bodies, and cholesterol is efficiently delivered from cell bodies to axons by anterograde transport. Cholesterol that is required for axonal growth can also be exogenously supplied from lipoproteins to axons of cultured neurons. Several studies have suggested a role for apolipoprotein E in lipid delivery for growth and regeneration of axons after a nerve injury. Alternatively, or in addition, apolipoprotein E has been proposed to be a ligand for receptors that mediate signal transduction cascades. Lipids are also transported from axons to myelin, although the importance of this process for myelination is not clear.

Protein synthesis in axons and its possible functions.

Proteins synthesized in neuronal cell bodies are transported along axons by fast and slow axonal transport. Cytoskeletal proteins and cytosolic proteins that travel by slow axonal transport could take years to reach the terminals of meter-long axons, and it is difficult to see how proteins could last long enough to make this journey. How then are proteins supplied to the distal regions of long axons? Evidence has accumulated indicating that axons contain specific mRNAs and ribosomes and can synthesize cytoskeletal proteins and some other proteins. This review considers the direct evidence that proteins can be synthesized in axons and considers the possible functional significance of axonal protein synthesis. It remains unclear whether local protein synthesis could supply the cytoskeletal proteins and other slow-transported proteins required for the maintenance, plasticity, and regeneration of long axons.

Synthesis of beta-tubulin, actin, and other proteins in axons of sympathetic neurons in compartmented cultures.

The proteins needed for growth and maintenance of the axon are generally believed to be synthesized in the cell bodies and delivered to the axons by anterograde transport. However, recent reports suggest that some proteins can also be synthesized within axons. We used [35S]methionine metabolic labeling to investigate axonal protein synthesis in compartmented cultures of sympathetic neurons from newborn rats. Incubation of distal axons for 4 hr with [35S]methionine resulted in a highly specific pattern of labeled axonal proteins on SDS-PAGE, with 4 prominent bands in the 43-55 kDa range. The labeled proteins in axons were not synthesized in the cell bodies, because they were also produced by axons after the cell bodies had been removed. Two of the proteins were identified by immunoprecipitation as actin and beta-tubulin. Axons synthesized <1% of the actin and tubulin synthesized in the cell bodies and transported into the axons, and 75-85% inhibition of axonal protein synthesis by cycloheximide and puromycin failed to inhibit axonal elongation. Nonetheless, the specific production by axons of the major proteins of the axonal cytoskeleton suggests that axonal protein synthesis arises from specific mechanisms and likely has biological significance. One hypothetical scenario involves neurons with long axons in vivo in which losses from turnover during axonal transport may limit the availability of cell body synthesized proteins to the distal axons. In this case, a significant fraction of axonal proteins might be supplied by axonal synthesis, which could, therefore, play important roles in axonal maintenance, regeneration, and sprouting.

Effects of the neurotrophins nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures.

We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth.

Rapid retrograde tyrosine phosphorylation of trkA and other proteins in rat sympathetic neurons in compartmented cultures.

According to the current theory of retrograde signaling, NGF binds to receptors on the axon terminals and is internalized by receptor-mediated endocytosis. Vesicles with NGF in their lumina, activating receptors in their membranes, travel to the cell bodies and initiate signaling cascades that reach the nucleus. This theory predicts that the retrograde appearance of activated signaling molecules in the cell bodies should coincide with the retrograde appearance of the NGF that initiated the signals. However, we observed that NGF applied locally to distal axons of rat sympathetic neurons in compartmented cultures produced increased tyrosine phosphorylation of trkA in cell bodies/ proximal axons within 1 min. Other proximal proteins, including several apparently localized in cell bodies, displayed increased tyrosine phosphorylation within 5-15 min. However, no detectable 125I-NGF appeared in the cell bodies/proximal axons within 30-60 min of its addition to distal axons. Even if a small, undetectable fraction of transported 125I-NGF was internalized and loaded onto the retrograde transport system immediately after NGF application, at least 3-6 min would be required for the NGF that binds to receptors on distal axons just outside the barrier to be transported to the proximal axons just inside the barrier. Moreover, it is unlikely that the tiny fraction of distal axon trk receptors located near the barrier alone could produce a measurable retrograde trk phosphorylation even if enough time was allowed for internalization and transport of these receptors. Thus, our results provide strong evidence that NGF-induced retrograde signals precede the arrival of endocytotic vesicles containing the NGF that induced them. We further suggest that at least some components of the retrograde signal are carried by a propagation mechanism.

Spatial regulation of neuronal gene expression in response to nerve growth factor.

To examine the cellular mechanisms whereby distally derived growth factors regulate nuclear responses in neurons, we have utilized compartmented cultures of sympathetic neurons to examine the regulation of two nerve growth factor (NGF)-inducible genes, tyrosine hydroxylase (TH) and p75 neurotrophin receptor (p75NTR). These studies demonstrate that NGF can signal retrogradely to mediate the induction of TH and p75NTR mRNAs. However, quantitative differences occurred as a function of the spatial localization of NGF exposure; application of NGF to cell bodies and proximal axons elicited peak levels of neuronal gene expression that were two- to threefold higher than when NGF was applied to distal axons alone. Furthermore, neurons responding maximally to NGF on distal axons were still able to respond to NGF administered to cell bodies and proximal axons. Biochemical analysis indicated that this difference in responsiveness was not due to differences in the number of TrkA/NGF receptors in the two compartments. Thus, although NGF signals retrogradely to mediate nuclear responses, the magnitude of these responses differs as a function of the spatial location of the activated NGF receptor:ligand complex. Moreover, these data suggest that neurons may be able to respond to a second cellular source of neurotrophins, even when target-derived neurotrophins are not limiting.

Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.

We have used compartmented cultures of rat sympathetic neurons to quantitatively examine the retrograde transport of 125I-nerve growth factor (NGF) supplied to distal axons and to characterize the cellular events that maintain steady-state levels of NGF in cell bodies. In cultures allowed to reach steady-state 125I-NGF transport, cell bodies contained only 5-30% of the total neuron-associated 125I-NGF, whereas 70-95% remained associated with the distal axons. This was true over an 8 pM to 1.5 nM 125I-NGF concentration range, indicating that saturation of high affinity receptors could not account for the large fraction of 125I-NGF remaining in axons. Dissociation assays indicated that 85% of 125I-NGF associated with distal axons was surface-bound. At steady-state, only 2-25% of the distal axon-associated 125I-NGF was retrogradely transported each hour, with higher transport rates associated with younger cultures and lower 125I-NGF concentrations. The velocity of 125I-NGF retrograde transport was estimated at 10-20 mm/hr. However, as in a previous report, almost no 125I-NGF transport was observed during the first hour after 125I-NGF administration, indicating a significant lag between receptor binding and loading onto the retrograde transport system. During 125I-NGF transport through axons spanning an intermediate compartment in five-compartment cultures, little or no 125I-NGF was degraded or released from the axons. After transport, 125I-NGF was degraded with a half-life of 3 hr. In summary, although some cellular events promoted NGF accumulation in cell bodies, distal axons represented by far the principal site of NGF-receptor interaction at steady-state as a result of a low retrograde transport rate.

Elevation of ceramide within distal neurites inhibits neurite growth in cultured rat sympathetic neurons.

Sphingolipids are abundant constituents of neuronal membranes and have been implicated in intracellular signaling. We show that two analogs of glycosphingolipid biosynthetic intermediates, fumonisin B1 (which inhibits dihydroceramide synthesis) and DL-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (which inhibits glucosylceramide synthesis) decrease glycosphingolipid synthesis in rat sympathetic neurons. Although both fumonisin and PPMP inhibit glycosphingolipid synthesis, these inhibitors have differential effects on ceramide metabolism in axons. threo-PPMP, but not erythro-PPMP or fumonisin, induces an accumulation of [3H]palmitate-labeled ceramide and impairs axonal growth. Moreover, exogenously added, cell-permeable C6-ceramide, but not C6-dihydroceramide, mimicks the effect of PPMP. Our studies suggest that the lipid second messenger ceramide acts in distal axons, but not cell bodies, as a negative regulator of neurite growth.

Role of lipoproteins in the delivery of lipids to axons during axonal regeneration.

Nerve fiber elongation involves the input of lipids to the growing axons. Since cell bodies are often a great distance from the regenerating tips, alternative sources of lipids have been proposed. We previously demonstrated that axonal synthesis of phosphatidylcholine is required for axonal growth (Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) J. Cell Biol. 128, 913-918; Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) Biochem. J. 312, 411-417). In contrast, cholesterol is not made in axons. We now show that when compartmented cultures of rat sympathetic neurons are incubated with pravastatin, in the absence of exogenously supplied lipids, cholesterol synthesis is inhibited and axonal growth is impaired. The addition of cholesterol to the axons or cell bodies of neurons treated with this inhibitor restores normal axonal elongation. Similarly, a supply of cholesterol via lipoproteins restores normal axonal growth. In contrast, lipoproteins do not provide axons with sufficient phosphatidylcholine for normal elongation when axonal phosphatidylcholine synthesis is inhibited. Thus, our studies support the idea that during axonal regeneration lipoproteins can be taken up by axons from the microenvironment and supply sufficient cholesterol, but not phosphatidylcholine, for growth. We also show that neither apoE nor apoA-I within the lipoproteins is essential for axonal growth.

Alkylphosphocholines inhibit choline uptake and phosphatidylcholine biosynthesis in rat sympathetic neurons and impair axonal extension.

At least 50% of the major axonal membrane lipid, phosphatidylcholine, of rat sympathetic neurons is synthesized in situ in axons [Posse de Chaves, Vance, Campenot and Vance (1995) J. Cell Biol. 128, 913-918]. In the same study we reported that, in a choline-deficient model for neuron growth, phosphatidylcholine synthesis in cell bodies is neither necessary nor sufficient for growth of distal axons. Rather, the local synthesis of phosphatidylcholine in distal axons is required for normal axon growth. We have now used three alkylphosphocholines (hexadecylphosphocholine, dodecylphosphocholine and octadecylphosphocholine) as inhibitors of PtdCho biosynthesis in a compartmented model for culture of rat sympathetic neurons. The experiments reveal that alkylphosphocholines decrease the uptake of choline into these neurons and inhibit PtdCho synthesis, but not via an effect on the activity of the enzyme CTP: phosphocholine cytidylyltransferase. We also show that when the distal axons, but not the cell bodies, are exposed to alkylphosphocholines, axonal elongation is inhibited, which is consistent with the hypothesis that phosphatidylcholine synthesis in axons, but not in cell bodies, is required for axonal elongation. The inhibitory effect of alkylphosphocholines on axon growth is most likely not mediated via a decrease in the activity of protein kinase C, since when this enzyme activity is down-regulated by treatment of the cells with phorbol ester, the alkylphosphocholines retain their ability to inhibit axonal growth.

Reciprocal regulation of choline acetyltransferase and choline kinase in sympathetic neurons during cholinergic differentiation.

The regulation of the synthesis of acetylcholine and phosphatidylcholine in rat sympathetic neurons was examined in the context of cholinergic differentiation. We demonstrate that the activities of choline acetyltransferase (ChAT) and choline kinase (CK) are inversely affected by treatment of sympathetic neurons with retinoic acid, utilized as an agent that induces cholinergic differentiation. Whereas ChAT specific activity increased 2- to 4-fold after 12 days of treatment with 5 microM retinoic acid, CK specific activity decreased by 25-30%. These changes in enzyme activities were essentially reflected in the incorporation of [methyl-3H]choline into ACh and the metabolites of the CDP-choline pathway for phosphatidylcholine synthesis. When sympathetic neurons were treated under high potassium conditions (50 mM) for 12 days, the specific activity of CK increased 1.3-fold whereas the activity of ChAT decreased by up to 90%. Furthermore, experiments in which the incorporation of [methyl-3H]choline into ACh and the metabolites of the CDP-choline pathway was measured in the absence of Na+ or in the presence of hemicholinium-3 (HC-3), demonstrate that CK has access to the same pool of choline utilized by ChAT. These results provide evidence that the activities of ChAT and CK may be inversely regulated during the process of cholinergic differentiation.

Role of axons in membrane phospholipid synthesis in rat sympathetic neurons.

The axonal synthesis of phospholipids has been demonstrated in compartmented cultures of rat sympathetic neurons. In this model of neuron culture, metabolic events occurring in distal axons were studied independently of those occurring in cell bodies. Using radiolabeled tracers the axonal biosynthesis of the major membrane phospholipids and fatty acids but not cholesterol was detected. The capacity of axons for synthesis of phosphatidylcholine (PC), the major membrane lipid, was confirmed by the demonstration that key enzymes of PC biosynthesis were present in distal axons. A double-labeling experiment showed that at least 50% of axonal PC was synthesized locally in axons, with the remainder being made in cell bodies and transported into axons. The requirement of axonal PC synthesis for axonal elongation was investigated. When PC biosynthesis in distal axons alone was inhibited by two independent approaches (deprivation of choline or addition of the inhibitor hexadecylphosphocholine) axonal growth was markedly retarded. Our experiments demonstrated that PC synthesis in cell bodies was neither necessary nor sufficient for growth of distal axons, whereas local synthesis of PC in distal axons was required for normal axonal elongation.

Axonal synthesis of phosphatidylcholine is required for normal axonal growth in rat sympathetic neurons.

The goal of this study was to assess the relative importance of the axonal synthesis of phosphatidylcholine for neurite growth using rat sympathetic neurons maintained in compartmented culture dishes. In a double-labeling experiment [14C]choline was added to compartments that contained only distal axons and [3H]choline was added to compartments that contained cell bodies and proximal axons. The specific radioactivity of labeled choline was equalized in all compartments. The results show that approximately 50% of phosphatidylcholine in distal axons is locally synthesized by axons. The requirement of axonal phosphatidylcholine synthesis for neurite growth was investigated. The neurons were supplied with medium lacking choline, an essential substrate for phosphatidylcholine synthesis. In the cells grown in choline-deficient medium for 5 d, the incorporation of [3H]palmitate into phosphatidylcholine was reduced by 54% compared to that in cells cultured in choline-containing medium. When phosphatidylcholine synthesis was reduced in this manner in distal axons alone, growth of distal neurites was inhibited by approximately 50%. In contrast, when phosphatidylcholine synthesis was inhibited only in the compartment containing cell bodies with proximal axons, growth of distal neurites continued normally. These experiments imply that the synthesis of phosphatidylcholine in cell bodies is neither necessary nor sufficient for growth of distal neurites. Rather, the local synthesis of phosphatidylcholine in distal axons is required for normal growth.

Evidence that protein kinase C activities involved in regulating neurite growth are localized to distal neurites.

Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by > 50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca(2+)-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 microM PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca(2+)-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC50 value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.

NGF and the local control of nerve terminal growth.

It is generally believed that the mechanism of action of neurotrophic factors involves uptake of neurotrophic factor by nerve terminals and retrograde transport through the axon and back to the cell body where the factor exerts its neurotrophic effect. This view originated with the observation almost 20 years ago that nerve growth factor (NGF) is retrogradely transported by sympathetic axons, arriving intact at the neuronal cell bodies in sympathetic ganglia. However, experiments using compartmented cultures of rat sympathetic neurons have shown that neurite growth is a local response of neurites to NGF locally applied to them which does not directly involve mechanisms in the cell body. Recently, several NGF-related neurotrophins have been identified, and several unrelated molecules have been shown to act as neurotrophic or differentiation factors for a variety of types of neurons in the peripheral and central nervous systems. It has become clear that knowledge of the mechanisms of action of these factors will be crucial to understanding neurodegenerative diseases and the development of treatments as well as the means to repair or minimize neuronal damage after spinal injury. The concepts derived from work with NGF suggest that the site of exposure of a neuron to a neurotrophic factor is important in determining its response.