Concentration-dependent regulation of neuronal gene expression by nerve growth factor.

NGF is a neurotrophic protein that promotes the survival, growth, and differentiation of developing sympathetic neurons. To directly determine the effects of different concentrations of NGF on neuronal gene expression, we examined mRNAs encoding the p75 low-affinity NGF (LNGF) receptor, T alpha 1 alpha-tubulin (T alpha 1), and tyrosine hydroxylase (TH) in pure cultures of rat sympathetic neurons from postnatal day 1 superior cervical ganglia. Studies of the timecourse of gene expression during 2 wk in culture indicated that a 5-d incubation period would be optimal for the concentration-effect studies. Analysis of RNA isolated from neurons cultured in 2-200 ng/ml 2.5S NGF for 5 d revealed that, as the NGF concentration increased, neurons expressed correspondingly increased levels of all three mRNAs. Both LNGF receptor and TH mRNAs increased seven-fold, and T alpha 1 mRNA increased four-fold in neurons cultured in 200 versus 10 ng/ml NGF. In contrast, T26 alpha-tubulin mRNA, which is constitutively expressed, did not alter as a function of NGF concentration. When neurons were initially cultured in 10 ng/ml NGF for 5 d, and then 200 ng/ml NGF was added, LNGF receptor, T alpha 1, and TH mRNAs all increased within 48 h. The timecourse of induction differed: T alpha 1 mRNA was maximal by 5 h, whereas LNGF receptor and TH mRNAs first began to increase at 12 h after the NGF increase. These experiments show that NGF regulates expression of a subset of mRNAs important to neuronal growth and differentiation over a broad concentration range, suggesting that the effects of NGF may be mediated by more than just a single receptor operating at one fixed affinity. These results also suggest a mechanism for coupling neuronal synthesis of axonal proteins to increases in size of the innervated target territory during growth of the organism.

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.

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.

Biosynthesis of membrane lipids in rat axons.

Compartmented cultures of sympathetic neurons from newborn rats were employed to test the hypothesis that the lipids required for maintenance and growth of axonal membranes must be synthesized in the cell body and transported to the axons. In compartmented cultures the distal axons grow into a compartment separate from that containing the cell bodies and proximal axons, in an environment free from other contaminating cells such as glial cells and fibroblasts. There is virtually no bulk flow of culture medium or small molecules between the cell body and axonal compartments. When [methyl-3H]choline was added to the cell body-containing compartment the biosynthesis of [3H]-labeled phosphatidylcholine and sphingomyelin occurred in that compartment, with a gradual transfer of lipids (less than 5% after 16 h) into the axonal compartment. Surprisingly, addition of [methyl-3H]choline to the compartment containing only the distal axons resulted in the rapid incorporation of label into phosphatidylcholine and sphingomyelin in that compartment. Little retrograde transport of labeled phosphatidylcholine and sphingomyelin (less than 15%) into the cell body compartment occurred. Moreover, there was minimal transport of the aqueous precursors of these phospholipids (e.g., choline, phosphocholine and CDP-choline) between cell compartments. Similarly, when [3H]ethanolamine was used as a phospholipid precursor, the biosynthesis of phosphatidylethanolamine occurred in the pure axons, and approximately 10% of the phosphatidylethanolamine was converted into phosphatidylcholine. Experiments with [35S]methionine demonstrated that proteins were made in the cell bodies, but not in the axons. We conclude that axons of rat sympathetic neurons have the capacity to synthesize membrane phospholipids. Thus, a significant fraction of the phospholipids supplied to the membrane during axonal growth may be synthesized locally within the growing axon.

Regeneration of neurites on long-term cultures of sympathetic neurons deprived of nerve growth factor.

Sympathetic neurons from newborn rats, cultured for 1 month or longer in the virtual absence of nonneuronal cells, were capable of regenerating neurites after neuritotomy. Regeneration occurred even after nerve growth factor was withdrawn from the cultures, although it was much less extensive and appeared limited to a few days following neuritotomy. Even after 29 days of nerve growth factor deprivation, reintroduction of the protein prompted a resumption of neurite growth. Possible roles of both nerve growth factor-independent and -dependent components in adult nerve regeneration are discussed.

Growth of sympathetic nerve fibers in culture does not require extracellular calcium.

A compartmented culture system in which distal neurites from newborn rat sympathetic neurons entered a fluid environment separate from that bathing the cell bodies and proximal neurites was used to investigate effects of extracellular Ca2+ deprivation on nerve fiber growth. Neurites readily grew into, elongated for many days within, and regenerated after neuritotomy within distal compartments substantially deprived of Ca2+ (0 added Ca2+, 0.5-5 mM EGTA), provided Ca2+ was supplied to the cell bodies. The Ca2(+)-deprived neurites generally extended at rates 20%-35% slower than controls. Growth of neurites did, however, cease within 2 days when the cell bodies were deprived of Ca2+, and the neurites and cell bodies eventually degenerated. These results show that neither extracellular Ca2+ nor the influx of Ca2+ at or near the growth cone is required for sustained neurite growth. They also rule out the possibility that the promotion of neurite growth by nerve growth factor is mediated, by the influx of extracellular Ca2+.

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.

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.

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.

Inhibition of nerve fiber regeneration in cultured sympathetic neurons by local, high potassium.

Distal regeneration of neurites from cultured sympathetic neurons of newborn rats was stunted by exposure to local, high K+ (20 mM), only when more proximal neurite regions and the cell bodies were exposed to a normal K+ concentration (5 mM). Neurites grew luxuriantly and extensively when exposure to high K+ was uniform over the entirety of the neurons. Thus, neurite growth can be strongly influenced by regional variations in K+ which are probably within the naturally occurring range, especially during development and regeneration.

The regulation of nerve fiber length by intercalated elongation and retraction.

Cell body movements were observed in cultures of rat sympathetic neurons that were accommodated by the elongation of neurite segments intercalated between the cell bodies and distal neurites. The elongation may represent growth of the neurites in response to mechanical tension arising from the cell body movements. Preliminary observations also suggest that neurites made slack may actively shorten. Control of nerve fiber length by mechanical tension could allow the nervous system to readily accommodate changes in the size and shape of the organism that occur during development and growth. It is possible that nerve growth factor promotes nerve fiber elongation indirectly through tension generated by growth cone movements.

Local control of neurite sprouting in cultured sympathetic neurons by nerve growth factor.

Sprouting of neurites by sympathetic neurons from newborn rats was studied in compartmentalized cultures. The neuronal cell bodies resided in proximal compartments, and neurites penetrated silicone grease barriers and elongated within distal compartments. Nerve growth factor (NGF) was initially supplied at 1 microgram/ml in all compartments, but was subsequently withdrawn from proximal compartments and for a time was only supplied to distal neurites. Little or no neurite growth was observed in proximal compartments after NGF withdrawal, but reintroduction of NGF resulted in substantial neurite growth over the next few days which was shown to have originated from local sprouting within the proximal compartments. This result is distinct from previous work on NGF-enhanced nerve fiber elongation in demonstrating that quiescent, NGF-deprived regions of sympathetic neurons sprout neurites in response to local reexposure to NGF.

Retraction and degeneration of sympathetic neurites in response to locally elevated potassium.

Sympathetic neurons from superior cervical ganglia of newborn rats were plated into center compartments of 3-compartment culture dishes, allowing exposure of distal neurites to media of different composition than provided to cell bodies and proximal neurites. Cultures were maintained initially with an external potassium concentration ([K+]o) of either 5 mM in all compartments or 50 mM in all compartments. After neurites had elongated into distal compartments, the culture medium was changed such that: the cell bodies and proximal neurites were exposed to 5 mM [K+]o; the distal neurites in one side compartment of each culture were also exposed to 5 mM [K+]o; but the distal neurites in the opposite side compartment were exposed to 50 mM [K+]o. During the next 7-10 days, the distal neurites locally exposed to 50 mM [K+]o degenerated. Many neurites developed a stretched appearance before degenerating, and detailed observations suggest that the neurites retracted to the point where mechanical tension exceeded their strength and then abruptly disintegrated. Neurites in opposite side compartments exposed to 5 mM [K+]o were normal in appearance and did not degenerate. These results suggest that a proximo-distal increase in [K+]o causes an extreme retraction of neurites distal to the increase. These results raise the possibility that K+ released by active nerve endings might cause the retraction of inactive nerve endings, thus providing a possible mechanism for the influence of activity on competition for synaptic sites, a pervasive phenomenon in the developing nervous system.

Influence of the extracellular potassium environment on neurite growth in sensory neurons, spinal cord neurons and sympathetic neurons.

The influence of the extracellular potassium concentration ([K+]o) on neurite growth in rat sensory neurons, spinal cord neurons and sympathetic neurons was investigated. Experiments carried out in 3-compartment culture dishes showed that although neurites from sensory and spinal cord neurons were capable of growing in both 5 mM [K+]o and 20 mM [K+]o, they were virtually unable to grow from a region of 5 mM [K+]o into a region of 20 mM [K+]o. Neurites from sympathetic neurons behaved similarly although [K+]o exceeding 20 mM was required to exclude sympathetic neurites. We suggest the possibility of a negative chemotaxis to [K+]o by growth cones in these neurons. Neurite regeneration following axotomy in sensory neurons was partially inhibited distal to a proximo-distal increase in [K+]o. The nature of this inhibition was somewhat different from that described previously in sympathetic neurons. The possibility is raised that [K+]o plays a role in the development of the nervous system.

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.

Local control of neurite development by nerve growth factor.

A three-chamber culture system was devised in which neurites growing from small clusters of somas of sympathetic neurons penetrated a virtually fluid-impermeable barrier; thus the local fluid environment of the distal portions of the neurites could be controlled independently of the local fluid environment of the somas and proximal portions of the neurites. Neurites regularly penetrated the barriers if a high concentration of nerve growth factor was present on both sides, but never penetrated into chambers to which no nerve growth factor had been added. After neurites crossed the barrier, local removal of nerve growth factor from the distal portions of the neurites caused the growth of these portions to stop, and they eventually appeared to degenerate even though nerve growth factor was continuously present in the chamber that contained their somas and proximal portions. In contrast, local nerve growth factor was not required at the somas and proximal portions of the neurites; many neurons survived its withdrawal provided their somas were associated with neurite bundles that crossed into a chamber containing nerve growth factor. These results show that the growth, and probably the survival, of neurites depends upon nerve growth factor in their local environment, regardless of the nerve growth factor concentrations to which other portions of the neuron are exposed. This is entirely consistent with the notion that nerve growth factor released by sympathetic target tissues promotes the establishment and maintenance of appropriate neuron-target connections during development.

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.