The Function of Mitochondria in Presynaptic Development at the Neuromuscular Junction

Abstract

Mitochondria with high membrane potential (⌬⌿ m ) are enriched in the presynaptic nerve terminal at vertebrate neuromuscular junctions, but the exact function of these localized synaptic mitochondria remains unclear. Here, we investigated the correlation between mitochondrial ⌬⌿ m and the development of synaptic specializations. Using mitochondrial ⌬⌿ m -sensitive probe JC-1, we found that ⌬⌿ m in Xenopus spinal neurons could be reversibly elevated by creatine and suppressed by FCCP. Along naïve neurites, preexisting synaptic vesicle (SV) clusters were positively correlated with mitochondrial ⌬⌿ m , suggesting a potential regulatory role of mitochondrial activity in synaptogenesis. Indicating a specific role of mitochondrial activity in presynaptic development, mitochondrial ATP synthase inhibitor oligomycin, but not mitochondrial Na ؉ /Ca 2؉ exchanger inhibitor CGP-37157, inhibited the clustering of SVs induced by growth factor-coated beads. Local F-actin assembly induced along spinal neurites by beads was suppressed by FCCP or oligomycin. Our results suggest that a key role of presynaptic mitochondria is to provide ATP for the assembly of actin cytoskeleton involved in the assembly of the presynaptic specialization including the clustering of SVs and mitochondria themselves. INTRODUCTIONMitochondria play multiple roles in cellular functions including cellular metabolism, calcium homeostasis, and programmed cell death. At both peripheral and central synapses, mitochondria are concentrated within the presynaptic nerve terminal as demonstrated by electron microscopic studies and they are presumably related to the transmitter release machinery (Herrera et al., 1985;Robitaille and Tremblay, 1987;Peters et al., 1991). Our previous study found that local synaptogenic signals induce the clustering of not only synaptic vesicles (SVs; Dai and Peng, 1995), but also mitochondria (Lee and Peng, 2006), thus suggesting that the clustering of this organelle is an integral part of presynaptic differentiation. The function of mitochondria at mature neuromuscular junctions (NMJs) has recently been explored. For example, the transmitter release under intense stimulation fails in a Drosophila NMJ mutant lacking presynaptic mitochondria (Guo et al., 2005;Verstreken et al., 2005). The calcium release from mitochondria has been shown to modulate synaptic potentiation at the vertebrate NMJ (Yang et al., 2003). However, the function of presynaptic mitochondria in the development of the NMJ is not yet known.Within the axon, mitochondria undergo bidirectional movement and accumulate at sites of high metabolic activity (Hollenbeck, 1996). The direction of mitochondrial transport is correlated with axonal outgrowth (Morris and Hollenbeck, 1993) and its activity (Miller and Sheetz, 2004), reflected by the electrical potential (⌬⌿ m ) across the inner membrane produced by oxidative phosphorylation-mediated electrochemical equilibrium of H ϩ ions. Previously, we found that mitochondria with high ⌬⌿ m are predominantly localized at the presyna...

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