oa Nonphosphorylated Alphasynuclein at Serine 129: A Potential Biomarker for Parkinson's Disease

Author and co-authors’ details: Muneera Fayyad1, Nour Majbour2, Mercy A. Thomas2, N. Vaikath2, and Omar M. A. El-Agnaf1,2 1Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar 2Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar. Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. It is characterized by a progressive loss of dopaminergic neurons and an accumulation of Lewy Bodies (LBs) and Lewy Neurites (LNs), protein-rich inclusions that deposit in the neurons. These inclusions are mainly composed of alpha-synuclein (α-syn), a small protein with a propensity to aggregate. Evidence from genetic, biochemical, pathological and animal studies suggest that the aggregation of α-syn plays a key role in the pathogenesis of PD and related disorders, termed synucleinopathies. To date, the diagnosis of PD primarily depends on the clinical criteria, however, this approach has its limitations since the neurodegeneration starts several years before symptoms manifest. In other words, the onset of typical motor symptoms is mostly preceded by 50-70% loss of dopaminergic neurons, which explains the irreversibility of the disease at the time of diagnosis. This necessitates the development of reliable biomarkers that would allow early diagnosis of PD at the preclinical stage. Accumulative evidence suggests that the majority of α-syn from LBs in the brains of PD patients is found to be phosphorylated at Serine 129 (pS129-α-syn). It is unknown whether phosphorylation of α-syn promotes or prevents its aggregation and toxicity. Nevertheless, this highlights the importance measuring the levels of pS129-α-syn in biological fluids. We highlighted that CSF oligomeric-/total-α-syn and p-S129-/total-α-syn ratios improved the discrimination between PD and healthy subjects. However, little is known about a the potential diagnostic role for non- phosphorylated α-syn at S129 (npS129-α-syn). Recently, we developed a novel mouse monoclonal antibody (4B1) that is specific for npS129-a-syn (i.e. does not recognize pS129-α-syn). Here we describe the generation of 4B1 using hybridoma technology. The antibody purification was done using Protein-G agarose affinity chromatography, and later characterized using a wide range of biochemical assays. More importantly, we describe the development of the first ELISA assay to reliably quantify concentrations of npS129-α-syn in biological samples. We also assessed the usefulness of our assay using CSF samples from PD patients and age-matched healthy controls. We report that the discrimination power between PD and healthy controls was improved by including npS129-/ t-α-syn ratio. We highlight that the combination of multiple CSF biomarkers, improved the diagnostic accuracy of PD. Such immunoassays would not only aid in the discovery of ideal CSF biomarkers but may also serve as research tools to facilitate a better understanding of the underlying role of α-syn phosphorylation in PD and related disorders. In addition, our efforts to identify biomarkers for PD may reveal molecular species that could serve as therapeutic targets for the development of new disease modifying therapies.