Binding of Noradrenaline to Native and Intermediate States during the Fibrillation of α-Synuclein Leads to the Formation of Stable and Structured Cytotoxic Species.

Abstract

Parkinson's disease is characterized by the deterioration of dopaminergic neurons of substantia nigra pars compacta along with a substantial loss of noradrenergic neurons of the locus coeruleus, which is the major source of noradrenaline (NA) in the brain. We have investigated the interaction of NA with α-synuclein (α-syn), the major protein constituent of Lewy bodies that are the pathological hallmark of Parkinson's disease (PD). It is expected that NA, like dopamine, could bind to α-syn and modulate its aggregation propensity and kinetics, which could also contribute to the onset of PD. We have, thus, evaluated the thermodynamic parameters of interaction of NA with α-syn monomer as well as species formed at different stages during its fibrillation pathway and have investigated the conformational and aggregation properties using various spectroscopic and calorimetric techniques. Binding isotherms of NA with α-syn species formed at different time points in the pathway have been observed to be exothermic in nature, suggesting hydrogen bonding interactions and weak affinity with binding constants in the millimolar range in all the cases. The interaction site of NA for α-syn was determined using Förster resonance energy transfer measurements that resulted in its binding in close proximity (23 Å) of an Alexa-labeled A90C mutant of α-syn. Docking studies further suggested binding of NA to the C-terminal as well as the non-amyloid-β component (NAC) region of α-syn. We have shown that α-syn oligomerization into sodium dodecyl sulfate resistant, higher-order, β-sheet-rich species is dependent on the oxidation of NA. Under non-reducing conditions, NA was also found to disaggregate the intermediates, populated during the fibrillation pathway, which are more cytotoxic compared to amyloid fibrils, as observed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide cytotoxicity assay using a human neuroblastoma cell line. On the basis of these and earlier data, we propose that NA-induced formation of α-syn oligomers may contribute to the progressive loss of the noradrenergic neuronal population and the pronounced Lewy body deposition observed in patients with PD.