Metallothionein I/II induces growth cone chemotaxis via an LRP­1 and LRP­2 dependent mechanism

Introduction: Axon guidance is essential in the developing nervous system as well as in regeneration and re­innervation of target tissues after neuronal injury. It is likely that both traditional and novel receptor­ligand systems are necessary to guide axon growth cones post nerve injury. We examined a novel guidance cue­receptor mechanism: metallothionein I/II (MTI/II) chemoattraction, mediated via the lipoprotein receptor­related proteins 1 and 2 (LRP­1 and LRP­2).

Methods: The turning response of growth cones from embryonic (E16­18) rat dorsal root ganglia were measured in vitro, using the growth cone turning assay 1 . Immunohistochemistry and western blotting were used to quantify protein levels in control and knockdown cultures.

Results: Immunocytostaining established that LRP­1 and LRP­2 are found at the leading edge and on filopodia, suggesting that they are part of the environment­sensing machinery of growth cones. We tested a range of LRP ligands for chemotactic properties using the growth cone turning assay, including MTI/II, ApoE3, tissue plasminogen activator, alpha­2­ macroglobulin and transthyretin. The only LRP ligands tested that elicited a turning response in growth cones were MTI/II and MTIII. Growth cones were attracted to MTI/II (turning angle 9.8°±1.7°, n=11, p<0.0001, compared to control ­1.8° ± 1.1°). Interestingly, the structurally related MTIII protein induced robust growth cone repulsion (­13.8°±1.9°, n=14, p<0.0001). Growth cone turning towards a gradient of MTI/II was abolished by LRP­receptor 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). This data demonstrates that both LRP­1 and LRP­2 are necessary for MTI/II­mediated chemotactic signal transduction. MTI/IImediated chemotaxis was found to be dependent on calcium ion concentration, removal of extracellular calcium abolished chemoattraction. Furthermore, pharmacological inhibition of calcium/calmodulin­dependent kinase II suggests that LRP1 and LRP2 signal via established downstream effectors. The LRP­MTI/II chemotactic system represents a novel, nonclassical axon guidance system that may be exploited in repairing the injured nervous system.