Figure 4. The amino terminus of APP is a regulated DR6 ligand.
(a)Hypothesis: if DR6 is ligand-activated, then DR6-Fc might sequester the ligand and inhibit degeneration.
(b)DR6-Fc inhibits local degeneration of sensory axons in Campenot chambers 24hr after NGF deprivation. Quantification in (c).
(d, e) DR6-binding sites are lost from axons and released into medium upon trophic deprivation. (d) DR6-AP binding (purple) to Bax−/− sensory axons (left) is lost 24hr after NGF deprivation (right), (e) Medium conditioned by sensory axons (in Campenot chambers) or ventral spinal cord explants, maintained with or deprived of trophic factors for 24 hr (sensory: NGF; motor: BDNF and NT3; Bax inhibitor present), was resolved under non-reducing conditions and probed with DR6-AP.
(f)Results in (a–e) support a “Ligand Activation” model in which an inactive DR6 surface ligand is shed in active form upon tropic deprivation.
(g)APP immunostaining on sections of mouse embryos at indicated ages, showing neuronal and axonal expression, v, ventricular zone; drez, dorsal root entry zone; vlf, ventro-lateral funiculus; s, sensory ganglia; sn, spinal nerve.
(h) Domain structure of APP (short form, APP695), indicating beta- and gamma-secretase cleavage sites and antibody binding sites. KPI and OX2: alternatively spliced domains of longer form. Adapted from ref. (20).
(i) DR6 binding sites in sensory axon conditioned medium include APP ectodomain fragments. Left: anti-N-APP(poly) detects bands at ~35 kDa (N-APP, major) and ~100 kDa (minor), enriched after trophic deprivation. Middle: anti-sAPPβ detects bands at ~55kDa (major) and ~100 kDa minor). Right: Immunodepletion using anti-N-APP(poly) depletes DR6-AP binding sites.
(j) Direct interaction between purified APP [1–286] and DR6-Fc revealed by pull-down.
(k) Loss of surface APP in patches from sensory axons 12hr after NGF deprivation is blocked by the BACE inhibitor OM99-2 (10μM) but not the alpha-secretase inhibitor TAPI (20μM).
Scale bar: (b): 50 μm; (d): 50 μm; (g): 500 μm; (i) 50 μm.