Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-Cbinding integrin ␣9 promotes axon regeneration. We have therefore studied the mechanism by which ␣9 integrin and its partner ␤1 are trafficked along axons and at the growth cone using adult DRG neurons and PC12 cells. We have focused on the small GTPase Rab11 and its effector Rab coupling protein (RCP), as they are involved in the long-range trafficking of ␤1 integrins in other cells. Rab11 colocalizes with ␣9 and other ␣ integrins and with ␤1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain ␣9 and ␤1. Endocytosed ␤1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and ␣9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, ␣9 and ␤1 overlap with RCP, particularly at the growth cone periphery. We show that ␤1 integrin trafficking during neurite outgrowth involves Rab11 and RCP, and that manipulation of these molecules alters surface integrin levels and axon growth, and can be used to enhance ␣9 integrin-dependent neurite outgrowth. Our data suggest that manipulation of trafficking via Rab11 and RCP could be a useful strategy for promoting integrin-dependent axonal regeneration.

Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration.

Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking.

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.

No abstract