Development and characterisation of viral vectors to study the molecular mechanisms of Parkinson's disease

• Main Author: McMillan, Kirsty Jane
• Published: University of Bristol 2013
• Subjects: 616.833
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654440

MicroRNAs are a newly described class of short endogenous non-coding RNAs, which bind to the 3' untranslated region of a target mRNA molecule and result in either their degradation or inhibition of their translation. Recently microRNAs have been shown to play a role in neurogenesis in the adult brain and with neuronal deterioration in neurodegenerative disorders. In particular, microRNA-7 has been shown to bind to both alpha synuclein and the epidermal growth factor receptor (EGFR). Alpha synuclein is known to play a key role in the pathogenesis of Parkinson's disease (PD), which is a common neurodegenerative disorder, whilst the EGFR has been shown to be decreased in PD patients in the subventricular zone (SVZ). The SVZ is one of two areas of the adult brain thought to be involved in neurogenesis. Therefore the aim of this thesis has been to investigate the role of miRNA-7 in the regulation of alpha synuclein and the EGFR further. Two lentiviruses were produced, one to cause an overexpression of miRNA-7 and another to cause a loss of miRNA-7 by acting as a target/sponge sequence for miRNA-7 (miRNA-7T). These lentiviruses were firstly transduced into HEK293T cells where miRNA-7 was found to bind to the 3 'UTR of the SNCA gene and inhibit translation causing a decrease in alpha synuclein expression. The miRNA-7T lentivirus was found to effectively bind to endogenous miRNA-7 causing an increase in alpha synuclein expression in HEK293T cells. To investigate this further, the miRNA-7T lentivirus was injected into the SNpc of mice. Data showed that 2 injections of the virus were sufficient to cause an overexpression of the virus in the SNpc. This resulted in an upregulation of alpha synuclein 24 weeks after surgery and a significant loss of dopaminergic neurons. However, the animals did not show any motor impairment and the effects on striatal DA was only reduced to 30% at 8 weeks post surgery. There was therefore, a disparity between the loss of dopaminergic neurons and the levels of striatal DA. To investigate the effect of manipulating miRNA-7 levels on dopaminergic neurons the viruses were also transduced into iPSCs. Unfortunately, the viruses had no effect on alpha synuclein expression in these cells, which may have been due to limitations of the experiment.