Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of ␣9 integrin promotes axon regeneration, so we have investigated ␣9␤1 trafficking and transport in axons and at the growth cone. We have previously found that ␣9 and ␤1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied ␣9 and ␤1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of ␤1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodaminedextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE.

The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin‐II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter‐pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release.