SNPH is recruited to axonal mitochondria in response to neuronal activity. (A and B) Representative images (A) and quantitative analysis (B) showing the relative colocalization of SNPH-labeled axonal mitochondria with synapses. Neurons before and after the KCl treatment were fixed and coimmunostained with antibodies against SNPH and the synaptic marker synaptophysin (synapto). Note that action potential firing recruits SNPH-labeled mitochondria to synapses. Data were quantified from a total number of neurons (indicated within the bars) in three independent experiments. (C and D) Representative images (C) and quantitative analysis (D) showing increased SNPH enrichment into axonal mitochondria after 100-Hz field stimulation. Cultured hippocampal neurons were coimmunostained with anti-SNPH and anti–cytochrome c (cyto c). Arrows point to axonal mitochondria with low or no SNPH staining. Mean SNPH intensity on each mitochondrion was normalized against cytochrome c intensity. Relative SNPH enrichment on mitochondria was expressed as an intensity ratio (SNPH/cytochrome c; D, top) and the percentage of low SNPH-labeled axonal mitochondria (defined by the SNPH/cytochrome c ratio <0.5; D, bottom). Note that those tiny SNPH puncta not colabeled with mitochondria may represent shuttle cargoes delivering SNPH from axoplasm to mitochondria. Data were quantified from several axonal mitochondria (M) from the total number of neurons (N) examined in three independent experiments. Bars, 10 µm. Error bars show SEM. Student’s t test. (E) Proposed “Engine-Switch and Brake” model. SNPH plays a critical role in docking mitochondria via directly binding KIF5 and inhibiting motor ATPase activity. KIF5–SNPH molecular interplay is triggered by a Miro-Ca²⁺ sensing that releases KIF5 to bind to SNPH. Thus, SNPH turns off the “engine” (KIF5 motor) by sensing a “stop sign” (elevated Ca²⁺) and arrests mitochondria moving along the MT track. When in their stationary phase, mitochondria remain bound to the MT tracks, whereas KIF5 is in an inactive state.