Novel engineered nanobodies specific for N‐terminal region of alpha‐synuclein recognize Lewy‐body pathology and inhibit in‐vitro seeded aggregation and toxicity. (25th February 2022)

• Record Type: Journal Article
• Authors: Hmila, Issam; Vaikath, Nishant N.; Majbour, Nour K.; Erskine, Daniel; Sudhakaran, Indulekha P.; Gupta, Vijay; Ghanem, Simona S.; Islam, Zeyaul; Emara, Mohamed M.; Abdesselem, Houari B.; Kolatkar, Prasanna R.; Achappa, Devaya K.; Vinardell, Tatiana; El‐Agnaf, Omar M. A.
• Is Part Of: FEBS journal. Volume 289:Number 15(2022)
• Page Start: 4657
• Page End: 4673
• Publication Date: 2022-02-25
• Subjects: aggregated alpha‐synuclein -- Lewy body -- nanobody -- Parkinson's disease; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.16376
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Nanobodies (Nbs), the single‐domain antigen‐binding fragments of dromedary heavy‐chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha‐synuclein (α‐syn). Phage display screening of the library allowed the identification of a nanobody, Nbα‐syn01, specific for α‐syn. Unlike previously developed nanobodies, Nbα‐syn01 recognized the N‐terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα‐syn01 and the engineered bivalent format BivNbα‐syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα‐syn01 and its bivalent format recognized preferentially α‐syn fibrils compared to the monomeric form. Nbα‐syn01 and BivNbα‐syn01 were also able to inhibit α‐syn‐seeded aggregation in vitro and reduced α‐syn‐seeded aggregation and toxicity in cells showing their potential to reduce α‐syn pathology. Moreover, both nanobody formats were able to recognize Lewy‐body pathology in human post‐mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα‐syn01 binds the N‐terminal region of the α‐syn aggregated form. Overall, these results highlight the potential of Nbα‐syn01 and BivNbα‐syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders. Abstract : A nanobody library specific to the monomeric form of α‐synuclein was constructed. The identified Nbα‐syn01 recognizes the α‐syn N‐terminal region prone to mutation. Nb α‐syn01 and its bivalent format recognized preferentially α‐syn fibrils compared to the monomeric form and they inhibited the α‐syn seeded aggregation in vitro and reduced α‐syn seeded toxicity in cells. The nanobodies also recognized Lewy bodies in human post‐mortem brain tissue from PD and DLB cases.