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Abstract

Chemotactic axon guidance has an essential role in development and is important for re-innervation of target tissues after neuronal injury. The aim of the work described in this thesis was to determine whether the low density lipoprotein receptor-related protein 1 (LRP1) and LRP2 receptors mediate neurite chemoattraction, and to assess their therapeutic potential in a model of peripheral neuropathy. I found that LRP1 and LRP2 were expressed on growth cones at the leading edge and on filopodia, suggesting that they are part of the environment-sensing machinery. I used the growth cone turning assay to test whether a range of LRP ligands were chemotactic to growth cones in vitro. The ligands tested included metallothionein II (MTII), apolipoproteinE3, tissue plasminogen activator (tPA), alpha-2-macroglobulin (α2M), vitamin D and transthyretin. E16-18 embryonic rat sensory neuron growth cones grew towards a microgradient of MTII (+11.6°±2.1°, p<0.0001, cf. control -1.8°±1.1°), and 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). I used siRNA knock down and pharmacological inhibition to demonstrate that LRP1 and LRP2 were both required for growth cone responses to MTII and MTIII. Growth cone turning towards a gradient of MTII or MTIII was abolished by LRP-receptor inhibition with receptor associated protein and siRNA knockdown of LRP1 or LRP2. In addition, pharmacological inhibition of TrkA receptors significantly augmented the turning response. These data demonstrate that both LRP1 and LRP2 are necessary for MTII and MTIII-mediated chemotactic signal transduction, and potentially form part of a signalling hub, recruiting other co-receptors such as TrkA. MTII and MTIII-mediated chemotaxis was found to be dependent on calcium ion concentration: when neurons were depleted of extracellular calcium, the turning response underwent a complete switch in direction. Furthermore, pharmacological inhibition of calcium/calmodulin-dependent kinase II suggested that LRP1 and LRP2 signal via established downstream effectors. I then asked whether LRP-mediated chemotaxis could be exploited to guide axon regeneration in vivo. I used a model of small-fibre neuropathy in rats. Ten age-matched rats had topical 8% capsaicin cream and placebo cream applied to contralateral areas on the lumbar dorsum. 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, compared to contralateral control skin, however MTII treated rats had complete regeneration by 7 days. LRP-mediated chemotaxis represents a novel, non-classical axon guidance signalling system. MTII has therapeutic potential as a disease-modifying agent for the injured nervous system.

Item Type:	Thesis - PhD
Authors/Creators:	Landowski, LM
Keywords:	Nerve regeneration, therapeutics, low-density-lipoprotein-receptor-related protein (LRP) axon guidance, neuropathy, novel chemotactic
Copyright Holders:	Copyright the Author
Copyright Information:	Copyright 2014 the Author
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