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.

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.

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.

Leukemia inhibitory factor and nerve growth factor are retrogradely transported and processed by cultured rat sympathetic neurons.

How neurons convert the presence of factors at their axon terminals into signals that affect mechanisms in their cell bodies is unknown, but retrograde axonal transport of the factors themselves may be involved. Nerve growth factor (NGF) and leukemia inhibitory factor (LIF) have previously been shown to produce changes in cell bodies of sympathetic neurons when applied to their peripheral neurites, and it is well established that NGF is retrogradely transported along sympathetic axons. In this study we show that 125I-LIF applied to terminal neurites of rat sympathetic neurons in compartmented cultures is retrogradely transported, but at a much lower level compared to the retrograde transport of 125I-NGF. Transport of 125I-LIF was competed by cotreatment with unlabeled LIF and was blocked by cotreatment with dinitrophenol. The rate of 125I-LIF transport was independent of NGF concentration. However, both 125I-LIF and 125I-NGF transport was reduced by pretreating neurons with LIF. SDS-PAGE analysis showed that retrogradely transported radiolabel which accumulated in cell body-containing extracts following transport of both 125I-LIF and 125I-NGF consisted of intact as well as partially processed species. Radiolabel also accumulated in the medium bathing the cell bodies and migrated near the dye front on SDS-PAGE, implying that both factors were extensively degraded and released by the neurons. These results are consistent with the suggestion that the retrograde transport of LIF, as thought for NGF, may be important for retrograde signaling mechanisms.

Evidence that the major membrane lipids, except cholesterol, are made in axons of cultured rat sympathetic neurons.

Membrane lipids and proteins required for axonal growth and regeneration are generally believed to be synthesized in the cell bodies of neurons and transported into the axons. However, we have demonstrated recently that, in cultured rat sympathetic neurons, axons themselves have the capacity to synthesize phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine. In these experiments, we employed a compartment model of neuron culture in which pure axons grow in a fluid environment separate from that containing the cell bodies. In the present study, we again used compartmented cultures to confirm and extend the previous results. We have shown that three enzymes of phosphatidylcholine biosynthesis via the CDP-choline pathway are present in axons. We have also shown that the rate-limiting step in the biosynthesis of phosphatidylcholine by this route in neurons, and locally in axons, is catalyzed by the enzyme CTP:phosphocholine cytidylytransferase. The biosynthesis of other membrane lipids, such as phosphatidylserine, phosphatidylethanolamine derived by decarboxylation of phosphatidylserine, phosphatidylinositol, and fatty acids, also occurs in axons. However, the methylation pathway for the conversion of phosphatidylethanolamine into phosphatidylcholine appears to be a quantitatively insignificant route for phosphatidylcholine synthesis in neurons. Moreover, our data provided no evidence for the biosynthesis of another important membrane lipid, cholesterol, in axons.

Cholinergic differentiation of rat sympathetic neurons in culture: effects of factors applied to distal neurites.

Cholinergic properties are induced in sympathetic neurons by several factors applied to entire neurons in culture. Evidence from work with the rat sweat gland model indicates that factors located in target tissues can induce cholinergic differentiation in vivo. We now report that when leukemia inhibitory factor (LIF), heart cell-conditioned medium (HCCM), or dermal fibroblast-conditioned medium (DFCM) is applied to only distal neurites in compartmented cultures of rat sympathetic neurons, the neurons exhibit an increase in specific choline acetyltransferase activity and a concomitant decrease in levels of tyrosine hydroxylase. LIF, HCCM, and DFCM also induce neurite fasciculation, thus suggesting an additional role of cholinergic switching factors in regulating axon-axon and/or axon-substrate adhesion. These results demonstrate that rat sympathetic neurons have the cellular machinery to respond to cholinergic differentiation cues located in peripheral targets, analogous to the response to nerve growth factor.

Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands.

In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.