Emtins : novel peptides derived from metallothionein-II as potential therapeutics in Alzheimer's disease

Alzheimer's disease (AD) is a common neurodegenerative disease that is characterised by the extracellular accumulation of plaques, comprised predominantly of the ˜í‚â§-amyloid (A˜í‚â§) peptide. These A˜í‚⧠peptides have demonstrated toxic effects on neurons under a variety of experimental conditions. Metallothioneins (MTs) are a family of low molecular weight, metal binding proteins that have demonstrated neuroprotective and neuroregenerative properties. Recently MTs have been shown in neuronal cultures to be protective against a toxic, copperbound form of A˜í‚⧠(CuA˜í‚â§). MTs have been proposed as a potential therapeutic for the treatment of AD, however, MT does not cross the blood brain barrier (BBB) to an appreciable degree, which limits its capacity as an AD therapeutic. More recently, synthetic peptides based on the MT sequence, termed emtins, have been developed. Emtins have not only reproduced some of the properties of MT but, importantly, one peptide has been demonstrated to cross the mouse BBB, indicating that emtins may represent a novel therapeutic for the treatment of AD. This thesis investigates the capacity of two emtins, EmtinB and EmtinAc, to protect cultured rat hippocampal neurons against CuA˜í‚⧠with the aim of determining the most effective peptide for ongoing studies. EmtinB was the more effective of the two emtins tested in this toxicity model. Furthermore, in a comparison of two EmtinB forms; a tetrameric, 4 -peptide form and a dimeric, 2-peptide form, the dimeric EmtinB peptide was shown to be considerably more protective than the tetrameric form. Three potential mechanisms by which EmtinB might protect cultured rat neurons against CuA˜í‚⧠were investigated: removal of copper from CuA˜í‚⧠to prevent formation of free radicals, direct scavenging of free radicals by EmtinB, and improved neuronal survival via activation of the LRP family of receptors. While no conclusion was reached on its primary mechanistic action, this thesis will show that EmtinB is able to bind metals such as copper and zinc, and can remove copper from the CuA˜í‚⧠complex. Further investigations into mechanism show that EmtinB is also able to protect cultured rat neurons against hydrogen peroxide (which can be generated by CuA˜í‚â§) and that EmtinB is unable to protect against CuA˜í‚⧠in the presence of an inhibitor of the LRP family of receptors suggesting that EmtinB may potentially be interacting with hippocampal neurons via the LRP family of receptors. The in vivo protective capacity of EmtinB was also investigated in this thesis using the APPswe/PS1˜ívÆ E9 mouse model of AD. In this model, EmtinB treatment was shown to improve cognitive outcomes, as measured by Y-maze, and also reduced astroglial activation, as measured by GFAP levels, but did not reduce levels of activated microglia. Although EmtinB treatment appeared to reduce plaque load, these changes were not statistically significant and soluble levels of A˜í‚⧠remained unchanged. This thesis investigates the activity of emtin peptides to protect cultured hippocampal neurons against a toxic copper-bound form of A˜í‚⧠and the ability of EmtinB to reduce cognitive deficits and alter pathological markers of AD in the APPswe/PS1˜ívÆE9 mouse model of AD. Emtin peptides have demonstrated beneficial outcomes in both in vivo and in vitro models of AD, but as yet a single definitive mechanism cannot be ascribed.