The role of extracellular metallothioneins in the cellular response to neuronal injury

Metallothioneins (MTs) are unusual, cysteine rich proteins, which can sequester heavy metals (including zinc and cadmium), and also have free radical scavenging properties, which allow them to protect cells from cytotoxicity induced by reactive oxygen species. In the last 10 years, interest in the potential roles of these proteins has shifted from metal homeostasis and free radical scavenging to the neurological functions which they may possess. In this regard, it is the brain specific, MT-III isoform, which has been of most interest. This protein exhibits neuronal growth inhibitory properties upon cultured cortical neurons, and also has been proposed to be involved in the development of Alzheimer's disease. The aim of this thesis is to further investigate the proposed neuroactive properties of MTs, at both a functional and biological level, to determine their possible role within the brain. This study firstly investigated the relationship between structure and biological function of MT-III by investigating the neuronal growth inhibitory activity of a variant MT-III protein previously identified in this laboratory, namely sheep MT-III. This protein was produced recombinantly, and by comparison with recombinant human MT-III, reductions in its metal binding and neuronal growth inhibitory activity upon cultured cortical rat neurons were found. These results indicate the importance of protein structure to MT-III's inhibitory activity, and may also partly explain the susceptibility of sheep to heavy metal induced toxicity. MT-III has. also been proposed to inhibit neurite outgrowth, although all available studies in the literature have alternatively focused upon its ability to inhibit neuronal survival. Using recombinant human MT-III, this study found that MT-III does indeed inhibit initial neurite formation and growth when applied to cultured cortical rat neurons. Furthermore, following axonal transection in culture, MT-III inhibited reactive (or regenerative) neurite sprouting. These results support the hypothesis in the literature that reduced levels of MT-III in the brain allow the aberrant neurite sprouting observed in Alzheimer's disease. Rather surprisingly, it was found that another MT isoform, human MTIA, promoted neurite elongation, reactive sprouting, and growth following injury in the same culture models. At the same time, several reports in the literature demonstrated that MT-I and ‚ÄövÑvÆII knockout mice had significantly reduced cortical wound healing capacity and that exogenous application of MT-II promoted cortical wound healing. To elucidate whether the neuroactive properties observed in culture within this study were involved in this response, human MT-IA was applied following focal cortical brain injury in the adult rat. MT-IIA promoted marked neural recovery following injury, suggesting that MT-I and MT-II might act in an extracellular capacity to promote cortical wound healing, as well as their better investigated intracellular roles. Based upon the demonstration, both within the literature and this thesis, that MTs can modulate neural recovery following injury, it was hypothesized that these properties might relate to their physiological function within the brain. In this regard, this hypothesis would explain the observation within the literature that MT-I and ‚ÄövÑvÆII are up-regulated within astrocytes in response to various forms of brain injury and neurodegenerative disorders. Using neuron/astrocyte co-cultures, this study found a similar pattern of MT-I and ‚ÄövÑvÆII up-regulation following scratch wound injury. However, injury to pure astrocyte cultures did not result in changes in MT-I and ‚ÄövÑvÆII expression, suggesting that MT-I and ‚ÄövÑvÆII respond specifically to neuronal injury. Based upon these results, .this thesis proposes a potential model/hypothesis for MT action within the CNS, where they are upregulated in astrocytes in response to neuronal injury, released, and subsequently promote/inhibit neuronal recovery. In summary, this thesis presents data suggesting an important role for extracellular MTs in the cellular response to neuronal injury. Furthermore, the neuroactive properties of MTs discovered in this work reveal the possibility of metallothionein based therapeutics in the context of brain injury.