Generation and Charactarization of Mouse Monoclonal Antibodies Against Phosphorylated Alphasynuclein at Serine 129

Generation and Charactarization of Mouse Monoclonal Antibodies Against Phosphorylated Alpha-synuclein at Serine 129. Author and co-authors> details: Heba Al- Tarawneh1, Muneera Fayyad2, Nishant Vaikath3, Nour Majbour3, and Omar M. A. El-Agnaf2,3 1Weill Cornell Medical College in Qatar, Education City, Qatar. 2Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar 3Neurological 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 disease after Alzheimer>s disease and one of the leading causes of disability worldwide. It manifests itself through several clinical symptoms, with the most prominent being motor impairment, resting tremors, rigidity and bradykinesia. PD is mainly characterized by a progressive degeneration of dopaminergic neurons in a region within the midbrain known as the Substantia Nigra. The major pathological hallmark of Parkinson’s disease is the appearance of proteinaceous inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs). These depositions are largely composed of a small 140-amino acid protein called alpha synuclein (α-syn). In fact, aggregation of α-syn has been implicated in a number of neurodegenerative diseases, collectively termed synucleinopathies. These include PD, Dementia with Lewy bodies and multiple system atrophy. Given the central role of α-syn in PD and related disorders, extensive efforts have been devoted in investigating the mechanisms that may regulate the aggregation and toxicity of this protein. In the last decade, an increasing number of studies has indicated that while only a small fraction of α-syn ( 90%), raising the possibility that phosphorylation may play an active role in α-syn aggregation. However, findings are contradictory and this hypothesis remains unclear. To date, there are no diagnostic tools that enable early detection of PD. The only Confirmed diagnosis is based on clinical criteria, which are preceded by a prolonged phase of neurodegeneration and an irreversible loss of dopaminergic neurons. This has driven several studies to explore α-syn as a candidate biomarker for PD. Given the strong association of pS129-α-syn with LB pathology, measuring levels of pS129-α-syn in biological samples could serve as a potential biomarker for diagnosis of PD. In this study, we describe the generation and characterization of three mouse monoclonal antibodies (5B9, 6H5, and 9G1) that are specific for pS129 α-syn. These were generated using hybridoma technology and purified by protein-G agarose chromatography. Using a wide range of biochemical assays, we demonstrated the specificity of our new antibodies to pS129 α-syn. This highlights that our highly specific antibodies represent excellent research tools to investigate the role of α-syn phosphorylation in LB pathology. Moreover, our antibodies can be used to develop different quantitative and qualitative immunoassays. Also our antibodies can help answer whether phosphorylation at S129 suppresses or enhances α-syn aggregation and toxicity, which is crucial for the understanding of synucleinopathies pathogenesis and thus the development of new disease-modifying therapies for PD and related disorders.