Interactions between metallothionein and neurons of the CNS

The primary function of metallothioneins (MTs) remains elusive since their discovery 50 years ago. These small proteins are characterised by their metal-binding capability, implying roles in metal ion homeostasis and free radical scavenging. This study focuses on MT in the central nervous system (CNS), where MT-I and -II are the topic of much recent research into their activity in promoting neurite growth and enhancing wound healing in various in vivo and in vitro models. This thesis aims to investigate the interaction of extracellular MT-I/II with neurons and the molecular mechanism through which it impacts neurite outgrowth. The physiological role of MT-I/II in CNS development will also be examined. Within the CNS, MT-I/II expression is primarily non-neuronal, yet MT-I/II alters the growth of neurons, suggesting an extracellular action. In order to investigate the mechanism by which extracellular MT-I/II interacts with neurons, rat cortical neurons were exposed to substrate-bound or soluble exogenous MT-IA. An increase in process length was noted only in the presence of soluble exogenous MT-IIA, suggesting that a specific interaction of MT-I/II and neurons is required for neurotrophic activity. Indeed, extracellular MT-I/II was internalised by neurons, mediated by an interaction with the endocytic receptor megalin. Moreover, MT-I/II treatment resulted in activation of the MAPK/ERK signalling pathway, providing a downstream target of MT-I/II. When MT-I/II was ectopically expressed in neurons, neurite outgrowth was augmented, implying an intracellular action. Further, the effect of exogenous MT-IIA on a population of growing, immature cortical neurons was examined. Neurite growth was not observed in this model of neurite elongation in contrast to that seen in neurite outgrowth models and possible reasons for this were addressed. Finally, MT-I/II null mice display no major phenotype; however subtle alterations in cognitive function and axonal structure have recently been reported. Within this study a reduction in axonal calibre at one month of age was observed in mice lacking MT-I/II genes. Moreover, the expression and phosphorylation of neurofilaments was delayed in MT-I/II null mice, suggesting that MT-I/II may contribute to normal axonal development. In summary, this thesis provides some insight into the possible molecular mechanisms through which extracellular MT-I/II interacts with neurons to promote neurite growth and suggests that MT-I/II expression contributes to the normal development of axons. Understanding of the physiology of MT-I/II and mechanisms through which it operates may contribute to development of MT-I/II-based therapeutics applicable to CNS injury and neurodegenerative diseases