Abstract
Application of nerve growth factor (NGF) covalently cross-linked to beads increased the phosphorylation of TrkA and Akt, but not of mitogen-activated protein kinase, in cultured rat sympathetic neurons. NGF beads or iodine-125–labeled NGF beads supplied to distal axons resulted in the survival of over 80% of the neurons for 30 hours, with little or no retrograde transport of iodine-125–labeled NGF; whereas application of free iodine-125–labeled NGF (0.5 nanograms per milliliter) produced 20-fold more retrograde transport, but only 29% of the neurons survived. Thus, in contrast to widely accepted theory, a neuronal survival signal can reach the cell bodies unaccompanied by the NGF that initiated it.
The literatures of neuronal development, neurotrauma, degenerative neurological disease, and neuronal regeneration are pervaded by the concept that the survival and function of neurons depend on retrograde transport of neurotrophic factors released from the target cells that they innervate. This idea began with the discovery that nerve growth factor (NGF) is retrogradely transported from axon terminals to neuronal cell bodies (1–7). The current theory, that NGF complexed with its receptor, TrkA, is endocytosed, trafficked to signaling endosomes (8, 9), and retrogradely transported to the cell bodies, has been supported by results of studies with compartmented cultures (10–14). However, Senger and Campenot (15) observed retrograde phosphorylation of TrkA and other proteins occurring 1 to 15 min after NGF application, preceding the arrival of detectable 125I-NGF by at least 30 min.
We examined this issue using NGF covalently cross-linked to beads to prevent internalization (16). Mass cultures of rat sympathetic neurons (17) that had been deprived of NGF (18) displayed a similar level of phosphorylation of TrkA (19) after 1 hour of application of free NGF (50 ng/ml) or NGF beads (50 μl/ml) (Fig. 1A). NGF and NGF beads also induced the phosphorylation of Akt (Fig. 1B), the latter suggesting that phosphorylated TrkA (pTrkA) at the plasma membrane can activate phosphatidylinositol 3-kinase (PI 3-kinase). In contrast, little mitogen-activated protein kinase (MAPK) phosphorylation was observed in response to NGF beads (Fig. 1B), suggesting that internalization of pTrkA is necessary for the activation of MAPK. This result is consistent with reports that the blockage of endocytosis of TrkA, by means of pharmacological inhibitors or dominant-negative dynamin, inhibits NGF-induced MAPK phosphorylation in dorsal root ganglion neurons (20) and PC12 cells (20–22). Thus, the lack of NGF-induced MAPK phosphorylation that we observed suggests that neither NGF nor TrkA is internalized when NGF is presented in bead-linked form.
Phosphorylation of TrkA, Akt, and MAPK in response to NGF and NGF beads. Mass cultures (17) were deprived of NGF (18) and incubated for 1 hour with medium lacking NGF, medium containing free NGF, or NGF beads at the concentrations indicated. Cell extracts were analyzed by immunoblot (19). All lanes are from the same PVDF membrane. Blots are representative of three experiments with similar results. (A) Proteins >100 kD were probed with antibody to phosphotyrosine (pTyr), stripped, reprobed with antibody to phosphoTrkA Y490 (pTrkA), stripped, and reprobed with antibody to TrkA (TrkA). (B) Proteins <100 kD were probed with antibody to phosphoAkt (pAkt), reprobed with antibody to phosphoMAPK (pMAPK), stripped, and reprobed with antibody to p44 MAPK (MAPK).
To determine whether NGF beads can produce retrograde signals, distal axons of sympathetic neurons in NGF-deprived compartmented cultures (17, 18) received different NGF treatments for 30 hours, and neuronal survival was assayed (23). Treatment with NGF beads resulted in 81% neuronal survival (Fig. 2, A and B), which approaches the survival in cultures given free NGF (50 ng/ml) (95%) and is almost four times the survival of cultures given no NGF (22%). Control beads to which NGF was not covalently cross-linked did not support survival (Fig. 2B). Retrograde survival could conceivably have been achieved by the release of NGF from the beads into the culture medium, followed by internalization and retrograde transport. However, supernatant media from NGF beads that had supported 30 hours of retrograde survival of one set of cultures (Fig. 2C, solid bars) failed to support survival of a second set of cultures (Fig. 2C, hatched bars), whereas free NGF from the first set again supported the survival of a second set. Supernatant media from NGF beads that were preincubated for 30 hours without neurons present also failed to support neuronal survival (Fig. 2C, open bars), ruling out the possibility that NGF released from the beads may have been depleted from the medium by retrograde transport.
NGF beads support retrograde survival of sympathetic neurons. Compartmented cultures were deprived of NGF (18), and distal axons (DAx) were given the indicated treatments for 30 hours while cell bodies/proximal axons (CB/PAx) were exposed to antibody to NGF. Then the nuclei were stained with Hoechst DNA stain to assess neuronal survival (23). (A) Confocal micrographs of representative neurons treated with no NGF, free NGF (50 ng/ml), and NGF beads (50 μl/ml). (B) A minimum of 1000 neurons were categorized per treatment group as surviving (diffusely labeled DNA) or dying/dead (condensed fragmented DNA or unlabeled). The percentage of live neurons in each treatment group (±SEM, n = 3 cultures) is plotted. The two bead control groups were given NGF beads (50 μl/ml) prepared without EDAC cross-linking or beads (50 μl/ml) cross-linked without NGF present (16). Results are representative of three experiments. (C) Stage 1: Distal axons of NGF-deprived cultures were treated for 30 hours with no NGF, free NGF, or NGF beads as in (B), and neuronal survival was determined (black bars). Concurrently, aliquots of these media were incubated in dishes under culturing conditions, but without neurons present. Stage 2: media from the distal axons of all groups were collected at the end of stage 1, beads were removed from the NGF-bead medium by centrifugation, and these media were used to treat distal axons of NGF-deprived neurons for 30 hours in a second set of cultures (gray bars). The supernatants from aliquots incubated without neurons during stage 1 were also tested (white bars). Data are combined from two experiments (n= 6 cultures ± SEM).
To directly assess possible retrograde transport of NGF from beads, we compared the retrograde transport of 125I originating from a range of concentrations of free 125I-NGF with transport from bead-linked 125I-NGF (50 μl/ml) (24). Iodination did not affect the ability of free NGF or bead-linked NGF to induce TrkA phosphorylation (Fig. 3A). Because 90% of the 125I transported to the cell bodies over 30 hours had been released into the center compartment medium (7, 15), we assessed retrograde transport by gamma counts of the center compartment medium and assayed the survival of the same neurons by Hoechst staining (23). Provision of 125I-NGF beads to distal axons supported the survival, on average, of 84% of the neurons, whereas retrograde transport by these neurons was barely detected at 0.57 pg of NGF per culture (Fig. 3B). Cultures given 50 ng/ml of free125I-NGF displayed 96% survival associated with transport of over 1000-fold more NGF (657 pg per culture), and even cultures given 0.5 ng/ml of free 125I-NGF, which displayed only 29% survival, transported over 20-fold more NGF (12 pg per culture). The high-performance liquid chromatography (HPLC)–purified125I-NGF was more than 97% pure iodinated species (24). Therefore, the possibility that unlabeled NGF was present, selectively released from the beads, and retrogradely transported, resulting in neuronal survival, can be ruled out. Thus, by providing NGF in bead-linked form, we have supported neuronal survival while virtually eliminating the retrograde transport of NGF observed when survival is supported by free NGF.
Relationship between NGF retrograde transport and neuronal survival for free NGF and NGF beads. (A) The ability of 125I-NGF beads (50 μl/ml),125I-NGF (50 ng/ml), and unlabeled NGF (50 ng/ml) to induce tyrosine phosphorylation of TrkA in NGF-deprived mass cultures was tested as described in Fig. 1. (B) NGF-deprived neurons in compartmented cultures (18) were provided with the indicated concentrations of free 125I-NGF or125I-NGF beads (50 μl/ml) applied to distal axons (DAx) for 30 hours. Then medium bathing the cell bodies/proximal axons was collected, and transported radioactivity was determined (24). Neuronal survival was determined in the same cultures by examination of Hoechst-stained nuclei of at least 250 neurons per culture (23). The percent survival is plotted against transported 125I-NGF for each individual culture (three or four per group) from three experiments.
Because NGF beads activate Akt, presumably via PI 3-kinase, we investigated whether blockage of PI 3-kinase activity with 50 μM LY294002 (LY) (25) could block retrograde survival signaling from NGF and NGF beads. In cultures given NGF in all compartments, LY applied to distal axons blocked NGF-induced Akt phosphorylation in the distal axons without effect on Akt phosphorylation in the cell bodies/proximal axons and vice versa as previously shown (14), indicating that LY can be used to locally block PI 3-kinase. NGF-deprived cultures displayed 40% survival, whereas provision of free NGF to distal axons increased survival to 99%, giving a free NGF–induced survival component of 59% of the population of neurons (Fig. 4). When LY was applied to cell bodies/proximal axons, the NGF-induced survival component was only 6%; but when LY was applied to distal axons, little inhibition was observed, with 47% NGF-induced survival. These results are consistent with a previous report (14). Provision of NGF beads to distal axons resulted in total survival of 79% of the neurons, giving an NGF bead–induced survival component of 39%. Application of LY to cell bodies/proximal axons reduced this to 8%, similar to the inhibition of free NGF–induced survival. However, application of LY to the distal axons inhibited the survival induced by NGF beads by about half, to 20% (paired sample t test, P < 0.002). This suggests that PI 3-kinase activity in the distal axons may be more important in generating retrograde signals from NGF beads, possibly because this pathway can be activated without internalization, whereas other survival pathways may require internalization.
Effects of LY294002 (LY) on retrograde survival supported by NGF and NGF beads. Compartmented cultures were deprived of NGF (18), and distal axons were given no NGF, NGF (50 ng/ml), or NGF beads (50 μl/ml) for 30 hours. Concurrently, the cell bodies/proximal axons (CB/PAx) or distal axons (DAx) were given 50 μM LY as indicated. Then the neuronal survival of at least 250 neurons per culture was assayed as in Fig. 2 (23). The percentage of live neurons in two replicate experiments is plotted (±SEM,n = 6 cultures). LY from Sigma was made as a 50 mM stock in dimethylsulfoxide. All compartments not given LY received equivalent dimethylsulfoxide, which was 0.1% of culture medium.
What possible mechanisms of retrograde signaling are consistent with our results? Because 50% of sympathetic neurons commit to apoptosis after 18 hours of NGF deprivation (26), and the survival signal must travel at least 1.5 mm to reach the cell bodies, diffusion of proteins 15 kD or larger is ruled out (27). Another possibility is that phosphorylation of TrkA bound to NGF beads propagated to neighboring unbound TrkA at the plasma membrane, as observed for EGF activation of ErbB1 receptors (28). Serial TrkA phosphorylation could conceivably propagate along the proximal axons to the cell bodies, as suggested by Senger and Campenot (15). Also, NGF beads could induce the internalization and retrograde transport of pTrkA in the absence of the NGF that initiated it, although it is unclear how TrkA would remain phosphorylated. Transport of activated downstream signaling molecules (29) and ionic propagation mechanisms involving release of calcium into the cytosol (15) have also been suggested. Our results raise the possibility that retrograde transport of NGF may not be required for any mechanism of retrograde signaling, but other studies present evidence that neurotrophin transport is required (10–14). Although we find this evidence inconclusive (30), different mechanisms of retrograde signaling are not mutually exclusive. Perhaps propagated signals operate early in neuronal development and/or during axonal regeneration after injury. At these times, axon terminals consist of a few growth cones, and the neurotrophic factors they encounter may have to produce amplified signals to be effective. There are many speculative scenarios, but clearly the assumption of over 25 years that retrograde transport of NGF (and all other neurotrophic factors) is the only way that retrograde signals can reach the cell bodies needs continued reexamination.
↵* To whom correspondence should be addressed. E-mail: bob.campenot{at}ualberta.ca
