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We report a novel role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of post-synaptic specializations on dendrites of sympathetic neurons. This, in turn, is prerequisite for formation of pre-synaptic specializations but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF–TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF–TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.
The nervous system relies on an enormous number of synaptic connections between neurons to propagate, integrate and store information. Thus, a major challenge during neural development is the establishment of the correct number, location and type of synaptic connections between neurons. Functional excitatory synapses require precise apposition of pre-synaptic vesicle release zones and specialized receptive zones on post-synaptic neurons referred to as the post-synaptic density (PSD). PSDs are composed of a dynamic combination of neurotransmitter receptors, scaffolding proteins, and effectors that serve to control the composition, structure, and function of the synapse. The number and activity of neurotransmitter receptors within PSDs can be modulated by signals originating from the pre- and/or postsynaptic neuron (Sheng and Hoogenraad, 2007). While much is known about synaptic modifications and plasticity, the signals governing the initial establishment of pre-synaptic vesicle release zones, PSDs, and synaptic connections between neurons are not well characterized.
Synapses between cholinergic axons of preganglionic sympathetic neurons emanating from the intermediolateral nucleus of the spinal cord and postganglionic sympathetic neurons within the superior cervical ganglia (SCG; Figure 1A) represent a tractable model to study the molecular and cellular events controlling synapse assembly (Forehand, 1985; Rubin, 1985). Postganglionic sympathetic neurons send primarily noradrenergic axons to target organs such as salivary glands and the heart to control a wide variety of exocrine, endocrine, cardiovascular, and other homeostatic functions (Glebova and Ginty, 2005). During the period of developmental cell death of sympathetic neurons (E17-P0), synapses between the terminals of preganglionic sympathetic neurons and the cell bodies/proximal dendrites of postganglionic sympathetic neurons begin to form. After this period, synapses are found almost exclusively on dendrites of postganglionic sympathetic neurons (Forehand, 1985; Rubin, 1985). The mechanisms controlling assembly, distribution, plasticity, and maintenance of these synapses are largely unknown.
Neurotrophins represent attractive candidates to influence neural connectivity in the peripheral nervous system (PNS), including the sympathetic nervous system. Work initiated decades ago has shown that these growth factors are synthesized and released by target cells in the periphery and serve as key regulators of growth, maturation, and survival of distinct populations of PNS neurons. Indeed, the archetypal neurotrophin, NGF, which is produced by target-organs innervated by sympathetic neurons (Glebova and Ginty, 2004), controls both axonal extension into the target fields and survival. Neurotrophins are also implicated in synaptic maintenance, proliferation, and competition (Poo, 2001; Snider and Lichtman, 1996). Whether neurotrophins control synapse formation during the assembly of neural circuits is not known.
Here we demonstrate that target-derived NGF regulates synapse formation between preganglionic and postganglionic sympathetic neurons. We found that NGF is both necessary and sufficient for clustering of pre-existing components of the PSD on postganglionic neurons. This is directly mediated by trafficking of the NGF–TrkA signaling endosome from distal axons to within close proximity of synaptic assembly sites in dendrites. Finally, we find that in order for long distance NGF–TrkA signaling to induce PSD formation, it must inhibit p75 signaling. Thus, neuronal innervation of target tissues and the acquisition of target-derived neurotrophic factors initiate the formation of synapses with presynaptic partners through retrograde endosomal signaling.
The roles of NGF in postganglionic sympathetic neuron survival and target innervation have been studied extensively (Cosker et al., 2008; Howe and Mobley, 2005; Markus et al., 2002). To determine whether NGF also regulates the formation of synapses between preganglionic and postganglionic sympathetic neurons in vivo we assessed the number of these synapses using markers of post-synaptic specializations in superior cervical ganglia (SCGs) of P0 NGF−/−;Bax−/− mice. These experiments were performed in a Bax−/− genetic background in order to circumvent the massive cell death normally associated with loss of NGF (Deckwerth et al., 1996; Glebova and Ginty, 2004; Patel et al., 2000). We chose to analyze the P0 time point, which captures the initial stages of synapse formation (Forehand, 1985; Rubin, 1985), because double null animals do not survive to later ages. Remarkably, compared to Bax−/− controls, the SCGs of NGF−/−;Bax−/− animals display a near complete absence of post-synaptic specializations as shown by immunohistochemical analysis of Membrane-Associated Guanylate Kinases (pan-MAGUK) and Shank, prominent components of the PSD (Figure 1B,C and Figure S1).
Because of the dramatic disruption of post-synaptic specializations in SCGs of mice lacking NGF, we next sought to assess the integrity of pre-synaptic specializations using immunohistochemistry for synaptophysin, a synaptic vesicle-associated protein. Interestingly, in the absence of NGF, we observed a near complete loss of presynaptic specializations (Figure 1B,C). Since preganglionic neurons do not express TrkA and therefore do not respond to NGF (Oppenheim et al., 1982), we reason that this effect is not a direct result of NGF ablation, but rather it is an indirect effect caused by changes to postganglionic neurons. One possibility is that NGF-induced PSD formation is a prerequisite for the establishment of presynaptic specializations. Another plausible scenario is that NGF signaling in postganglionic neurons is a prerequisite for the growth of preganglionic axons into the SCG. This latter scenario seems unlikely, however, since we observed no difference in the density of preganglionic sympathetic axons in SCGs from P0 Bax−/− and NGF−/−;Bax−/− mice as visualized by neurofilament-200 (NF-200) immunohistochemistry (Figure 1D,E). Therefore, while NGF is dispensable for preganglionic axon growth into the SCG, it is absolutely essential for the formation of synapses between preganglionic and postganglionic sympathetic neurons in vivo.
We next asked whether NGF is sufficient to directly promote PSD formation in sympathetic neurons. Toward this end, long-term cultures of postganglionic sympathetic neurons (12–18 DIV) grown in the presence of NGF were established. These cultured sympathetic neurons develop both dendrites and post-synaptic specializations, as visualized by immunostaining for MAP2 and a variety of post-synaptic scaffolding proteins, respectively (Figure 2A, D). The majority of MAGUK clusters were not colocalized with the pre-synaptic marker synapsin in these cultures (Figure S2A–C). Using this paradigm, we next examined post-synaptic specializations in neurons following NGF deprivation for 12 or 48 hours. Reminiscent of what was observed in vivo, at both deprivation time points there was an approximately 80% reduction in the number of PSDs as measured by immunostaining for MAGUK (Figure 2A, B). Twelve hours of NGF withdrawal also led to a dramatic loss of clusters containing Shank, Guanylate Kinase-Associated Protein (GKAP), and nAChR proteins (Figure 2D,E). Importantly, we did not observe a change in dendrite length 12 hours after NGF withdrawal while at 48 hours post-withdrawal, dendrites appear shorter (Figure 2C). This indicates that the dramatic loss of MAGUK+ clusters observed following NGF removal is not due to changes in dendrite morphology. Furthermore, treatment of 12-hour-NGF-deprived neurons with NGF for two hours led to reemergence of PSDs (Figure 2F,G). Interestingly, we did not observe differences in the number of synapsin+ clusters under similar NGF withdrawal/treatment conditions (Figure S2A–B). Thus, NGF can act directly on sympathetic neurons in vitro to promote PSD formation and maintenance.
In vivo, NGF is thought to act on the distal axons of sympathetic neurons. Therefore, the absence of PSDs on sympathetic neurons of the SCG in NGF−/−;Bax−/− mice is likely caused by a lack of retrograde NGF–TrkA signaling. To directly ask whether NGF acting on distal axons and subsequent retrograde NGF–TrkA signaling can support PSD formation on dendrites, we turned to an in vitro compartmentalized microfluidic culture system, which allows for differential treatment of distal axons (DA) versus cell bodies/dendrites (CB/D) (Park et al., 2006) (Figure S3). Within two days of plating, cultured sympathetic neurons grown in microfluidic devices extend axons through small channels between the cell body/dendrite/proximal axon and distal axon compartments (Figure S3B). These long (300 micron), narrow (10 micron) channels and a volume differential between the cell body and distal axon compartments ensure unidirectional diffusion resulting in fluidic isolation, thereby allowing for exclusive treatment of either cell bodies/dendrites/proximal axons or distal axons. We first asked if NGF applied to distal axons is capable of supporting PSD formation on dendrites in a retrograde manner. Indeed, we observed MAGUK+ clusters on dendrites of neurons grown in the presence of NGF exclusively in the distal axon compartment. While the number of MAGUK+ clusters present on these dendrites was comparable to neurons grown in mass cultures, few of these MAGUK+ clusters were associated with synapsin+ clusters (Figure S2D–F). Moreover, similar to what was observed in mass cultures, the MAGUK+ PSDs were rapidly lost following withdrawal of NGF from distal axons (Figure 2H). In addition, re-addition of NGF exclusively to distal axons for 6 hours led to reemergence of dendritic PSDs (Figure 2H,I). Taken together with the in vivo observations, we conclude that long-range, retrograde NGF–TrkA signaling is both necessary and sufficient for the assembly of post-synaptic specializations on dendrites of postganglionic sympathetic neurons.
Retrograde NGF–TrkA signaling may control PSD formation through de novo synthesis of PSD components, through the clustering of pre-existing components, or both. To test the possibility that NGF regulates the level of expression of one or more PSD component(s), immunoblot analyses were performed using extracts from cultures that had been deprived of NGF for 12 or 48 hours. While 12 hours of NGF deprivation led to near complete loss of PSDs (Figure 2A–D), neither 12 nor 48 hour NGF deprivation led to a loss of PSD-93 protein (Figure 3A,B), which is the principal MAGUK family member expressed in postganglionic sympathetic neurons (Parker et al., 2004). Furthermore, we did not observe a decrease in the levels of Shank or GKAP proteins following 12 hours of NGF deprivation (Figure S4A). Therefore, the dramatic loss of PSDs following NGF deprivation cannot be explained by the regulation of expression or stability of these major PSD components.
The PSD is a structure containing a large number of proteins (Sheng and Hoogenraad, 2007) and therefore our findings showing stable levels of PSD-93, Shank and GKAP proteins do not exclude the possibility that NGF regulates synthesis or stability of other unidentified factor(s) that may be required for PSD clustering. An alternative possibility is that NGF controls clustering of resident, pre-existing synaptic proteins without regulating de novo synthesis of PSD components. To distinguish between these possibilities, PSDs were dispersed by depriving sympathetic neurons of NGF for 12 hours, and NGF was then reapplied to these neurons in the presence or absence of the translation inhibitor cycloheximide (CHX) to prevent de novo synthesis of proteins. The efficacy of CHX was confirmed in experiments that showed a complete loss of detectable levels of the labile protein c-Fos (Figure S4B). We observed robust reappearance of MAGUK-containing clusters at two and six hours following reapplication of NGF to CHX-treated neurons, albeit at a slightly reduced level (Figure 3C,D). We next asked whether this effect is recapitulated with application of NGF only to distal axons. Similar to mass culture experiments, neurons grown in microfluidic chambers were subjected to NGF deprivation for 12 hours to disperse synaptic clusters. Then, NGF was reapplied exclusively to distal axons for 6 hours in the presence or absence of CHX in both cell body/dendrites and distal axon compartments. Consistent with mass culture experiments, retrograde NGF signaling promoted PSD clustering on dendrites incubated with CHX, again at a slightly reduced level compared to control, untreated cultures (Figure 3E,F). Taken together, these findings show that, while there is a contribution of protein synthesis for PSD formation, retrograde NGF signaling initiates PSD assembly in dendrites in large part through clustering of pre-existing resident PSD components.
A major mechanism by which NGF mediates long distance signaling is through the formation and retrograde transport of NGF–TrkA containing signaling endosomes, which upon arrival in the cell body can support signaling events that promote gene expression changes and survival (Cosker et al., 2008; Howe and Mobley, 2005; Zweifel et al., 2005). Therefore, we next sought to determine where in relation to the sites of PSD formation the TrkA signaling endosomes reside. Localization of TrkA endosomes has been historically difficult to monitor using standard immunohistochemical techniques because of the abundance of newly synthesized, intracellular pools of immature TrkA and mature cell-surface TrkA found all along the plasma membrane of the neuron. Therefore, we generated a Flag-TrkA knock-in (TrkAFlag) mouse and an anti-Flag antibody-feeding assay that enables visualization of trafficking of endogenous, postendocytic TrkA in sympathetic neurons, as previously described for transfected receptors (Chen et al., 2005b; Tanowitz and von Zastrow, 2003; Vargas and von Zastrow, 2004) (Figure S5). The TrkAFlag mouse was made by inserting the coding determinants of the Flag epitope in frame with the first coding exon of TrkA by homologous recombination in mouse ES cells, which were then used to generate mutant mice harboring the TrkAFlag allele. Homozygous TrkAFlag mice are viable and fertile, and Flag-TrkA is expressed normally in these animals and functions to support NGF-dependent growth and survival of sympathetic neurons (data not shown). Importantly, neurons harvested from TrkAFlag mice, used in conjunction with microfluidic devices and the anti-Flag antibody-feeding assay, allows clear visualization of the subcellular distribution of TrkA endosomes (Figure S5C–F). Indeed, application of NGF to distal axons of compartmentalized TrkAFlag neurons that had been pre-treated with anti-Flag on distal axons led to the formation and retrograde transport of Flag-TrkA endosomes and their accumulation in cell bodies. Importantly, while a large number of Flag-TrkA endosomes were present in cell bodies of NGF-treated neurons, few if any Flag-TrkA endosomes were observed in cell bodies of neurons not exposed to NGF (Figure 4A,B). Moreover, the Flag antibody specifically recognizes Flag-TrkA in these neurons since anti-Flag binding to wild-type neurons is undetectable (data not shown). Using this assay, we asked whether TrkA endosomes move from distal axons into cell bodies and then into dendrites in proximity to newly formed PSD clusters. Strikingly, we observed that treatment of distal axons with NGF led to accumulation of Flag-TrkA endosomes within dendrites (Figure 4A,B), including distal regions of primary dendrites as well as secondary and tertiary branches (data not shown). Furthermore, time course experiments showed that the accumulation of Flag-TrkA endosomes in dendrites is temporally coincident with the formation of PSD clusters. (Figure 4C,D). Thus, TrkA endosomes are formed in distal axons and retrogradely trafficked in dendrites to within close proximity of sites of PSD formation.
The finding that NGF-TrkA signaling endosomes accumulate in cell bodies and dendrites coincident with the appearance of PSDs suggests that TrkA endosomes are carriers of retrograde NGF signals to mediate PSD formation. To test this idea and address the role of TrkA signaling in cell bodies and dendrites, we used to a chemical-genetic strategy employing neurons from TrkAF592A mice grown in microfluidic chambers. TrkAF592A mice harbor a single codon substitution introduced into the exon encoding the protein kinase subdomain V of TrkA; This phenylalanine-to-alanine substitution in endogenous TrkA renders its catalytic activity sensitive to specific inhibition by the membrane-permeable, small molecule PP1 analog, 1NMPP1 (Chen et al., 2005a). 1NMPP1 blocks the tyrosine kinase activity of TrkAF592A at concentrations that have no inhibitory effect on the activity of wild-type TrkA. Thus, 1NMPP1 blocks NGF–TrkA signaling in sympathetic neurons harvested from TrkAF592A mice but not in neurons from wild-type mice. Pharmacological specificity of the approach is determined through the use of 1NMPP1 treatment of wild-type neurons (Chen et al., 2005a). For these experiments, sympathetic neurons harvested from either TrkAF592A mice or wild-type mice were grown in microfluidic chambers for 12 days. Subsequently, neurons were deprived of NGF for 12 hours to disperse PSDs, the cell bodies/dendrite compartments were pretreated with either vehicle control or 1NMPP1 (0.5µM), and then distal axons were treated with NGF for three hours. We found that addition of 1NMPP1 to cell bodies and dendrites of neurons from TrkAF592A mice resulted in a dramatic reduction in the number of PSDs induced by NGF stimulation of distal axons (Figure 5A). The effect of 1NMPP1 was due to specific inhibition of TrkAF592A because 1NMPP1 treatment of wild-type neurons was without effect. Moreover, the 1NMPP1 effects are compartmentalized in the microfluidic chambers since NGF addition directly to the cell bodies and dendrites of TrkAF592A neurons that had been pretreated with 1NMPP1 on their distal axons was without effect. Thus, retrograde NGF control of PSD formation requires TrkA signaling in distal axons (to initiate the signal) and also in the cell body/dendrite compartment. Taken together with the abovementioned Flag-TrkA endosome transport measurements, these findings support a model in which TrkA signaling endosomes are the mediators of retrograde NGF signals that control PSD assembly.
We next sought to determine which signaling pathways downstream of NGF–TrkA mediate PSD formation. Toward this end, we asked whether pharmacological inhibition of either of two well-established downstream effectors of NGF signaling, MEK/MAPK and PI3K, could prevent PSD clustering induced by NGF. Interestingly, inhibition of either pathway eliminated the appearance of PSDs following NGF treatment of mass cultures of sympathetic neurons, suggesting that TrkA signaling requires both the MAPK and PI3K pathways for PSD assembly (Figure S6A,B). To determine the subcellular site of action of the MAPK and PI3K signaling pathways, we again turned to the compartmentalized microfluidic culture system. Neurons were deprived of NGF for 12 hours to disperse PSDs and then distal axons were treated with NGF for 6 hours. These neurons were pretreated with or without MEK/MAPK or PI3K inhibitors present in either the cell body/dendrite/ or distal axon compartments. When the MEK/MAPK inhibitor was applied exclusively to the cell body/dendrite compartment, NGF acting on distal axons failed to induce PSD clustering. In contrast, the PI3K inhibitor added exclusively to the cell body/dendrite compartment was without effect on retrogradely induced PSD clustering (Figure 5C,D). The PI3K inhibitor did, however, prevent the formation of PSD clusters when applied to distal axons. Thus, two main TrkA effector pathways are required in distinct subcellular compartments for proper retrograde control of PSD assembly. While MEK/MAPK signaling is required in cell bodies for PSD formation, the PI3K effector pathway is required in distal axons, presumably reflecting its role the initiation of retrograde TrkA signaling, consistent with previous observations (Kuruvilla et al., 2000). Taken together, these findings support a model in which NGF acting on distal axons exerts control over PSD formation in dendrites through retrograde NGF-TrkA endosomal signaling platforms, which transport to within close proximity of sites of PSD formation.
Several studies have demonstrated that NGF–TrkA signaling and p75 signaling are functionally antagonistic with respect to sympathetic neuron survival, axonal growth, and axonal pruning (Bamji et al., 1998; Deppmann et al., 2008; Singh et al., 2008). Additionally, p75 is implicated in the control of spine development in hippocampal neurons (Zagrebelsky et al., 2005) as well as synaptic efficacy at hippocampal synapses of adult mice (Woo et al., 2005). Therefore, we next asked whether an interplay between the NGF–TrkA and p75 signaling pathways modulates synapse formation in the SCG. To test this idea, we examined post-synaptic specializations at the peak of preganglionic-postganglionic sympathetic neuron synapse establishment, P10, in p75−/− and wild-type littermate control animals. At this age, p75−/− mice displayed a dramatic increase in the number of post-synaptic specializations compared to wild-type controls (Figure 6A,B). Concurrent with an increase in post-synaptic specializations, we also observed an increase in pre-synaptic specializations in p75−/− animals (Figure 6A,B). Therefore, as with other developmental processes, TrkA and p75 may be functionally antagonistic with respect to synapse establishment in vivo. Furthermore, these findings suggest that the role of p75 in the process of synapse establishment is to repress the formation of post-synaptic specializations.
To further examine the interplay between NGF–TrkA and p75 signaling pathways, we used sympathetic neuron cultures derived from neonatal wild-type or p75−/− mice. Consistent with our in vivo analysis, p75−/− neurons display a substantially greater number of PSDs compared to wild-type controls (Figure 6C,D). We next examined the number of PSD clusters as a function of NGF concentration in the culture media. We found that at all concentrations of NGF tested, p75−/− neurons displayed a marked increase in the number of PSDs (Figure 6C,D). Even in the absence of NGF–TrkA signaling, a condition in which wild-type neurons are devoid of PSD clusters, neurons from p75−/− mice exhibited a large number of PSD clusters compared to wild-type controls. These findings suggest that p75 signaling actively restricts NGF–TrkA dependent PSD formation. Finally, we sought to identify the cellular compartment in which p75 signaling interferes with NGF–TrkA signaling to control PSD assembly. Sympathetic neurons express BDNF which can result in paracine and autocrine signals through p75 in cultured sympathetic neurons to affect axon growth and survival (Bamji et al., 1998; Deppmann et al., 2008; Yoon et al., 1998). We neutralized endogenous BDNF and thus blocked BDNF–p75 signaling with a function-blocking BDNF antibody, added to either the cell body/dendrite or distal axon compartments of sympathetic neurons grown in microfluidic chambers and maintained with NGF acting exclusively on distal axons. Addition of anti-BDNF to the cell bodies and dendrite compartment led to a significant increase in the number of PSDs. In contrast, addition of anti-BDNF to distal axons was without effect (Figure 6E,F). Taken together with previous findings that show NGF–TrkA signaling suppresses p75 signaling in sympathetic neurons (Bamji et al., 1998; Deppmann et al., 2008; Yoon et al., 1998), our findings support a model in which NGF promotes PSD formation and synapse assembly through retrograde TrkA endosome signaling and the suppression of p75 signals emanating from cell bodies and dendrites.
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.
p75−/− mice (deletion of exon III) (Lee et al., 1992) were generated as previous described (Kuruvilla et al., 2004). NGF+/− and Bax+/− (Crowley et al., 1994; Knudson et al., 1995) mice were maintained on the C57BL/6 background. The genotyping of NGF−/−;Bax−/− double knockout has been previous described (Glebova and Ginty, 2004).
Essentially, neurons were cultured as described previously (Ye et al., 2003). In brief, neurons were obtained by enzymatic dissociation of P0–P4 rat or mouse superior cervical ganglia (SCG). These neurons were plated in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (1U/ml) and 40ng/ml NGF purified from mouse salivary glands. After 24–48 hours, 5uM Ara-C was added to the culture media for 48–96 hours to eliminate glial contamination. This resulted in long term cultures that remained essentially glia free for the duration of all experiments. Media was changed every 48 hours and all experiments commenced at 16–18 DIV for mass cultures and 10–12 DIV for microfluidic chambers (unless otherwise noted). Sensory, motor, and SCG neurons were cultured as previously described (Henderson et al., 1995; Wickramasinghe et al., 2008; Ye et al., 2003). Motor neurons were isolated from HB9-GFP mice which have been previously described (Wichterle et al., 2002).
Sections were produced by fresh frozen tissue that was cryosectioned at a thickness of 12um. Sections were rinsed in PBS, blocked for 30–60 minutes (5% Normal Goat Serum, 0.05% Triton-X in PBS), incubated in primary antibody diluted in block overnight at 4°C. They were subsequently washed in PBS, incubated with appropriate Alexa fluor fluorescent or Cy3-conjugated secondary antibodies for 60 minutes at room temperature, washed in PBS, and then mounted for confocal microscopy. Primary antibodies include Synaptophysin (1:500, Synaptic Systems), pan-MAGUK (2ug/ml, Neuromab), pan-Shank (2ug/ml, Neuromab) and Neurofilament-200 (1:500, Millipore).
Neurons were fixed in 100% methanol at −20C for 10 minutes. After fixation, they were thoroughly washed in PBS, blocked for 30–60 minutes in 0.2% Gelatin with 0.05% Triton-X in PBS at 4°C, rinsed in PBS, and incubated in primary diluted in block overnight at 4°C, rinsed in PBS, incubated with appropriate Alexa flour fluorescent or Cy3-conjugated secondary antibodies for 60 minutes at room temperature, rinsed again in PBS, and subsequently mounted for confocal microscopy. Primary antibodies include pan-Shank (2ug/ml, Neuromab), pan-MAGUK (2ug/ml, Neuromab), pan-GKAP/SAPAP (2ug/ml, Neuromab), synapsin (1:1000, Synaptic systems) and nAChR B2-subunit (4ug/ml, DSHB) and MAP-2 (1:5000, Millipore).
After indicated treatments, cells were lysed in boiling Laemmli buffer. Lysates were resolved by SDS-PAGE and immubloted with antibodies against PSD-93 (1ug/ml, Neuromab), pan-Shank (1ug/ml, Neuromab), pan-GKAP/SAPAP (1ug/ml, Neuromab) or c-Fos (1:1000). Normalization was done by reprobing blots with the neural marker Tuj1 (1:5000, Covance) or actin (1:2500).
Comparisons between two groups were done by the Student's t-test. Comparisons between more than one group were done using an ANOVA test followed by a Tukey's post-hoc test.
The FlagTrkA targeting vector comprised a 5.2kb long arm, a 2.2kb short arm, a 2.8kb targeted region carrying the Flag-TrkA sequence and a Neo cassette flanked by two loxP sites. To construct the vector, a 10.1 kb XhoI-NdeI fragment containing the targeted exon (exon 1) was obtained from the TrkA-BAC clone and inserted into pBK-CMV (Stratagene). Next, an inframe Flag-Tag was generated by PCR cloning of two fragments, Bst98I-SmaI and ATG-Flag-BssHII, into PUC18. This new Flag-containing Bst98I-BssHII fragment was used to replace the Bst98I-BssHII fragment in pBK-TrkA to generate pBK-FlagTrkA. Lastly, the lox-Neo-lox cassette was introduced into pBK-Flag-TrkA. The targeting vector was linearized with BsrGI and electroporated into 129S6SvEvTac mouse ES cells. ES cell clones were selected with G418 (300ug/ml) and screened by PCR and Southern blotting. Positive clones were karyotyped and injected into C57BL/6 blastocysts which were then introduced into pseudopregnant females. Chimeric mice were crossed with C57BL/6 to obtain heterozygous animals which were subsequently mated with mice expressing Cre recombinase in germ cells to excise the Neo cassette. Heterozygous mice were later crossed to each other to generate TrkAFlag/Flag mice which were used for all analyses. Genotyping of mouse progeny was done with primers flanking the Flag sequence, using a PCR program at 95°C for 30s, 59°C for 30s and 72°C for 30s, repeated for 30 cycles. Primers for Flag are as follows: 5’-CTGCGCGGCCCGAGCAA -3’and 5’-AGCAGACCTCGCGACAGGAT-3’, which generate a 122bp fragment from the targeted allele.
Neurons in mass culture were first incubated with the Fab fragment (6µg/ml) of the Flag M1 antibody (Sigma) at 4°C for 20 minutes to label cell surface TrkA. The Fab fragment was generated from Flag M1 IgG according to the manufacturer’s protocol (Pierce). Unbound antibody was then removed by rinsing plates with medium, and then medium containing NGF (50ng/ml) or medium alone was applied. Cells were then placed at 37°C to trigger internalization. After the indicated treatment times, cells were washed with a 0.5M NaCl/0.2M acetic acid solution to remove any surface bound antibody. Following fixation with 4% paraformaldehyde, internalized TrkA was visualized by anti-Flag immunostaining. Neurons in microfluidic chambers were processed in the same manner except with two exceptions: (1) The anti-Flag Fab fragment was added exclusively to the distal axon compartment; (2) Excess antibody was not removed with 0.5M NaCl/0.2M acetic acid since the fluidic separation of the two compartments prevented direct uptake of antibody by the cell bodies.
Microfluidic Chambers were generated as previously described (Park et al., 2006).
We thank members of the Ginty lab, Michael Greenberg, Larry Schramm, V. Denic, S. Margolis, and B. Bloodgood for valuable scientific discussions and comments on this manuscript, and Dori Reimert and Naren Ramanan for help generating the Flag-TrkA mice. This work was supported by ZYC--National Natural Science Foundation of China (No. 30671050, 30725020) and NIH grant NS34814 (DDG). D.D.G is an investigator of the Howard Hughes Medical Institute.
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