The ontogeny of specific retrograde transport of nerve growth factor by motoneurons of the brainstem and spinal cord.

Radiolabeled Nerve Growth Factor (NGF) was injected into either the mandibular process of the first visceral arch or the limb bud of chick embryos at Days 3.5-14 or Days 4-13 of incubation, respectively. Control embryos received injections of labeled cytochrome-C or labeled NGF plus an excess of unlabeled NGF. The tissues were then processed for autoradiography. The 125I-NGF was retrogradely transported by motoneurons of the trigeminal (V) motor nucleus on Days 3.5-8 of incubation, but not at later stages. Similar transport was seen in motoneurons of the spinal cord lateral motor column from Days 4-10 of incubation, but not at later stages. Sensory neurons of the V ganglion and of the dorsal root ganglia transported NGF at all injection ages. In no instance was the 125I-cytochrome-C transported by sensory or motor neurons. The injection of an excess of cold NGF along with labeled NGF resulted in no evidence of retrograde transport of the labeled NGF indicating that the transport was saturable. The time of transport by these brainstem and spinal cord motoneurons corresponds closely to the points during development at which they have been found to exhibit specific NGF binding. The present results, then, provide further evidence for a possible biological role for NGF during early developmental stages of these motoneuron populations.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.     

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.     

Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth factor receptor

We have demonstrated in vitro and in vivo the specific binding of a monoclonal antibody to the rat nerve growth factor (NGF) receptor. Previous work had shown that this antibody, designated 192-IgG, does not compete with NGF for binding to the NGF receptor of PC12 cells, but instead interacts with the receptor to increase NGF binding to PC12 cells (Chandler, C. E., L. M. Parsons, M. Hosang, and E. M. Shooter, 1984, J. Biol. Chem., 259:6882-6889). In the present study, a solid-phase separation assay verified the specific formation of a ternary complex of 192-IgG, the NGF receptor, and NGF: 125I-labeled 192-IgG precipitated from solution only when incubated with both solubilized NGF receptor and NGF covalently linked to a solid phase (Sepharose 4B). Filtration assays using plasma membrane preparations of various tissues showed strict correlation of 125I-192-IgG and 125I-labeled NGF binding; only membranes obtained from superior cervical ganglion bound significant amounts of the monoclonal antibody and NGF. Injection of 125I-192-IgG into the rat anterior eye chamber led to accumulation of intact antibody molecules in the ipsilateral superior cervical ganglion, indicating retrograde axonal transport of 125I-192-IgG from the neuronal termini, located at the iris, to the cell bodies situated in the ganglion. The time course and saturation characteristics of 125I-192-IgG retrograde transport were very similar to those previously reported for 125I-NGF transport, indicating that 192-IgG can be internalized and transported by the same mechanisms as is NGF. Consistent with results of the in vitro binding assays, 192-IgG and NGF failed to compete for retrograde transport and were actually co-transported. Retrograde axonal transport of 192-IgG appears to be species specific, since 125I-192-IgG was transported in the rat, but not in mice, gerbils, hamsters, or guinea pigs. These results establish monoclonal antibody 192-IgG as a specific probe for the rat NGF receptor in vitro and in vivo.     

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to sub-populations of sensory neuron cell bodies in the L4/L5 DRG of neonatal rats. The size distribution of ¹²⁵I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 μm²). ¹²⁵I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 μm². Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 μm², respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 22–32, 1997.     

Retrograde transport of NGF by early chick embryo spinal cord motoneurons

Neuronal retrograde transport of nerve growth factor (NGF) was examined in chick embryos at 5, 6, and 7 days of incubation. Radiolabeled NGF was injected in the target limb muscle and the retrograde transport was viewed following processing for autoradiography. Silver grains were localized in the peripheral nerve, in the ventral root, in neuronal cell bodies within the dorsal root ganglion, and in motoneurons of the lateral motor column. Comparable injections of 125I-cytochrome c resulted in the presence of label at the peripheral injection site only. The possible developmental significance of these observations is discussed.     

NGF-stimulated retrograde transport of trkA in the mammalian nervous system

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.     

A quantitative study of the developmental expression of nerve growth factor (NGF) receptor in rats

The developmental expression of nerve growth factor (NGF) receptor was quantitated in either homogenates or plasma membrane-enriched preparations from whole rat embryos or from isolated tissues. The assay involved crosslinking 125I-NGF to receptors followed by immunoprecipitation with a monoclonal antibody to rat NGF receptor. In some cases, the pellet was resuspended and subjected to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiographic analysis. The NGF receptor was found in whole embryo homogenates as early as embryonic Day 10 (E10) (earliest age examined). The NGF receptor content in whole embryos per milligram protein increased about 3-fold from E11 to E18 and decreased slightly at E20. SDS-PAGE autoradiography showed that the molecular weights of 125I-NGF-bound receptors did not vary with age. The NGF receptor content in sciatic nerve homogenates decreased 23-fold from newborn to adulthood. The change of NGF receptor level in hindleg muscle had a profile similar to that seen in sciatic nerve. The NGF receptor content in superior cervical ganglion (SCG) or dorsal root ganglion (DRG) homogenate preparations was expressed in two ways. On a per milligram protein basis, in SCG, the receptor density was decreased slightly from E20 to adulthood; in DRG, it was relatively constant from E15 through postnatal Day 0 (PND-0) and then dropped 6.7-fold in adults. On a per ganglion basis, in SCG, it increased 4.4-fold from E20 to adult; in DRG, it increased 9-fold from E15 to PND-0 and then stayed constant through adulthood. In brain membrane preparations, the NGF receptor level decreased 11-fold from E15 to adulthood. In spinal cord membrane preparations, it decreased 7-fold from E18 to adulthood. Levels of receptor in cord were always greater than in brain. These data suggest that alterations in the NGF receptor density may have a role in changes in tissue responsiveness to NGF during development.     

Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes

Spleen cells from BALB/c mice immunized with a plasma membrane-enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I-NGF to its receptor in sympathetic or sensory neurons or PC12 cells.     

The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor

Dissociated neurons from the trigeminal (V) region of the metencephalic basal plate or the ventral spinal cord from chick embryos of Day 4 (V basal plate) or Day 5 (spinal cord) were cultured on a laminin substratum either in the presence of nerve growth factor (NGF) or in control medium. Assessment was made of neuronal survival, the amount of neurite elaborated, and the percentage of neurons initiating neurites. The presence of motoneurons was verified by retrograde labeling with the fluorescent dye diI. NGF was found to significantly increase the quantity of neuritic processes produced by the spinal cord dissociates at both 24 and 48 hr in vitro. The percentage of neurons initiating neuritic processes was significantly increased by NGF in the trigeminal population at 48 hr in vitro. Neuronal survival was not enhanced by NGF in either group. Both trigeminal and spinal cord neurons were also found to specifically bind 125I-NGF in culture. These results provide direct evidence for an influence of NGF on process formation of early embryonic motoneurons in culture.     

On Trk for retrograde signaling.

Target-derived neurotrophins like nerve growth factor (NGF) mediate biological effects by binding to and activating Trk neurotrophin receptors at nerve terminals. The activated Trk receptors then stimulate local effects at nerve terminals, and retrograde effects at neuronal cell bodies that often reside at considerable distances from the terminals. However, the nature of the retrograde signal has been mysterious. Recent experiments suggest that the major retrograde signal required for survival and gene expression consists of activated Trk itself. Remarkably, signaling by Trk may differ at the terminal versus the neuronal cell body as a consequence of the retrograde transport mechanism, thereby allowing NGF to not only promote growth locally, but to specifically support survival and gene expression retrogradely.     

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. 1994 John Wiley & Sons, Inc.     

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF)

Adult rat retinal ganglion cells (RGC) undergo degeneration after optic nerve transection. Studies have shown that exogenously applied neurotrophic factors such as brain-derived neurotrophic factor (BDNF) can attenuate axotomy-induced as well as developmental RGC death. Here, we examined whether glial cell line-derived neurotrophic factor (GDNF), a known neurotrophic factor for dopaminergic neurons and motor neurons, could provide neurotrophic support to RGC in adult rats. We determined whether RGC could retrogradely transport GDNF from their target tissue. After injection into the superior colliculus of adult rats, 125I-GDNF was retrogradely transported to contralateral eyes but not to ipsilateral eyes. The transport of 125I-GDNF could be blocked by coinjection of excess unlabeled GDNF, indicating that it was receptor mediated. We tested whether intravitreally applied GDNF could prevent axotomy-induced RGC degeneration. The RGC were prelabeled with Fluorogold (FG) and axotomized by intraorbital optic nerve transection. GDNF, BDNF (positive control), cytochrome c (negative control), or a GDNF/BDNF combination was injected intravitreally on days 0 and 7. On day 14, FG-labeled RGC were counted from whole-mount retinas. We found that, similar to BDNF, GDNF could significantly attenuate the degeneration of RGC in a dose-dependent fashion. Furthermore, the combination treatment of GDNF and BDNF showed better protection than either factor used individually. Our data indicate that GDNF is a neurotrophic factor for the adult rat RGC. GDNF, like BDNF, may be useful for the treatment of human RGC degenerative diseases.     

Role of nerve growth factor in plasticity of forebrain cholinergic neurons

Neuronal plastic rearrangements during the development and functioning of neurons are largely regulated by trophic factors, including nerve growth factor (NGF). NGF is also involved in the pathogenesis of Alzheimer’s disease. In the brain, NGF is produced in structures innervated by basal forebrain cholinergic neurons and retrogradely transported along the axons to the bodies of cholinergic neurons. NGF is essential for normal development and functioning of the basal forebrain; it affects formation of the dendritic tree and modulates the activities of choline acetyltransferase and acetylcholinesterase in basal forebrain neurons. The trophic effect of NGF is mediated through its interactions with TrkA and p75 receptors. Experimental and clinical studies have shown that brain levels of NGF are altered in various pathologies. However, the therapeutic use of NGF is limited by its poor ability to penetrate the blood–brain barrier, adverse side effects that are due to the pleiotropic action of this factor, and the possibility of immune response to NGF. For this reason, the development of gene therapy methods for treating NGF deficit-associated pathologies is of particular interest. Another approach is creation of low molecular weight NGF mimetics that would interact with the corresponding receptors and display high biological activity but be free of the unfavorable effects of NGF.     

Effect of aging on the rate of axonal transport of choline-phosphoglycerides

The anterograde axonal transport of choline-phosphoglycerides was studied in sciatic nerve motoneurons of adult (3-month-old) and aged (24-month-old) rats. After the spinal cord injection of [2-³H]glycerol, choline-phosphoglycerides; the major phospholipid class was transported along the nerve. The axonal transport rate was determined by plotting the distance covered by the front of transported radioactivity as a function of the time employed. In aged animals the rate of the choline-phosphoglyceride anterograde axonal transport was about 68% lower than that of adults; furthermore, the rate slowed down along the nerve in the proximal-distal direction. This alterated axonal transport mechanism might contribute to the degenerative processes observed in distal regions of peripheral nerve fibers of aged animals.     

Evidence in support of signaling endosome-based retrograde survival of sympathetic neurons

The mechanism by which target-derived Nerve Growth Factor (NGF) signaling is propagated retrogradely, over extremely long distances, to cell bodies to support survival of neurons is unclear. Here we show that survival of sympathetic neurons supported by NGF on distal axons requires the kinase activity of the NGF receptor, TrkA, in both distal axons and cell bodies. In contrast, disruption of TrkA activity exclusively in proximal axonal segments affects neither retrograde NGF-TrkA signaling in cell bodies nor neuronal survival. Ligand-receptor internalization is necessary for survival of neurons supported by NGF on distal axons. Furthermore, antibody neutralization experiments indicate that retrogradely transported NGF, within cell bodies, is critical for neuronal survival but not for growth of distal axons. Taken together, our results indicate that retrogradely transported NGF-TrkA complexes promote sympathetic neuron survival.     

Retrograde axonal transport of specific macromolecules as a tool for characterizing nerve terminal membranes

The uptake of macromolecules by nerve terminals which is followed by retrograde axonal transport seems to occur by two different mechanisms, a specific and a nonspecific one. The nonspecific uptake depends on the presence of macromolecules (e.g., horseradish peroxidase) in the vicinity of the nerve terminals at very high concentrations and is enhanced by neuronal activity. In contrast, the specific uptake and subsequent retrograde axonal transport becomes apparent at much lower concentrations of the appropriate macromolecules, depends on the affinity of these ligands for specific binding sites on the surface of the neuronal membrane, and is independent of neuronal activity. The fact that lectins and some bacterial toxins bind to specific membrane glycoproteins or glycolipids allows conclusions to be drawn regarding qualitative and even quantitative aspects of the composition of the plasma membrane of the nerve terminals. ¹²⁵I-labelled nerve growth factor (NGF), tetanus toxin, cholera toxin, wheat germ agglutinin (WGA), ricin II, phytohemagglutinin (PHA), and concanavalin A (ConA) were injected into the anterior eye chamber of rats where they were taken up by adrenergic nerve terminals and transported retrogradely to the superior cervical ganglion. The saturation of the uptake-transport found for NGF, WGA, choleragenoid and an atoxic binding-fragment of tetanus toxin indicates that limited numbers of binding sites, which showed also different affinites, are present for each ligand on the membrane of the nerve terminals. Competition experiments showed that the binding sites for the ligands investigated are largely independent. Two different classes of binding sites (high affinity-low capacity and intermediate affinity-intermediate capacity) seem to be involved in the saturable retrograde axonal transport of NGF. In contrast, WGA seems to have only a single class of binding-uptake sites with high capacity and relatively low affinity. Strong evidence for positive cooperativity was obtained for the uptake and subsequent transport of the tetanus toxin fragment.     

The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.     

Retrograde transport of neurotrophins: fact and function

Retrograde signals generated by nerve growth factor (NGF) and other neurotrophins promote the survival of appropriately connected neurons during development, and failure to obtain sufficient retrograde signals may contribute to neuronal death occurring in many neurodegenerative diseases. The discovery over 25 years ago that NGF supplied to the axon terminals is retrogradely transported to the cell bodies suggested that NGF must reach the cell body to promote neuronal survival. Research during the intervening decades has produced a refinement of this hypothesis. The current hypothesis is that NGF bound to TrkA at the axon terminal is internalized into signaling endosomes, with NGF in their lumens bound to phosphorylated TrkA in their membranes, which are retrogradely transported to the cell bodies, where TrkA activates downstream signaling molecules that promote neuronal survival and regulate many aspects of neuronal gene expression. This model has been extrapolated to retrograde signaling by all neurotrophins. We consider the evidence for this model, focusing on results of experiments with neurons in compartmented cultures. Results to date indicate that while the transport of signaling endosomes containing NGF bound to TrkA may carry retrograde signals, retrograde survival signals can be carried by another mechanism that is activated by NGF at the axon terminal surface and travels to the cell body unaccompanied by the NGF that initiated it. It is hypothesized that multiple mechanisms of retrograde signaling exist and function under different circumstances. The newly discovered potential for redundancy in retrograde signaling mechanisms can complicate the interpretation of experimental results.