Mitochondrial membrane potential in axons increases with local nerve growth factor or semaphorin signaling

Neurons concentrate mitochondria at sites in the cell that have a high demand for ATP and/or calcium buffering. To accomplish this, mitochondrial transport and docking are thought to respond to intracellular signaling pathways. However, the cell might also concentrate mitochondrial function by locally modulating mitochondrial activity. We tested this hypothesis by measuring the membrane potential of individual mitochondria throughout the axons of chick sensory neurons using the dye tetramethylrhodamine methylester (TMRM). We found no difference in the TMRM mitochondrial-to-cytoplasmic fluorescence ratio (F-m/F-c) among three functionally distinct regions: axonal branch points, distal axons, and the remaining axon shaft. In addition, we found no difference in Fm/Fc among stationary, retrogradely moving, or anterogradely moving mitochondria. However, F-m/F-c was significantly higher in the lamellipodia of growth cones, and among a small fraction of mitochondria throughout the axon. To identify possible signals controlling membrane potential, we used beads covalently coupled to survival and guidance cues to provide a local stimulus along the axon shaft. NGF- or semaphorin 3A-coupled beads caused a significant increase in F-m/F-c in the immediately adjacent region of axon, and this was diminished in the presence of the PI3 (phosphatidylinositol-3) kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or the MAP (mitogen-activated protein) kinase inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2- amino-phenylthio] butadiene), demonstrating that signaling pathways downstream of both ligands affect the Delta Psi(m) of mitochondria. In addition, general inhibition of receptor tyrosine kinase activity produced a profound global decrease in F-m/F-c. Thus, two guidance molecules that exert different effects on growth cone motility both elicit local, receptor-mediated increases in membrane potential.