LRP receptors in a novel mechanism of axon pathfinding and peripheral nerve regeneration

Chemotactic axon guidance has an essential role in re-­innervation of target tissues after neuronal injury. We determined whether the low density lipoprotein receptor-­related protein 1 (LRP1) and LRP2 receptors mediate neurite chemoattraction, and assessed therapeutic potential in a model of peripheral denervation. The growth cone turning assay was used to test whether a range of LRP ligands could induce growth cone chemotaxis in vitro (in E16-­18 rat sensory neurons). The ligands tested included metallothionein II (MTII), MTIII, apolipoprotein E3, tissue plasminogen activator (tPA), α­-2-­macroglobulin (α2M), vitamin D and transthyretin. Growth cones grew towards a microgradient of MTII (+9.8° ± 1.7°, p < 0.0001, cf. control ­1.8° ± 1.1°), turned away from a microgradient of MTIII (­13.8° ± 1.9, p < 0.0001), α2M (­11.9° ± 3.4, p < 0.01) and tPA (­11.1° ± 2.1 p < 0.001). Growth cone turning towards a gradient of MTII was abolished by LRP-­receptor inhibition with receptor associated protein (RAP, 0.6° ± 1.2) and siRNA knockdown of LRP1 (3.5° ± 1.9) or LRP2 (3.6° ± 2.5), confirming LRP requirement. Pharmacological inhibition of TrkA receptors significantly augmented the turning response. LRP1 and LRP2 colocalised on growth cones at the leading edge and on filopodia, suggesting that they are part of the environment-­sensing machinery. These data demonstrate that both LRP1 and LRP2 are necessary for MTII-mediated chemotactic signal transduction, and may form part of a signalling hub, recruiting other co­receptors such as TrkA. MTII-­mediated chemotaxis was found to be dependent on intracellular and extracellular calcium ions. Furthermore, pharmacological inhibition of calcium/calmodulin­-dependent kinase II suggested that LRP1 and LRP2 signal via established downstream effectors. The regenerative ability of LRP­MTII­mediated chemotaxis was examined in vivo in a model of chemical denervation in rats. Ten age-­matched rats had topical 8% capsaicin cream and placebo cream applied to contralateral areas of the paraspinal lumbar region. Treated areas were given 3x weekly intradermal injections of MTII or saline. Punch and epidermal roof biopsies were harvested weekly to monitor regeneration. At 14 days, regeneration was observed in saline treated capsaicin regions (42.1 ± 4.0 ENFs/mm), compared to contralateral control skin (49.6 ± 1.5 ENFs/mm), however MTII treated rats had complete regeneration by 7 days (45.0 ± 4.9 ENFs/mm, vs. 18.4 ± 3.8 ENFs/mm in saline treated capsaicin tissue). LRP-MTII­-mediated chemotaxis represents a novel, non­-classical signalling system, and has therapeutic potential as a disease-­modifying agent for the injured nervous system.