Design and characterization of Nanobodies to dementia-related α-synuclein strains in Parkinson’s disease

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Project Summary Parkinson's disease (PD) is the second most common neurodegenerative disorder. The typical hallmark of PD is the abnormal accumulation of misfolded α-synuclein (α-syn) and dopaminergic neuron loss in the substantia nigra. This results in the classic motor manifestations of PD, including tremor, bradykinesia, rigidity, and postural instability. Braak's theory described the disease progression as prion-like α-syn spreading, and the PD-related cognitive impairment is due to the increased burden caused by α-syn spreading in the cortex of afflicted individuals. During the disease progression, 50-80% of PD patients develop dementia (PDD), showing increased morbidity and mortality above those with PD alone. To study the roles of α-syn in PD disease progression, we have amplified α-Syn from PD and PDD patients using PMCA (protein misfolding cyclic amplification). We have determined that the amplified α-syn from PD and PDD patients are different strains and the PDD strain α-syn exhibits more severe neurotoxicity than the PD strain. These two distinct α-Syn strains could potentially be used as biomarkers for PD disease progression and drug targets. However, further studies on the roles of these two α-syn strains during the PD progression have been impeded by a lack of reagents for direct recognition of these two α-syn strains. In response, we have designed a new library of synthetic nanobody variants that are redox stable, by removal of the disulfide bond, and have identified a number of nanobodies that recognize α-syn preformed fibrils (PFF), but not the monomeric form. Two out of the five nanobodies that we characterized showed strain selectivity for the phosphorylated α-syn at serine 129 PFF over wild type PFF. The overall objective of this proposal is to design and characterize α-syn strain-specific nanobodies that can recognize either PD- or PDD-strain α-syn, and to test the efficacy of PFF-specific nanobodies in preventing the cell-to-cell transmission of α-syn PFF. Accomplishing this objective will contribute to our long-term goal to understand the roles of α-syn strains in the progression from PD to PDD, which is critical for the development of α-syn strains as biomarkers for PD progression, and to develop new therapeutics for PD. These nanobodies could potentially provide new therapeutic treatment for PD by preventing α-syn PFF spreading. Lastly, nanobodies recognizing distinct strains can also be applied to study other α-synucleinopathies, including dementia with Lewy body and multiple system atrophy.