WAVE1 controls depolarization-induced trafficking of the mitochondria to dendritic spines. (A) Hippocampal neurons were cotransfected with EGFP and Mito-DsRed plus control plasmid vectors (−), WAVE1 RNAi (RNAi), or WAVE1 RNAi plus RNAi-resistant WAVE1 (Rr-WAVE1). Neurons were stimulated with NaCl or KCl as in Fig. 1 (4× KCl or 4× NaCl). Arrowheads indicate the mitochondria in spine heads. (Scale bar: 10 μm.) (B and C) Quantification of percentage of dendritic protrusions (spines) containing mitochondria (B) and the percentage of dendritic mitochondria in dendritic protrusions (C). The depolarization protocol increased the number of filopodia while reducing the number of spines. Thus, the total number of protrusions was not significantly affected by the depolarization protocol. Mitochondria were equally distributed in filopodia and spines. Data represent means ± SEM (n = 10–19 dendrites, 3–5 neurons per culture, three independent cultures). Multiple comparison by two-way ANOVA with Bonferroni's posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Depolarization induces WAVE1 dephosphorylation through NMDA receptor activation in primary hippocampal neurons. Primary cultured hippocampal neurons were stimulated with four repeated NaCl (4× NaCl) or KCl (4× KCl) treatments in the absence or presence of 100 μM APV (NMDA receptor antagonist). Levels of total p35 (A), Cdk5 (B), and WAVE1 (C) and the phosphorylation of WAVE1 at P-S310 (D), P-S397 (E), and P-S441 (F) were measured by immunoblotting, and the results were quantified by densitometry. Data normalized to the value of 4× NaCl in the absence of antagonist represent means ± SEM for four experiments. *, P < 0.05; **, P < 0.01 versus the value of 4× NaCl in the absence of antagonist (one-way ANOVA with Neuman–Keuls posttest).

Localization of WAVE1 in the mitochondria. Primary hippocampal neurons were transfected with Mito-DsRed (DsRed2 fused to the mitochondrial targeting sequence of cytochrome c oxidase). Neurons were stimulated with four repeated depolarizations (4× KCl) (50 mM KCl in CM for 3 min, followed by a 10-min interval with CM). NaCl (50 mM) replaced KCl in the controls (4× NaCl). Endogenous WAVE1 was detected with anti-WAVE1 antibody, and Mito-DsRed was detected with anti-DsRed antibody. (Scale bar: 10 μm.)

Depolarization induces WAVE1 dephosphorylation via NMDA receptor activation in primary cortical neurons. Primary cortical neurons were stimulated with 4× NaCl or 4× KCl in the absence or presence of 100 μM APV (NMDA receptor antagonist) and/or 20 μM CNQX (AMPA/kainate receptor antagonist). Levels of total p35 (A), Cdk5 (B), and WAVE1 (C) and the phosphorylation of WAVE1 at P-S310 (D), P-S397 (E), and P-S441 (F) were measured by immunoblotting (Upper), and the results were quantified by densitometry (Lower). Data that were normalized to the value of 4× NaCl in the absence of antagonists represent means ± SEM for four experiments. **, P < 0.01;***, P < 0.001 versus the value for 4× NaCl in the absence of antagonists (one-way ANOVA with Neuman–Keuls posttest).

WAVE1 controls depolarization-induced spine growth. Primary hippocampal neurons were cotransfected with EGFP plus control plasmid vector, WAVE1 RNAi plasmid (RNAi), or WAVE1 RNAi plus RNAi-resistant WAVE1 (Rr-WAVE1). Neurons were stimulated with NaCl or KCl as in Fig. 1. (A) Representative images of neurons cotransfected with EGFP plus control plasmid vector showing repetitive depolarization-induced generation of filopodia and multiheaded protrusions. Arrowhead, filopodia; arrows, multiheaded protrusions. (Scale bar: 10 μm.) (B) Quantification of protrusions with multiple heads (number per 10-μm dendritic length; means ± SEM, n = 10–17 dendrites, 3–5 neurons per culture, three independent cultures). *, P < 0.05; ***, P < 0.001 versus control (4 × NaCl) (two-way ANOVA with Bonferroni's posttest).