Amyloid Precursor Protein family as unconventional Go-coupled receptors and the control of neuronal motility

Abstract

Cleavage of the Amyloid Precursor Protein (APP) generates amyloid peptides that accumulate in Alzheimer Disease (AD), but APP is also upregulated by developing and injured neurons, suggesting that it regulates neuronal motility. APP can also function as a G protein-coupled receptor that signals via the heterotrimeric G protein Gαo, but evidence for APP-Gαo signaling in vivo has been lacking. Using Manduca as a model system, we showed that insect APP (APPL) regulates neuronal migration in a Gαo-dependent manner. Recently, we also demonstrated that Manduca Contactin (expressed by glial cells) induces APPL-Gαo retraction responses in migratory neurons, consistent with evidence that mammalian Contactins also interact with APP family members. Preliminary studies using cultured hippocampal neurons suggest that APP-Gαo signaling can similarly regulate growth cone motility. Whether Contactins (or other APP ligands) induce this response within the developing nervous system, and how this pathway is disrupted in AD, remains to be explored.

Keywords: APP; APPL; G protein; Gαo; Manduca; growth cone; hippocampal neuron; neuronal migration.

Figure 1.

Neuronal migration in Manduca is regulated by MsContactin-dependent activation of APPL-Gαo signaling. (A), Schematic representation of neuronal and glial cell migration during the formation of the Enteric Plexus in Manduca. Each panel shows a dorsal view of the embryonic ENS near the foregut-midgut boundary (FG/MG); embryos raised at 25°C complete their development in 100 hr post-fertilization (HPF). (A₁), Embryo at 55 HPF: Enteric Plexus neurons (EP cells; magenta) have delaminated from a neurogenic placode in the foregut epithelium and spread bilaterally to encircle the foregut. Subsets of EP cells align with one of eight longitudinal muscle bands (“b;” curved arrows) on the midgut surface; only the 4 dorsal bands are shown. Homophilic interactions mediated by the Ig-CAM Fas II (Fasciclin II; expressed by both the neurons and the bands) subsequently promote EP cell migration and outgrowth specifically along the bands, during which the neurons typically avoid the adjacent interband regions (“ib”). Concurrently, proliferating glial cells (green) closely follow the migratory neurons, rapidly surrounding their somata and processes. (A₂), By 70 HPF, the EP cells have transitioned from migration to a prolonged period of axon outgrowth, during which they extend processes posteriorly along the bands (beyond the field of view). Glial ensheathment of the neurons is typically complete by this stage (arrows). Only once axon elongation is complete (80 HPF) will the neurons extend terminal synaptic processes onto the interband regions (not shown). (A₃), Manipulations that inhibit APPL expression (by the EP cells) or MsContactin expression (by the glial cells; illustrated by fainter green shading) permit the neurons and their processes to travel inappropriately onto the adjacent interband regions (open arrowheads). Inhibiting Gαo activity in the EP cells also induces the same distinctive pattern of ectopic migration and outgrowth. By comparison, glial migration is unaffected by reduced MsContactin expression. (A₄), Overstimulating APPL-Gαo signaling in the EP cells (with MsContactin-Fc fusion proteins) induces a dramatic collapse/stall response in the neurons, resulting in the premature termination of migration (arrows) and reduced axonal outgrowth. (B), Enteric Plexus in a filleted Manduca embryo (at 60 HPF) immunostained with anti-APPL (blue), anti-GPI-Fas II (green), and anti-MsContactin (red). Anti-APPL specifically labels the migratory EP cells (arrows), while anti-MsContactin and anti-GPI-Fas II co-label adjacent glial cells (arrowheads). (C), Higher magnification of the boxed region in panel B. Arrowheads indicate areas of colocalization of MsContactin (red) with GPI-Fas II (green) in glial processes ensheathing the migratory neurons (“n”) expressing APPL (blue). Scale bar = 45 μm in (A); 30 μm in (B); 10 μm in (C). (D), Model of how Gαo-dependent signaling might be regulated by APP family proteins. In the migratory EP cells of Manduca, glial MsContactin functions as a ligand for neuronal APPL, inducing the local activation of Gαo in their leading processes; in turn, activated Gαo can regulate their motile behavior (potentially via both Ca²⁺-dependent and independent effectors), resulting in local retraction responses that prevent ectopic migration and outgrowth. In cultured hippocampal neurons, activation of APP signaling by antibodies against its extracellular domain, including crosslinked 22C11 (double arrows) or non-crosslinked nAPP-3 (single arrow), also induces collapse/stall responses in a Gαo-dependent manner, whereas antibodies against the cytodomain (cAPP) do not. Whether physiologic activation of APP-Gαo signaling regulates the behavior of developing neurons in the mammalian nervous system remains to be explored. Different candidate ligands (including other Contactins and sAPPα ectodomain fragments) might also promote or restrict motile responses in a context-specific manner, depending on the presence of additional co-receptors or Gαo effectors.

Figure 2.

Growth cone motility in cultured rat hippocampal neurons may also be regulated by APP-Gαo signaling. (A-F), Embryonic rat hippocampal neurons after 24 hr that had established normal polarity (Stage 3), with a single elongating axon (arrows) and a variable number of smaller dendrites extending from their somata. (A₁), Neuron that was immunolabeled with anti-nAPP (22c11; green) and anti-Gαo (magenta). (A₂), Enlarged view of boxed region in (A₁₎, showing that APP and Gαo colocalize within the axonal growth cone, particularly at the leading lamellipodial margin and in some of the larger filopodial extensions (arrowheads; colocalized immunolabeling appears white). (B-F), Representative examples of neurons that were treated with reagents targeting APP or Gαo, then immunolabeled with anti-β-III tubulin (Tuj; green) and counterstained with Phalloidin-Tetramethylrhodamine B (Rhod-Phalloidin; green) to label polymerized actin. Enlarged regions (indicated by white boxes in each panel) show only Rhod-Phalloidin staining in gray scale to highlight growth cone morphologies. (B₁), Neuron treated with control medium, exhibiting anti-Tuj1 immunolabeling along the length of its axon and dendrite, and strong Rhod-Phalloidin staining in its growth cones (associated with active motility). (B₂), The axonal growth cone in this neuron exhibited a typical “extended” morphology. (C₁), Neuron treated with the Gαo/Gαi activator Mastoparan 7 (Mas 7, 50 μM; Enzo Life Sciences) exhibited a dramatic reduction in Rhod-Phalloidin staining and a more moderate reduction in anti-Tuj1 immunoreactivity. (C₂), The axonal growth cone in this neuron exhibited a typical “collapsed” morphology. (D₁), Neuron treated with the Gαo/Gαi inhibitor PTX (100 ng/ml; List Biological Labs) exhibited an expansion of its axonal and dendritic growth cones. (D₂), The axonal growth cone in this neuron exhibited an “extended” morphology. (E₁), Neuron treated with non-crosslinked anti-nAPP antibodies (nAPP-3; 0.1–0.5 μM; Aves Laboratories) exhibited a collapsed/retracted morphology (highlighted in panel E₂). (F₁), Neuron that was pre-incubated with PTX before treatment with anti-nAPP antibodies showed morphological features similar to control neurons. (F₂), The axonal growth cone in this neuron exhibited an “extended” morphology, similar to the control neuron shown in panel (B). Scale bar = 30 μm in low-magnification images and 3 μm in highlighted boxed regions. (G), Quantification of the proportion of neurons in each treatment group that possessed axonal growth cones with either extended (green bars) or collapsed/retracted morphologies (orange bars). P-values above each histogram are derived from pairwise comparisons with controls after applying the Bonferroni correction; NS = not significantly different. Compared to controls, treatment with Mas 7 induced a significant increase in neurons exhibiting collapsed/retracted growth cones, whereas PTX treatment had the opposite effect, increasing the number of growth cones with extended morphologies. Treatment with anti-nAPP-3 induced a concentration-dependent increase in the proportion of growth cones with collapsed/retracted morphologies, with a maximal response similar to the effects of Mas 7. Pre-incubation with PTX prevented the effects of anti-nAPP treatment. The percentage of extended growth cones in neurons treated with PTX plus anti-nAPP (0.1 μM) was significantly greater than in cultures treated with 0.1 μM anti-nAPP alone (p = 0.004), but was not significantly different from controls after applying the Bonferroni correction. Likewise the percentage of extended growth cones in neurons treated with PTX plus anti-nAPP (0.5 μM) was significantly greater than in cultures treated with 0.5 μM anti-nAPP alone (p = 0.010), and was not significantly different from controls. By comparison, growth cone morphologies were unaffected by treatment with antibodies targeting the cytoplasmic domain of APP (cAPP; see Fig. 1D).