Long-distance control of synapse assembly by target-derived NGF

Abstract

We report a 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 postsynaptic specializations on dendrites of sympathetic neurons. This, in turn, is a prerequisite for formation of presynaptic 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.

Figure 1. Requirement of NGF for SCG synapse formation but not pre-synaptic axonal innervation in vivo

(A) Diagram of the preganglionic-postganglionic synapse. (B) Immunostaining for synaptophysin and pan-MAGUK, markers of presynaptic and post-synaptic specializations, respectively, in the SCG of P0 Bax−/− and NGF−/−;Bax−/− animals. (C) Quantification of (B). Results are represented as means +/− SEM. n = 3 for all genotypes. (D) Immunostaining for Neurofilament-200 (NF-200), which is used to visualize axons of preganglionic sympathetic neurons, in P0 Bax−/− and NGF−/−;Bax−/− animals. Scale bar in all images represents 10um. (E) Quantification of (D). Data are represented as the level of fluorescence intensity of immunostained sections. Results are represented as means +/− SEM. n = 3 for all genotypes. * p < 0.05 using ANOVA followed by Tukey’s post hoc test.

Figure 2. Retrograde NGF signaling controls post-synaptic organization on postganglionic neurons in vitro

(A) Immunohistochemistry for pan-MAGUK (Green) and MAP-2 (Blue) on 16–18 DIV postganglionic sympathetic neurons at 0, 12, and 48 hours following removal of NGF and concomitant addition of anti-NGF. Boxed regions are shown at a higher magnification below. (B) Quantification of PSD clusters and (C) dendritic field length in (A). PSD clusters are represented as total number of MAGUK clusters per 10um of dendrite and dendritic field is represented as the sum of the lengths of all dendrites on one neuron. (D) Immunohistochemistry for nAChR, Shank, and GKAP on 16–18 D.I.V postganglionic sympathetic neurons at 0 and 12 hours after removal of NGF and concomitant addition of anti-NGF. (E) Quantification of (D). Data are represented as total number of synaptic clusters per 10µm of dendrite. (F) 12–14 DIV postganglionic sympathetic neurons are either left untreated, treated with anti-NGF for 12 hours, or treated with NGF (40ng/ml) for 2 hours following 12 hours in anti-NGF. Post-synaptic clustering is assessed by pan-MAGUK immunostaining. (G) Quantification of (F). Data are represented as a total number of MAGUK clusters per 10µm. (H) Postganglionic sympathetic neurons were grown in microfluidic chambers for 10–12 DIV and subsequently treated with anti-NGF for 12 hours or NGF (40ng/ml) on D.A. for 6 hours after 12 hours in anti-NGF. Postsynaptic clustering is assessed by pan-MAGUK immunostaining. (I) Quantification of (H). Data re represented as a total number of MAGUK clusters per 10µm of dendrite. All results are represented as mean +/− SEM. All conditions have n > 15. Scale bar in all images represents 10µm. * p < 0.05 using ANOVA followed by Tukey’s post hoc test.

Figure 3. Acute NGF-dependent PSD formation is largely independent of protein synthesis

(A) PSD-93 and Tuj1 protein levels as measured by Western blot (WB) using extracts prepared from 16–18 DIV sympathetic neurons following 0, 12, or 48 hours of NGF withdrawal and concomitant addition of anti-NGF. (B) Comparison of relative PSD-93 levels by quantification of WB. Results are normalized to Tuj1 levels and represent n = 3. (C) 16–18 DIV sympathetic neurons were treated with anti-NGF for 12 hours. NGF (40ng/ml) was then reapplied to the culture media in the presence or absence of cycloheximide (10µg/ml) for the indicated times and post-synaptic clustering was assessed by immunostaining for MAGUK. (D) Quantification of (C). Data are represented as a total number of MAGUK clusters per 10um of dendrite. (E) Postganglionic sympathetic neurons were grown in microfluidic chambers for 12–14 days and subsequently treated with anti-NGF in both CB/D and DA compartments for 12 hours. NGF (40ng/ml) was reapplied exclusively to DA for 6 hours of neurons in the absence or presence of cycloheximide (10µg/ml) in both compartments. Post-synaptic clustering was assesed by pan-MAGUK staining. (F) Quantification of (E). Data are represented as a total number of MAGUK cluster per 10um of dendrite. All results are represented as mean +/− SEM. n > 20 for all conditions unless otherwise indicated. Scale bar in all images represents 10µm. * p < 0.05 using ANOVA followed by Tukey’s post hoc test.

Figure 4. NGF–TrkA signaling endosomes are transported from distal axons to dendrites

(A) Postganglionic sympathetic neurons were grown in microfluidic chambers for 12–14 DIV and subjected to a Flag antibody labeling assay to visualize the appearance of Flag-TrkA endosomes in the cell body (see Supplemental Materials & Methods). NGF was present only on distal axons. Note that Flag-TrkA endosomes, which form on distal axons, appear in both the cell bodies and the dendrites of these neurons. Boxed regions are shown at higher magnifications below. (B) Quantification of the number of Flag-TrkA punctae with and without NGF on DA for 6 hours. Data are represented as the total number of Flag-TrkA punctae per 10um on dendrite. All results are represented as mean +/− SEM. n > 10 for all conditions (C) Postganglionic sympathetic neurons were grown in microfluidic chambers for 10–14 DIV. Neurons were either subjected to the Flag-TrkA assay, as above, or MAGUK staining for different NGF treatment times or were subjected to anti-NGF treatment followed by an acute reapplication of NGF for 1,2, or 6 hours. (D) Quantification of (C). Data are represented as total number of clusters/punctae per 10um of dendrite. n>15 for all conditions. Scale bar in all images represents 10µm. * p < 0.05 using ANOVA followed by Tukey’s post hoc test.

Figure 5. NGF mediated PSD organization requires TrkA signaling in the cell body and dentrite compartment as well as MEK/MAPK and PI3K signaling in distinct subcellular locales

(A) Postganglionic sympathetic neurons were isolated from TrkA^F592A or wild-type mice and grown in microfluidic chambers for 12–14 DIV and subsequently treated with anti-NGF in both the cell body/dendrite and distal axon compartments for 12 hours. NGF (40ng/ml) was reapplied for three hours exclusively to DA compartments in the absence or presence of 1NMPP1 (0.5µM) or DMSO in the cell body/dendrite compartment. Post-synaptic clustering was assessed by MAGUK staining. (B) Quantification of (A). Data are represented as a total number of MAGUK clusters per 10um of dendrite. (C) Postganglionic sympathetic neurons were grown in microfluidic chambers for 12–14 DIV and subsequently treated with anti-NGF in both the cell body/dendrite and distal axon compartments for 12 hours. NGF (40ng/ml) was reapplied exclusively to DA compartments in the absence or presence of PD98089 (50µM) or LY294002 (50µM) in the indicated compartments. Post-synaptic clustering was assessed by MAGUK staining. (D) Quantification of (B). All results are represented as mean +/− SEM. n > 20 for all conditions unless otherwise indicated. Scale bar in all images represents 10µm. * p < 0.05 using ANOVA followed by Tukey’s post hoc test.

Figure 6. p75 restricts NGF mediated control of synaptic organization in vitro and in vivo

(A) Immunostaining for synaptophysin and pan-MAGUK, markers of presynaptic and postsynaptic specializations, respectively, in SCGs of P10 wild-type and p75−/− animals. (B) Quantification of (A). Data are represented as clusters/unit area. n=3 for all genotypes. All results are represented as mean +/− SEM. (C) 16–18 DIV sympathetic neurons were established from wild-type of p75−/− animals. The concentration of NGF was changed as indicated for 48 hours and MAGUK clusters were evaluated by immunostaining. Boxed regions are shown at higher magnifications below. (D) Quantification of (B). Data are represented as MAGUK clusters per 10µm of dendrite. n > 20 for all conditions. (E) Postganglionic sympathetic neurons were grown in microfluidic chambers for 10–11 DIV and either treated with anti-NGF for 12 hours, NGF (40ng/ml) on DA with anti-BDNF (1ug/ml) on CB/D after 12 hours anti-NGF, or NGF (40ng/ml) on DA with anti-BDNF (1ug/ml) on DA after 12 hours anti-NGF. (F) Quantification of (E). Data are represented as MAGUK clusters per 10um of dendrite. n>15 for all conditions. Scale bar in all images represents 10µm. * p < 0.05 using ANOVA followed by Tukey’s post hoc test.