Neuroprotective effect of metallothionein

Abstract

Non-mammalian vertebrates such as teleosts have the ability to regenerate brain parts after injury. Previously, we identified significant up-regulation of metallothionein (MT) in the zebrafish brain after brain injury. MT is a relatively small molecular weight peptide rich in cysteine (Cys), and is thought to maintain essential metal homeostasis, heavy metal detoxification and provides protection against oxidative stress. Studies in mammalian models have demonstrated changes of MT expression in several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Hence, this study was designed to investigate the distribution of MT and its possible neuroprotective effect in mechanical and neurotoxin-induced brain injury in the adult zebrafish. The thesis has three experimental chapters with three main objectives as described in below. In objective 1, we localized two MT homologous genes (mt2 and smtb) and their receptor gene (lrp2a) in the brain of zebrafish to investigate their functions by in situ hybridization (ISH). ISH showed mt2, smtb and lrp2a are expressed in different parts of the brain, especially near to the ventricular surface of the brain, most of which are cell proliferating regions. Double-labelling staining further confirmed that mt2-, smtb- and lrp2a-mRNA are expressed in neurons and not in astrocytes, and some cells are co-localized with a cell proliferating marker protein. These results suggest possible role of MTs in neurogenesis, or protection of new born cells in the brain of zebrafish. In objective 2, we assessed the expression of mt2, smtb and lrp2a, and the effect of exogenous MT (human MT2 peptide, hMT2) in the zebrafish following mechanical brain injury to the telencephalon. Upon brain injury, mt2 and smtb expression were up-regulated and remained high until 4 day post-injury (dpi), however, lrp2a expression were down-regulated. ISH revealed increased number of mt2 and smtb mRNA expressing cells in the injured telencephalon at 4-dpi. Exogenous administration of hMT2 up-regulated mt2 expression and increased the number of proliferating cells but reduced apoptotic cells. This effect was blocked by administration of antibody against hMT2. These results demonstrated neuroprotective effect of MT following mechanical brain injury in the zebrafish. In objective 3, we examined the role of MT in a neurotoxin-induced brain injury. Fish were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce degeneration and death of the dopaminergic cell. MPTP administration reduced the total numbers of dopaminergic neurons and the locomotor activity as seen in Parkinson’s disease. Intracranial MPTP administration up-regulated mt2, smtb, lrp2a and dopamine related genes (th1 and th2) expression at day 14 post-injection, which were attenuated by hMT2 treatment. Furthermore, hMT2 treatment induced reduction of dopaminergic neuronal loss and recovery of locomotor activity, suggesting the protective effect of MT against MPTP-induced brain injury. In conclusion, I have presented the possible role of zebrafish MTs in neuronal proliferation in the brain. The up-regulation of mt2 and smtb genes following mechanical and neurotoxin-induced brain injury suggest neuroprotective roles of MTs. Furthermore, administration of exogenous MT successfully promoted recovery of brain injury. Our findings suggest that MT plays an important role in strong neuroregenerative capability of non-mammalian vertebrate brain, which provides a novel insight into the MT as a novel therapeutic target for human brain injury.