Monoamine oxidase (MAO) is an oxidative enzyme that deaminates a variety of amine substrates, including the neurotransmitter dopamine. The enzymatic reaction requires molecular oxygen and produces hydrogen peroxide as a by-product. MAO is localised in the outer mitochondrial membrane and exists as two isoforms, MAO-A and MAO-B, which are differentially expressed in the body and differ in their substrate and inhibitor specificities. Previous studies have suggested that MAO-generated reactive oxygen species (ROS) contribute to oxidative stress in the cell and can directly inhibit electron transport, cause damage to mitochondrial DNA and enhance cell death signalling. In this study the role of MAO in cell death was investigated in dopaminergic neuroblastoma (SH-SY5Y) cells, in three diverse models of mitochondrially-mediated apoptosis. The relevance of MAO in cell death signalling was confirmed with the use of two unrelated MAO inhibitors and the creation of stable SH-SY5Y cell lines that either over express MAO-A or have reduced levels of MAO-A. The study is the first to over express MAO-A using recombinant technology and to use miRNA to stably knock-down MAO-A expression in human neuronal cells. Results confirm that MAO-A is involved in modulating cell death but the mechanism and extent of the involvement depends on the apoptotic inducer. In classical apoptosis induced by staurosporine (STS), cells undergo rapid morphological and biochemical changes indicative of mitochondrially-mediated apoptosis, which is partly dependent on ROS production by MAO-A and induction of mitogen-activated protein kinase (MAPK) signalling cascades. MAO-A protein and catalytic activity are increased in this model, however the mechanism by which this occurs is unknown and is not a result of increased gene transcription. In death induced by growth factor withdrawal, the MAO-A gene is up regulated via p38 and JNK MAPK pathways, which occurs downstream of caspase activation. In both the STS and growth factor withdrawal models, MAO inhibition reduced apoptosis. Most significantly reduced levels of MAO-A expression in 'knock down' cells protected against cell death induced by the complex I inhibitor rotenone, suggesting that MAO has an important role in mitochondrial function. Over expression of MAO-A resulted in stress and apoptosis, followed by a period of cellular senescence and eventually death by necrosis. These data compliment the effects of chronic exposure to oxidative stress in ageing and neurodegeneration. For the first time this work has shown that the MAO-A isoform is an important regulator of STS-induced apoptosis, that MAO-A gene expression is regulated by JNK signalling, and that MAO-A is significantly involved in mitochondrial dysfunction induced by complex I inhibition. These data raise important questions regarding predisposition to the development of neurodegenerative diseases such as Parkinson's disease and to approaches used for their treatment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:493065 |
| Date | January 2008 |
| Creators | Fitzgerald, Julia |
| Publisher | Nottingham Trent University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://irep.ntu.ac.uk/id/eprint/51/ |
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