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Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-C-binding integrin alpha9 promotes axon regeneration. We have therefore studied the mechanism by which alpha9 integrin and its partner beta1 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 beta1 integrins in other cells. Rab11 colocalizes with alpha9 and other alpha integrins and with beta1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain alpha9 and beta1. Endocytosed beta1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and alpha9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, alpha9 and beta1 overlap with RCP, particularly at the growth cone periphery. We show that beta1 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 alpha9 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.

Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The alpha9beta1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the alpha9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of alpha9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling alpha9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express alpha9 integrin in vivo in DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in alpha9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after alpha9 integrin treatment but remained elevated in control groups.

Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-Cbinding integrin α9 promotes axon regeneration. We have therefore studied the mechanism by which α9 integrin and its partner β1 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 β1 integrins in other cells. Rab11 colocalizes with α9 and other α integrins and with β1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain α9 and β1. Endocytosed β1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and α9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, α9 and β1 overlap with RCP, particularly at the growth cone periphery. We show that β1 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 β9 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. [ABSTRACT FROM AUTHOR]