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	Figure 1. Conduction velocities of compound action potentials (CAPs) are reduced in the spinal cords of APP KO mice.Representative CAPs from the spinal cords (SC, A) and the sciatic nerve (SN, B) of APP KO and WT mice. Representative CAPs recorded at 24 °C and 37 °C from spinal cords of APP KO and WT mice (upper panel). The conduction velocity measured in WT mouse spinal cord (open bars) was consistently higher than that seen in APP KO mice (closed bars) at both temperatures measured (lower panel). N numbers are indicated inside the bars. Student’s t-test, *p < 0.05; **p < 0.01. Error bars represent mean ± S.E.
	Figure 2. APP enhances of sodium current expression through modulation of α-subunit of Nav1.6.(A) Sodium current expression is reduced in hippocampal neurons from APP KO mouse. Whole cell recordings of voltage-gated sodium currents from hippocampal slice of APP KO and WT mice at 7–8 weeks old of age. Sodium current responses elicited by depolarizing voltage steps (from −60 mV to 60 mV, 10 mV increments) in a neuron from WT and APP KO mice. (a), Current-voltage relationships of sodium currents density in WT mice (open squares) and APP KO mice (solid circles). pF, picofarads. (b) Peak currents density in APP KO and their littermate WT mice. (B) Overexpression of APP enhances sodium currents in HEK293 cells stably expressing α-subunit of human Nav1.6 (HEK293-Nav1.6 cells). Whole cell recordings of voltage-gated sodium currents in APP- and empty vector (vector)-transfected HEK293-Nav1.6 cells. Sodium current responses elicited by depolarizing voltage steps (from −60 mV to 60 mV, 10 mV increments). (a), Current-voltage relationships of sodium currents density in vector-(open squares) and APP-transfected HEK293-Nav1.6 cells (closed circles). pF, picofarads. (b), Peak currents density in vector- and APP-transfected HEK293-Nav1.6 cells. (C) APP enhances sodium currents in Xenopus oocytes. Two-electrode voltage-clamp recordings in oocytes which were injected cRNA for Nav1.6 α subunits and together with APP. The oocytes injected cRNA for Nav1.6 α subunits alone were recorded as the control. Sodium current responses elicited by depolarizing voltage steps (−75 mV to 40 mV, 5 mV increments). (a), Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits alone (open squares) and together with APP (closed circles). (b), Sodium peak currents in oocytes expressing Nav1.6 α subunits alone and together with APP. (D) Normalized current-voltage relationships of sodium currents by two-electrode voltage-clamp recordings in oocytes injected cRNA for Nav1.6 α subunit alone (open squares) and those injected cRNA for Nav1.6 α subunitvand the reversed sequence of APP (closed circles). (E) Normalized current-voltage relationships of sodium currents by two-electrode voltage-clamp recordings in oocytes expressing Nav1.5 α subunit alone (open squares) and those expressing Nav1.5 α subunit together with APP (closed circles). Insert: Representative examples of sodium current responses elicited by depolarizing voltage steps. One-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent mean ± S.E.
	Figure 3. The effects of distinct APP intracellular domains on voltage-gated sodium channels.(A) Schematic representation of APP (full-length APP695), APP intracellular domain (AICD50), APP C31 domain (C31), APP Go protein binding domain (GoBD), Fe65 binding domain (Fe65BD) and VTPEER peptides (Val667-Arg672). (B) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunit and together with APP intracellular domain (AICD50; (a); closed circles), APP C31 domain (C31; (b); closed circles), APP Go protein binding domain (GoBD; (c); closed circles), Fe65 binding domain (Fe65BD; (d); closed circles), VTPEER peptide (VTPEER; (e); closed circles) and the reversed VTPEER sequence (reverse VTPEER; (f); closed circles). The oocytes expressing Nav1.6 α subunit alone were recorded as control (a–f; open squares). Insert: Representative examples of sodium current responses elicited by depolarizing voltage steps.
	Figure 4. APP Increases Nav1.6 currents in a Go Protein-dependent manner.(A–F), Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits and distinct APP intracellular domains alone (open squares; (A) Full-length APP; (B) AICD; (C) C31; (D) Go protein binding domain, GoBD; (E) Fe65 binding domain, FeBD; (F) VTPEER peptides together with a dominant active mutant of Go protein α subunit (Q205L, closed circles) or a dominant negative mutant of the Go protein α subunit (G203T; closed triangles). (G) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits alone (open squares) and together with a dominant active mutant of Go protein α subunit (Q205L, closed circles) or a dominant negative mutant of the Go protein α subunit (G203T; closed triangles). (H) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits and APP with (closed circles) or without (open squares) intracellular injection G protein inhibitor NF023. Insert: Representative examples of sodium current responses elicited by depolarizing voltage steps. Error bars represent mean ± S.E.
	Figure 5. Thr-668 phosphorylation of APP enhances sodium current expression.(A) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits alone (a; open squares) and together with APP (a; closed circles), APP plus CDK5 inhibitor (a; Roscovitine 10 μM; open triangles) and APP plus JNK inhibitor (a; JNK inhibitor III 7 μg/ml, closed triangles). The mean peak sodium current amplitude in oocytes injected with Nav1.6 and APP and applied with the indicated inhibitors (b). (B) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits alone (a; open squares) and together with wild-type APP (a; closed circles), APP T668E (a; open triangles), and APP T668A (a; closed triangles). The mean peak sodium current amplitude in oocytes injected with Nav1.6 and the indicated APP or APP mutant cRNAs (b). (C) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits with APP T668E alone (open squares) and together with a dominant active mutant of Go protein α subunit (Q205L, closed squares) or a dominant negative mutant of the Go protein α subunit (G203T; closed triangles) (a). The mean peak sodium current amplitude in oocytes injected with Nav1.6 and T668E and the Go mutant cRNAs (b). (D) Normalized current-voltage relationships of currents in oocytes expressing Nav1.6 α subunits with APP T668A alone (open squares) and together with a dominant active mutant of Go protein α subunit (Q205L, closed squares) or a dominant negative mutant of the Go protein α subunit (G203T; closed triangles) (a). The mean peak sodium current amplitude in oocytes injected with Nav1.6 and T668A and the Go mutant cRNAs (b). Insert: Representative examples of sodium current responses elicited by depolarizing voltage steps. One-way ANOVA. **p < 0.01; Error bars represent mean ± S.E.
	Figure 6. The cell surface expression of Nav1.6 sodium channels in APP, p35 and JNK3 knockout mice and Hek 293-Na1.6 cells for virous treatments.Analysis of cell surface expression of Nav1.6 protein in the white matter of spinal cord isolated from mutant mice and Hek 293-Na1.6 cells. Total proteins and cell surface proteins, biotinylated and pull-down by NeutrAvidin Gel, were subjected to Western blots (n = 3). (A–C) Total proteins and cell surface proteins isolated from the ventral white matter of spinal cords of APP KO (Aa), p35 KO (Ba), JNK3 KO (Ca) and their littermate WT mice were blotted using antibodies against Nav1.6, APP, phosphorylated APP at T668 (p-APP), F3, p35, JNK3 and γ-tubulin. Quantification of the level of protein expression in input and cell surface. The levels of Nav1.6 in the WT spinal cords were normalized to 1.0, the relative levels of Nav1.6 in KO mice were quantified (b). (D) HEK293-Nav1.6 cells were transfected APP siRNA or a scrambled siRNA (NC) and then treated with JNK inhibitor III (JNKi, 10 μg/ml), CDK5 inhibitor (CDK5i, 20 μM Roscovitine) or vehicle/DMSO (Control, Ctrl). Western blot analysis of the cell surface expression or the total protein of Nav1.6 using antibodies against Nav1.6 and APP. GAPDH and (transferrin receptor, TfR) were loaded as control. The levels of Nav1.6 in DMSO-treated NC-transfected cells were normalilzed to 1.0. The relative levels of Nav1.6 in other groups of the cells as indicated were quantified. Student t test. *, p < 0.05; **, p < 0.01. Error bars represent mean ± S.E.
	Figure 7. Conduction velocities of compound action potentials in the spinal cords of p35 and JNK3 knockout mice.Determination of conduction velocities of CAPs in p35 (A) and JNK3 (B) KO and WT mice. Representative CAPs from spinal cords of p35 (p35 KO; n = 6) and JNK3 (JNK3 KO; n = 7) KO and their wild-type mice (n = 6 for P35 WT; n = 10 for JNK3 WT). Representative CAPs recorded at 24 °C and 37 °C from spinal cords of APP KO and WT mice (upper panel). The conduction velocity measured in WT mouse spinal cord (open bars) was consistently higher than that seen in APP KO mice (closed bars) at both temperatures measured (lower panel). N numbers are indicated inside the bars. Student’s t-test, *p < 0.05; **p < 0.01. Error bars represent mean ± S.E.
	Figure 8. Schematic model for regulation of Nav1.6 by APP.APP through VTPEER activates Go protein, which enhances phosphorylation of APP (Thr668) by activating of JNK3. Phosphorylated APP (Thr668) binds to Nav1.6 and promotes cell surface expression of Nav1.6. In addition, activation of Goα further enhances the functional interaction between phosphorylated APP (Thr668) and Nav1.6. AICD, after cleavage from full-length APP, upon being phosphylated (Thr668), increases cell surface expression of Nav1.6 as well, which is also mediated by activity of Goα. Phosphorylated APP/AICD (Thr668) mediates cell surface expression of Nav1.6 may through promoting Nav1.6 insert into the cell surface or maintaining Nav1.6 on the cell surface.

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