Localisation of LRP1 in neurons and glia of the central nervous system

The low density lipoprotein receptor-related protein-1 (LRP1) receptor modulates neuronal survival, neurite outgrowth, regeneration and calcium signaling. Its effects are thought to be ligand and cell specific, but how LRP1 is able to differentiate between different signals to produce specific responses is unclear. The subcellular localisation of LRP1 in neurons and glia of the central nervous system (CNS) was investigated to gain insight into the receptor's role in specific cell types and during maturation. The role of LRP1 in calcium signaling and regeneration following injury was assessed using two of its ligands, metallothionein (MT) and emtinB. Expression and localisation of LRP1 in vivo and in vitro was determined using western blot analysis and immunochemistry. LRP1 was expressed in neurons and glia of post-natal day 2 (P2), P7 and adult rat brains. Greater immunoreactivity was demonstrated in P2 compared to adult brain, mainly due to increased expression in neurons and oligodendrocytes. Neuronal expression was greatest in cell bodies of hippocampal and cortical neurons in P2 and adult brain. Glial expression of LRP1 was greatest in oligodendrocytes of the corpus callosum and hippocampal fimbria, but LRP1-positive astrocytes and microglia were also present throughout the brain at both time points. These data suggest that LRP1 has a role in neuronal and glial function and that this changes during maturation. LRP1 was localised to cell bodies of hippocampal neurons 3, 7, 14 and 21 days in vitro (DIV). The receptor was also expressed on dendrites at all time points, and did not co-localise with synaptophysin, PSD-95 or the NMDA receptor subunit, NR2a, but did partially co-localise with NR2b. Addition of MT and emtinB did not induce calcium influx in live neurons demonstrating that LRP1 mediated synaptic regulation is not modulated by these ligands in this cell type. LRP1 was also expressed along and at the ends of 3DIV and some 7DIV axons, suggesting a role in neurite outgrowth. This hippocampal neuron model was used to investigate the role of LRP1 in response to injury. LRP1 was expressed at the ends of extending neurites 24 hours after scratch injury, suggesting that it is involved in neurite regeneration. 10˜í¬¿g/mL MT did not promote neurite extension but 25˜í¬¿M emtinB significantly increased the number of neurites that extended along or into the scratch injury site (57.8 ¬¨¬± 4.6 neurites/cm injury site) compared to saline controls (27.7 ¬¨¬± 3.3 neurites/cm injury site; p<0.005) and cultures treated with both emtinB and the LRP1 inhibitor, receptor associated protein (36.5 ¬¨¬± 3.2 neurites/cm injury site; p<0.005). These findings demonstrate the ability for LRP1 to promote neurite extension following injury through stimulation by emtinB. This thesis presents the temporal, regional and subcellular expression profile of LRP1 in neurons and glia of the CNS and how this may influence the receptor's function in specific cell types during normal and injury conditions. The data support a role for LRP1 in neuronal biology and provide the foundation for further investigation into how LRP1 regulates neuron function.