Development and characterization of monoclonal antibodies for Parkinson's Diseases and Related Disorders

Development and characterization of monoclonal antibodies for Parkinson’s Diseases and Related Disorders Najlaa Al-Thani1, Nour Majbour2, Muneera Fayyad3, N. Vaikath2, and Omar M. A. El-Agnaf2,3 1 Carnegie Mellon University in Qatar 2Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar. 3Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar Neurodegenerative diseases are the leading cause for disability in the world and one of the biggest burdens on our societies. Parkinson's Disease (PD) is the second most common neurodegenerative disease after Alzheimer's Disease (AD). PD is a chronic, progressive and irreversible disorder. The social and economic burden imposed by PD is significantly increasing as populations age. PD was first described by James Parkinson, an English physician, in his assay “Shaking Palsy” over 100 years ago. Although in less than a year PD will enter its next century, many aspects of the disease remain to be elucidated. PD can be sporadic or inherited, and in both cases it is accompanied by degeneration of dopaminergic neurons in the SN. Lewy bodies (LBs) and Lewy nurites (LNs) are the main pathological features of PD, where alpha-synuclein (α-syn) is the main component. α-Syn is a pre-synaptic neuronal protein and its aggregation and dysfunction is linked to a number of neurodegenerative disorders named as “synucleinopathies”. Synucleinopathies mainly refer to PD, dementia with Lewy bodies and multiple system atrophy. α-Syn is 140 amino-acid protein that is highly abundant in the brain and can also be found in red blood cells, plasma, cerebrospinal fluid (CSF) and saliva. The protein is natively unfolded, however, accumulating evidence indicate that enhanced oligomerization and aggregation of α-syn is associated with increased toxicity. Extensive efforts have been put into the elucidation of the mechanisms responsible for the polymerization and aggregation of α-syn. α-Syn undergoes several post-translational modifications such as phosphorylation, truncation or ubiquitination. A better understanding of the role of α-syn post-translational modifications will help to elucidate the exact role of α-syn in the pathogenesis of PD, paving the way to develop new diagnostic and therapeutic strategies for synucleinopathies. Truncated α-syn was found to be abundant in the brains of PD and DLB patients, suggesting that truncated α-syn may play a normal physiological role as well as a pathological one. C-terminal truncated α-syn as it exhibited a higher propensity to fibrilize in comparison with WT full length α-syn. In vitro studies that truncated α-syn specifically C-terminal residues 109-140 promoted aggregation presumably through nucleation formation. Antibodies that recognize both full length and c-terminally truncated a-synuclein are available, however, to study the role of c-terminally truncated a-synuclein in PD, specific antibodies are needed. In this project, we have generated and thoroughly characterized monoclonal antibodies against c-terminally truncated α-syn at Asn122. Following the selection of the stable clones, only the clones of IgG antibody were selected. All the clones were passaged multiple times to identify stable clones and subjected to single cell cloning to achieve monoclonality. The selected clones were cultured on a larger scale, mass culture, and the culture supernatant was then purified using Protein G affinity purification. The mAbs were highly selective for 122 α-syn, and didn't cross-react with 140 α-syn. The specificity and sensitivity of these antibodies were assessed by an array of biochemical methods including slot-blotting, western blotting, ELISA and immunohistochemical analysis. The potential of our antibodies can be explored as diagnostic or therapeutic tools for PD and related disorders.