Identification of factors in peripheral sensory neurons that contribute to the axonal targeting and functional coupling of G protein-coupled receptors such as opioid receptors

众所周知，外周感觉神经元中的G蛋白偶联受体（GPCR）既参与兴奋和敏化，也参与抑制。由于离子通道不仅分布在神经末梢，而且沿外周感觉神经元的轴膜分布，因此问题出现了，是否可以想象gpcr也位于轴突膜并在功能上偶联？这种gpcr，如阿片受体，整合到轴突膜的证明尚不清楚，这将鼓励临床使用此类受体的药物，如阿片。最初的临床试验似乎表明对神经损伤患者有临床益处。在坐骨神经损伤的动物模型中，尽管在炎症性疼痛的动物模型中没有观察到，但在神经周围应用阿片类药物可以引起有效的抗痛觉作用。在此，我们研究了在周围神经损伤和炎症性疼痛中，多阿片受体（MOR）轴突转运、膜靶向和功能偶联的机制不同的假设。我们的第一个目标是在患有完全弗氏佐剂性后爪炎症的动物和患有慢性收缩神经损伤的动物中，确定神经周阿片的不同抗伤害性作用与它们的轴突存在和MOR的功能偶联有关。第二个目标将确定MOR是否进入轴突初始段（轴突传递蛋白的过滤器），它们与囊泡相关膜蛋白（VAMP）共定位，它们通过运动蛋白（如运动蛋白）沿着轴突内微管运输，并最终通过肌凝蛋白Va运动蛋白沿着f -肌动蛋白丝传递到目的地。通过干扰这些机制，我们将评估这些组件的个别相关性。第三个目标将测试局部机制，如脂筏的产生增强，G蛋白调节的神经突生长诱导因子（GRIN1）的表达增加，或负责将MOR整合到轴突膜的神经细胞粘附分子L1 （L1- cam）的上调，以及它们是否依赖于生长因子（如NGF）的局部表达。了解gpcr（如阿片受体）在轴突膜上的靶向性和功能偶联的确切机制，可能会为调节这一过程提供新的激励和工具，以获得治疗优势。这对于因神经损伤或肿瘤浸润而遭受严重疼痛的患者尤其具有重要意义。

It is well known that G protein-coupled receptors (GPCR) in peripheral sensory neurons contribute to the excitation and sensitization as well as to their inhibition. Since ion channels are distributed not only at the nerve terminals, but also along the axolemma of peripheral sensory neurons, the question arises whether it is conceivable that GPCRs are also located and functionally coupled to the axonal membrane? The demonstration of such GPCRs, e.g. opioid receptors, integrated into the axonal membrane is yet unknown and would encourage the clinical use of drugs for such receptors, e.g. opioids. First clinical trials seem to indicate a clinical benefit for patients suffering from nerve injury. Consistently, in animal models of sciatic nerve injury perineural application of opioids elicited potent antinociceptive effects, although they were not observed in animal models of inflammatory pain. Here we investigate the hypothesis that mechanisms of mu-opioi receptors (MOR) axonal transport, membrane targeting and functional coupling are different in peripheral nerve injury versus inflammatory pain. Our first goal will identify in animals with complete Freunds adjuvant hindpaw inflammation versus animals with chronic constriction nerve injury the distinct antinociceptive effects of perineural opioids in relation to their axonal presence and functional coupling of MOR. The second goal will identify whether MOR enter the axonal initial segment, a filter for axonally delivered proteins, they colocalize with vesicle-associated membrane proteins (VAMP), they are transported via motor proteins such as kinesins along intraaxonal microtubules, and they are finally delivered to their destination via myosin Va motor proteins along F-actin filaments. By interfering with these mechanisms we will assess the individual relevance of these components. The third goal will test local mechanisms such as enhanced production of lipid rafts, increased expression of G protein-regulated inducer of neurite outgrowth (GRIN1), or up-regulation of neural cell adhesion molecule L1 (L1-CAM) of being responsible for the integration of MOR into the axonal membrane and whether they depend on the local expression of growth factors (e.g. NGF). To understand the exact mechanisms that determine the targeting and functionally coupling of GPCRs such as opiod receptors to the axonal membrane may give novel incentives and tools to regulate this process towards a therapeutic advantage. This would be of great relevance particularly for patients suffering from severe pain due to nerve injury or to tumor infiltration.