A central issue in developmental neurobiology is how synapses are established, maintained and modified. We report here that retrograde NGF signaling is both necessary and sufficient to support the formation of synapses between preganglionic and postganglionic sympathetic neurons of the SCG, and that TrkA endosomes, which are trafficked from their sites of formation on distal axons to within close proximity of sites of PSD assembly in dendrites, mediate this signal. Moreover, PSD assembly mediated by target-derived NGF is restricted by p75 suggesting that a balance between the activities of the TrkA and p75 signaling pathways determines the number of PSDs on postganglionic sympathetic neurons. Furthermore, we find that NGF control of PSD assembly is a prerequisite for the formation of pre-synaptic specializations. Thus, long-range, retrograde NGF–TrkA signaling promotes the formation of synapses between preganglionic and postganglionic sympathetic neurons through inhibition of p75 and the promotion of clustering of resident components of the post-synaptic density.
Our findings suggest a hierarchal regulation of axo-dendritic synapse establishment whereby a neurotrophic growth factor expressed in the target field of a neuron dictates the degree of connectivity with its presynaptic partner. This, in combination with recent studies of the neuromuscular junction (NMJ), supports a general model in which post-synaptic components exert control over the assembly of presynaptic components and synapse formation (Lin et al., 2001
; Yang et al., 2000
). In the case of the NMJ, post-synaptic specializations on muscle form prior to innervation by motor neurons. This 'pre-patterned' postsynaptic structure encodes an instructive signal for the appropriate location of pre-synaptic terminal differentiation (Kim and Burden, 2008
). Our data suggest a similar mechanism of control for neuron-neuron synapse establishment in the SCG. Moreover, there are mechanistic similarities between these systems, one being the requirement of a receptor tyrosine kinase in both systems; TrkA for PSD organization in sympathetic neurons (this study) and MuSK for PSD organization in the muscle (Dichiara et al., 1996
). It is interesting to note that the loss of PSD formation in MuSK
mutant mice is rescued by a chimeric protein in which the kinase domain of MuSK is replaced with that of TrkA (Herbst et al., 2002
Reminiscent of the period of developmental cell death when target-derived NGF defines the appropriate number of neurons that ultimately populate sympathetic ganglia, we propose that target-derived trophic factors additionally define the proper amount of synaptic connections with preysnaptic partners. How is the NGF signal propagated from distal axons to dendrites to control PSD formation and synapse development? Using TrkAFlag knockin mice and microfluidic chambers to monitor the localization of endogenous TrkA following its internalization in distal axons, we found that TrkA endosomes move retrogradely and localize not only to the cell body but, remarkably, throughout the entire dendritic arbor of sympathetic neurons. Moreover, specific inhibition of TrkA signaling within the cell body/dendrite compartment using a chemical-genetic strategy employing TrkAF592A mice prevented retrograde induction of PSDs. These observations serve to demonstrate the truly remarkable versatility of NGF–TrkA signaling. Indeed, in addition to NGF promoting local axonal growth as well as long-range retrograde transcriptional regulation and survival, it also controls synaptogenesis from a distance. Furthermore, the initial stages of PSD formation via retrograde NGF–TrkA signaling can occur, in large part, independent of its ability to support gene expression. Thus, a novel function of retrograde NGF–TrkA signaling is to promote the assembly of synaptic clusters from pre-existing PSD components.
Previous studies have implicated p75 in the modulation of hippocampal neuron spine number (Zagrebelsky et al., 2005
) and plasticity (Woo et al., 2005
). Moreover, antagonistic NGF–TrkA and p75 signaling pathways control neuronal survival, axonal growth and pruning (Bamji et al., 1998
; Deppmann et al., 2008
; Singh et al., 2008
). Our findings extend these observations and indicate that in sympathetic neurons retrograde NGF–TrkA promotes PSD assembly and synapse formation through inhibition of p75 signaling in cell bodies/dendrites. We propose that antagonistic NGF–TrkA “pro-synapse” and p75 “anti-synapse” pathways reach steady state levels of signaling to achieve synaptic homeostasis. We further suggest that if this “synaptic balance” is disrupted, re-engagement of competitive programs mediated by antagonistic pathways can occur. Such an imbalance could be triggered by a variety of physiological and pathological conditions, including changes in activity or size of the target field, NGF availability, or the levels of either NGF–TrkA or p75 signaling. A change in any one of these parameters could lead to reengagement of developmental synaptic assembly/disassembly programs and may serve to modify sympathetic neuron synapse number, strength, and location under normal physiological circumstances in the adult. In fact, NGF dependent changes in synaptic transmission in the SCG have been observed when communication between postganglionic sympathetic neurons and their target organs is severed via axotomy in adult animals (Purves, 1975
; Purves, 1976
). Of course, reengagement of such developmental programs may also occur in response to insult or injury as is observed when a muscle fiber is denervated and a neighboring motor unit assumes control (Ramon y Cajal, 1928
; Sanes et al., 1978
). However, if neurons cannot reestablish a balance between antagonistic synaptogenic signaling pathways, an entire population of neurons may be exposed to onset and perhaps spread of synaptic dysfunction leading to neural disease.
A growing body of evidence suggests that this may indeed be the case for Alzheimer’s disease (Morfini et al., 2009
). One recent and particularly relevant study shows that an N-terminal region of amyloid precursor protein (APP) binds directly to a p75 family member, death receptor 6 (DR6), which in turn promotes axonal degeneration in an autocrine/paracrine fashion (Nikolaev et al., 2009
). Similar to the role of p75 in survival (Deppmann et al., 2008
) and axonal pruning (Singh et al., 2008
), and as shown here in synaptic disassembly, DR6 promotes axonal degeneration in the absence of NGF–TrkA signaling, whereas in the presence of NGF–TrkA signaling, DR6 signaling is suppressed. The striking similarities between p75 and DR6 structure, function, and dominant suppression by NGF–TrkA signaling warrant further attention to all members of this family of receptors in the control of synaptic balance and perhaps synaptic dysfunction as well. Indeed, our findings suggest a model in which neurons that fail to acquire target-derived trophic cues, or for any reason undergo deficits in trophic factor signaling, will fail to inhibit endogenous PSD disassembly programs involving p75 family members. Such an alteration in favor of p75 family member signaling, and the positive feedback mechanisms that ensue (Deppmann et al., 2008
), may place neurons into a state of synaptic disassembly rendering them susceptible to neurodegeneration. Future work addressing the mechanisms by which p75 mediates synaptic disassembly, the sites of action for additional p75 ligands, perhaps including the N-terminal portion of APP, and the pathways by which TrkA suppresses p75 signaling will provide insights into the assembly of synapses during normal neural development as well as possibly lending fundamental insight into the onset and progression of neurodegenerative disease.
In summary, target-derived NGF supports the formation of synapses between postganglionic sympathetic neurons and their presynaptic partners. The establishment of PSDs by target-derived NGF can occur, in large part, independent of new protein synthesis, is restricted by p75, and is mediated by retrogradely transported TrkA signaling endosomes. Remarkably, these endosomes are trafficked from distal axons to cell bodies and then into dendrites where they move into close proximity of nascent PSDs. We propose that neuronal innervation of peripheral target tissues and the acquisition of target-derived NGF promotes “systems matching” not only through the support of neuronal survival during the period of developmental cell death, but also through the formation of the proper number of synapses with presynaptic partners. In this manner, NGF orchestrates the flow of neural information from the CNS to the periphery through its control of both the size of the sympathetic neuron population and the synaptic organization of sympathetic circuits.