Development and utilization of Luminex biomarker assays for diagnosis and monitoring of neurodegenerative disease

• Main Author: Ismail, Kurimun
• Other Authors: Allsop, David
• Published: Lancaster University 2016
• Subjects: 616.8
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.695919

A common pathological feature of various neurodegenerative disorders is the accumulation of misfolded proteins in the brain. Neurodegenerative disorders associated with protein misfolding include Alzheimer’s disease (AD), Parkinson’s disease (PD), dementia with Lewy bodies (DLB), fronto-temporal lobar degeneration (FTLD), motor neuron disease (MND), Huntington’s disease and the prion diseases. The incidence and prevalence of most of these diseases is rising, especially those that cause dementia, due to an increase in the average human life span. The diagnosis of neurodegenerative disorders is heavily reliant on physical examinations and assessment of clinical symptoms. The clinical symptoms of many of these neurodegenerative diseases overlap, which poses a huge difficulty for accurate diagnosis, especially in the early stages. This has led to an interest in identifying reliable and robust discriminatory molecular biomarkers. A successful biomarker test will not only provide a more accurate means of diagnosis, but will allow efficient tracking of disease progression, benefitting the process of developing therapeutic strategies. In this project, the development and validation of a bead based assay system that has multiplexing capabilities (simultaneously measure multiple analytes in a single sample via a single assay) has been described. This assay system uses the Luminex technology and has been developed to quantitatively measure phosphorylated α-synuclein, total α-synuclein, total DJ-1 and LRRK2 in human CSF and plasma. These proteins are predominantly implicated in diseases collectively termed α-synucleinopathies. The initial aim of the project was to develop assays for proteins that span a range of neurodegenerative disorders, however, for reasons discussed in the final chapter of this thesis, this was not possible. This project provides evidence on how the use of plasma as a possible matrix for potential markers associated with brain diseases can be justified, since levels of phosphorylated α-synuclein in matched plasma and CSF samples positively correlated with each other. Plasma would be an ideal sample source for biomarker studies, since it is less invasive than obtaining CSF, thus allowing longitudinal studies to be performed. It was also shown how the DJ-1 protein in plasma may carry diagnostic potential by allowing differentiation between PD patients and healthy controls (p=0.004) as well as between PD and MSA patients (p=0.005). The discrimination between PD and MSA is vital since the two diseases are symptomatically very similar, thus posing a greater issue with accurate diagnosis. There has been minimal research discussing the presence of LRRK2 in human biological fluids such as plasma and CSF. This thesis presents the use of western blotting, high performance liquid chromatography (HPLC) and the Luminex technology as a means of detecting this protein in human CSF and plasma. The data related to LRRK2 in this thesis, opens up avenues for further research into this protein; to definitively show whether it can be detected in such biological fluids and whether it has any value as a biomarker.