The role of non-coding regions of the alpha-synuclein gene in protein expression and the determination of alternatively spliced alpha-synuclein isoforms in oxidatively stressed cells

Thesis

Parkinson’s disease (PD) is a neurodegenerative disorder caused by substantial loss of dopaminergic neurons and has been extensively linked to alpha synuclein (α-synuclein). This thesis has investigated the non-coding regions and alternatively spliced isoforms of α-synuclein and an improved cell model of PD in which this might be studied. The results indicate that there are several different 5’ untranslated region isoforms of α-synuclein expressed and that these stimulate significantly different levels of protein expression when analysed using a luciferase assay. Real-time PCR data indicates that these isoforms are differentially expressed; particularly isoform 202 which was up-regulated in both oxidatively stressed cells and a sample of cingulate gyrus from a dementia with Lewy body (DLB) patient. Interestingly this α-synuclein 202 isoform stimulated the lowest levels of protein expression in the luciferase assay. Three of the four previously reported splice isoforms of α-synuclein were detected; 112, 126 and 140 and the expression levels determined by real-time PCR. Results indicate that isoform 112 expression is up-regulated in oxidatively stressed cells and a sample of cingulate gyrus from a DLB patient but this is not seen when levels of total α-synuclein are normalised. The three spliced isoforms were also cloned and used to determine that a novel antibody was able to detect specifically the 126 α-synuclein isoform by western blotting. Finally, progress was made in developing a new protocol for the differentiation of induced pluripotent stem cells (iPSCs) into dopaminergic neurons using modified messenger RNAs encoding dopamine-specific transcription factors (TFs). Five of required TFs were cloned and ligated to the UTRs needed to produce RNA. Additionally we discovered that iPSCs could not only survive plating onto neuronal substrate in neuronal medium with two daily transfections, but they start to express neuronal markers after four days of culture.

Authors: Williams, SE

• Publication date: 2013
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: alpha synuclein; 5' untranslated regions; Parkinson's disease
• Subjects: Biology