Binding of Noradrenaline to native and intermediate states during

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Binding of Noradrenaline to native and intermediate states during fibrillation of #-Synuclein leads to the formation of stable and structured cytotoxic species Priyanka Singh, and Rajiv Bhat ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00650 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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ACS Chemical Neuroscience

Binding of Noradrenaline to native and intermediate states during fibrillation of α-Synuclein leads to the formation of stable and structured cytotoxic species

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Priyanka Singh and Rajiv Bhat*

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School of Biotechnology, Jawaharlal Nehru University, New Delhi, India 110067

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, the major protein constituent of Lewy bodies that are the pathological hallmark of 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 FRET measurements that resulted in its binding in close proximity (23 Å) of Alexa labelled A90C mutant of α-syn. Docking studies further suggested binding of NA to the C-terminal as well as NAC region of α-Syn. We have shown that α-Syn oligomerization into SDS-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 MTT cytotoxicity assay using 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.

KEYWORDS: Parkinson’s disease, Aggregation, Amyloid oligomers, α-Synuclein, Noradrenaline

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INTRODUCTION

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Parkinson’s disease (PD) involves α-synucleinopathy that impairs central nervous system with characteristic clinical features that comprise bradykinesia, gait instability, rigidity and resting tremor (1,2). Apart from motor inability, PD also entails non-motor symptoms that lead to hallucinations, olfactory disturbances, pain, sleeping troubles, depression and dementia (3). Alpha synuclein (α-syn) is an intrinsically disordered, 140 amino acid long, 14 kDa protein which is the major constituent of Lewy bodies, the pathological hallmark of PD (4). It is composed of an amphipathic N-terminus (residues 1-61) which forms an alpha helical structure when associated with lipid vesicles (5,6), a central hydrophobic, aggregation prone NAC (non-Aβ-component) region (residues 62-95) and a negatively charged C-terminal region (residues 96-140) rich in acidic residues. The existence of physiological α-syn in either oligomeric (principally tetrameric) or unfolded monomeric form is currently being debated (7-11). The mechanism of aggregation of α-syn involves changes in its conformation from unfolded disordered state to partially folded conformation forming protofibrils and fibrils (12). Studies have shown that early intermediary oligomers, rather than the mature fibrils of αsyn, are toxic to the neurons (13-15).

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Although pathology of PD is primarily characterized by typical disturbances of the function of basal ganglia, evidently not only dopaminergic systems of the central nervous system (CNS) are involved in degenerative process but noradrenergic (16,17) neurons also show serious cytoskeletal damage in addition to the altered nigrostriatal dopaminergic pathway. It has also been reported that there is a prominent but less appreciated loss (> 80%) (16) of locus coeruleus (LC) neurons in PD brain, which is the foremost source of noradrenaline in the brain (18). In addition to the remarkable decrease in noradrenergic cell bodies (19), noradrenaline concentration has also been reported to be considerably declined in the PD brain (20) which might contribute not only to some of the motor symptoms but also to non-motor functions. In several studies, Neuromelanin (NM) has been reported to be involved in pathogenesis of neurodegenerative diseases like PD (21). NM accumulates in LC during aging (22) and predominantly consists of various catecholic metabolites including NA and its metabolites formed by the oxidative deamination by monoamine oxidase (23,24). The studies indicate that oxidised metabolites of NA play a role in disease pathogenesis. α-Syn is known to be expressed in both neuronal and non-neuronal cells. Very recently, overexpression of α-syn has been shown to inhibit neurotransmitter release (25). Also we have shown that different aggregating species of α-syn formed in the presence of Ca (II) and dopamine (DA) neurotransmitter are internalized into the human neuroblastoma cells with different rates and are responsible for the differential cytotoxicity (26). In a recent study, combination of the two catecholamines, dopamine and noradrenaline have been shown to both inhibit the formation of α-syn fibrils and disaggregate the existing fibrils, yielding oligomeric form of α-syn (27). Also, it has been reported that the loss of either noradrenaergic or dopaminergic neurons can affect the function of each other, indicating the importance of both the noradrenergic and dopaminergic systems in PD (28). There are also reports, where upregulation and accumulation of αsyn in the LC region has been observed (29,30). These studies thus point to a link between α-syn and the neurotransmitter noradrenaline and their possible interactions in the noradrenergic LC neurons that might play a critical role in disease pathogenesis.

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Keeping in view the relevance of noradrenergic system in PD, it is important to investigate in detail the effect of noradrenaline on the conformation and aggregation kinetics of α-syn as well as cytotoxicity of various aggregating species of α-syn formed in the presence of NA. Our results demonstrate that NA in its oxidised form effectively inhibits α-syn fibrillation in a concentration dependent manner as well as disassembles α-syn mature fibrils into higher order, structured amyloid oligomers which are SDS-resistant and cytotoxic to the neuronal cell line. Titration calorimetric 2 ACS Paragon Plus Environment

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studies showed that NA interacts weakly and non-specifically with species of α-syn corresponding to every stage of the fibrillation pathway. The studies signifythe role of NA in PD and could help in the development of alternative strategies to cure the disease.

RESULTS AND DISCUSSION

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Inhibition of fibril formation by NA. To study the effect of NA on α-syn fibrillation pathway, we incubated α-syn (70 µM) at 37°C for 48 h with increasing concentration of NA (10 µM - 200 µM). The kinetics of fibrilliationwere examined by the dye Thioflavin T (ThT), which exhibits fluorescence spectral changes when bound to amyloid fibrils but not to monomers or prefibrillar species (31). ThT fluorescence increased in the absence of NA following the pathway that involved an initial lag phase, exponential phase and a saturation phase characterized by the formation of mature fibrils. The addition of NA was found to inhibit fibril formation in a concentration dependent manner with a substantial suppression at 50 µM and complete suppression at higher concentrations as there was a significant decrease in ThT intensity with increasing concentration of NA (Figure 1A).

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Light scattering measurements at 90° using fluorescence spectrophotometer with excitation and emission wavelengths of 500 nm each, were carried out on the species formed during the fibrillation pathway in the absence and presence of NA (Figure 1B). A prominent decrease in scattering was observed in the presence of 200 μM NA that is the concentration at which maximum fibril suppression was observed, suggesting the accumulation of smaller aggregates as compared to control where mature fibrils contributed to large scattering. NA-mediated formation of α-syn aggregates was observed to be concentration dependent and supports earlier findings wherein excessive catecholamines in the cytoplasm of neurons have been proposed to lead to neurodegeneration (32).

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The α-syn species formed at the end of fibrillation were visualized by TEM. In the absence of NA, the sample was observed to be composed predominantly of dense network of large sized, entangled fibrils. The density of fibrils decreased to some extent in the presence of 50 μM NA that further led to the formation of smaller sized aggregates in the presence of 100 μM NA. At the final concentration of NA used (200 μM), heterogeneously sized, spherical and granular aggregates were the predominant species, again suggesting suppressed fibrillation in a concentration dependent manner (Figure 1C). This report validates the previous observation by Fischer et al (27) wherein α-syn has been found to readily form oligomers in the presence of NA.

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Disassembly of α-syn fibrils by NA. To gain further insight into the effect of NA on different stages of fibrillation kinetics of α-syn, ThT assay was performed by adding 200 µM of NA at different time points during the fibrillation pathway. It was found that, when NA was added at the beginning of fibrillation, there was almost complete inhibition of fibril formation, denoted by no further increase in ThT fluorescence even at the end of fibrillation. Intriguingly, the addition of NA at any other stage, whether exponential phase or stationary phase of fibrillation pathway, resulted in a rapid decrease in the ThT fluorescence intensity (Figure 2A). As the sudden drop in fluorescence signal appeared unusual, light scattering measurements were performed on species formed at the end of the fibrillation assay to further authenticate the effect of NA on fibrils. As compared to control, up to 60% decrease in the light scattering was noticed by species formed when NA was added at the initial stage of fibrillation pathway, whereas the decrease in light scattering was approximately 40% by species 3 ACS Paragon Plus Environment


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formed after NA addition at other stages which indicated the disassembly of preformed fibrils of αsyn to some extent by the addition of NA (Figure 2B). This suggested that NA was able to disaggregate the species into smaller aggregates, irrespective of the stage they were in and that, it could be binding to species corresponding to each and every stage of fibrillation pathway of α-syn.

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These results were further supported by TEM images which did not show any large sized fibrils in the samples where NA was added during the initial stage of fibrillation. On the other hand, mixed population of fibrils and oligomeric species were seen upon addition of NA at exponential and stationary phases suggesting the disassembly of α-syn fibrils into non-fibrillar oligomers (Figure 2C). Amyloid oligomers have also been reported earlier to have diversity in their structure, morphology and function (33). Disaggregation of samples was additionally confirmed by using AFM. Upon addition of NA at 0 h (Figure 3A Panel IV), large oligomers, clustered into ring like structures were observed with an average height of 15-20 nm unlike α-syn fibrils which were imaged as positive control (Figure 3A Panel III). Samples, where NA was added in exponential phase, exhibited a homogeneous distribution of disaggregated fibrils into comparatively smaller but again clustered oligomers, 7-8 nm in height (Figure 3A Panel V), whereas the corresponding 12 h control (Figure 3A Panel II) showed elongated fibrils with measured height of 6-7 nm. Monomeric α-syn showed a height of 3-4 nm (Figure 3A panel I) which is comparable with the hydrodynamic radius calculated using DLS (SI Figure 3). Interestingly, when NA was added at the fibrillar stage, oligomers with mean height of 2-3 nm could still be noticed along with few fibrils (Figure 3Apanel VI). The morphology of α-syn oligomers formed in the presence of NA revealed that NA promoted the formation of distinct clustered, higher order bead like structures assembled in spherical shapes of different sizes. The cross sectional analysis of the NA treated samples at different times of fibrillation by AFM (Figure 3A) also showed that the resulting species were clustered to form higher order oligomers as the cross sectional peaks were not being resolved. The disassembly of protofibrils and fibrils by NA is consistent with the previous report (27). However, the findings in the current study extend previous studies by demonstrating the morphology of species formed as a result of disassembly of α-syn species corresponding to different stages of fibrillation. From the data, it could be inferred that NA-α-syn interaction in the initial stages of α-syn aggregation pathway is sufficient to prevent the formation of amyloid fibrils. Nevertheless, species formed in all the three cases, although different, clearly displayed non-fibrillar morphologies indicative of disaggregation of fibrils or protofibrils suggesting that NE is capable of binding to species formed in the fibrillation pathway of α-syn. NA promotes formation of insoluble, structured and SDS-resistant oligomers. α-Syn is essentially intrinsically disordered in an aqueous environment and displays an ensemble of structural conformations. In order to structurally characterize the aggregates formed in the absence and presence of NA, far-UV CD spectroscopy that measures the average content of secondary structure of proteins was used. Whereas monomeric α-syn showed a spectrum typical of an intrinsically disordered structure, that of its amyloid fibrils was typical of β-sheets (SI Figure 4). For NA generated α-syn aggregates, we observed a significant increase in β-sheet structure as the fibrillation proceeded, evidenced by the increase in negative ellipticity at 220 nm and absence of 208-210 nm peak with time, pointing towards reduced conformational flexibility (Figure 3B). Similar results were observed when NA was added at different time intervals of α-syn aggregation, illustrating that the species formed after NA treatment also achieved β-sheet structure (Figure 3C). The data indicated a significant increase in β-sheet content of aggregates formed in the presence of NA during the fibrillation pathway, suggesting that NA-induced oligomers could probably be annular protofibrils as they share similar morphologies (34). On the other hand, only a slight reduction has been reported in 4 ACS Paragon Plus Environment

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the random coil content of α-syn in the presence of DA (35,36) signifying that DA forms nonamyloidogenic oligomers in contrast to NA-induced species which are amyloid oligomers.

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To analyse the oligomers and higher order α-syn species formed in the presence of NA, the samples incubated with and without 200 µM NA for 72h were ultracentrifuged at 100,000g for 30 min and the pellet was treated with urea. Interestingly, NA was observed to form SDS resistant dimers, trimers as well as higher order oligomers which were detected in the insoluble fraction of the samples where NA was added at lag phase, exponential phase and stationary phase of α-syn fibrillation pathway (Figure 4). SDS-PAGE analysis of both the fractions confirmed the presence of oligomeric species predominantly in the pellet. The stability of NA-mediated α-syn oligomers, which have been found to be resistant to SDS denaturation, suggests a possible covalent modification of α-syn by NA via lysine side chains as reported in the case of DA (37,38) except that unlike NA, DA was found to form soluble oligomers of α-syn upon incubation. As could be seen in the denaturing gel of soluble fraction, the monomeric band of 72h fibril sample of α-syn (Figure 4A lane 3) was found to have 7580% higher intensity as compared to the NA treated 0h, 12h and 24h samples (Figure 4A lane 4,5,6), demonstrating that significant ratio of NA treated samples were constituting the insoluble species. This was authenticated by SDS-PAGE of urea- treated insoluble content where prominent fibril control monomeric band was observed (Figure 4B lane 3) as compared to NA treated samples, which were 15-30 % less in intensity consisting additionally of dimers, trimers and higher order oligomers (Figure 4B lane 4, 5, 6). The lack of oligomers seen in the gel under same aggregating conditions in the absence of NA suggested that α-syn alone did not form stable oligomers as the fibrils yielded only monomers on the gel (Figure 4A lane 3). On the other hand, the aggregates formed at the end of fibrillation, that involved addition of NA at protofibrillar and fibrillar stages of pathway, were also found to form precisely similar kind of oligomers as noticed in the sample where NA was added at 0h, supporting the observation that NA oligomerizes α-syn as well as disaggregates the preformed fibrils to form similar oligomeric species.

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To further confirm the disaggregation of α-syn fibrils by NA, we measured the absorbance of urea treated pellet at 220 nm in order to minimize the interference by NA absorption at 280 nm and calculated the percent insoluble content which was higher by approximately 30% for NA treated time course samples than that of the fibril control (SI Figure 5). This increase in the concentration of protein in the pellet confirms the disaggregation of fibrils into insoluble oligomers which pelleted down upon ultracentrifugation.

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The soluble fraction obtained after ultracentrifugation was further evaluated by SEC in order to identify oligomeric species, if any. Representative chromatograms of α-syn species generated without addition of 200 µM NA are shown in SI Figure 6A. Monomeric α-syn eluted as a single peak with an apparent molecular weight of 44 kDa at elution volume of 15.7 ml consistent with the previous report by Follmer et al (39). α-Syn aggregates collected at different time points showed subsequent decrease in the intensity of monomeric peaks suggesting the conversion of monomers and oligomers into fibrils. On the other hand, NA treated α-syn samples showed distinguished chromatograms with declining monomeric peaks after 12h incubation, indicating that most of the monomers are converted into insoluble higher order species during the exponential phase of kinetics that last till the end of fibrillation (Figure 5A). Such oligomeric peaks could not be resolved on the chromatographic column as the samples were ultracentrifuged prior to analysis. These peaks could be seen in the samples that were simply centrifuged at 14,000 rpm for 30 min (SI Figure 6B, 6C) which were found to have a 5 ACS Paragon Plus Environment


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hydrodynamic radii of 60 nm as obtained by DLS measurements (SI Figure 6D). Chromatograms with diminishing monomeric peaks as compared to the control fibril sample were observed in each case where NA was added at different time points of fibrillation, suggesting the formation of similar kind of species upon its addition at any stage of the aggregation process (Figure 5B) authenticating the observed differences in the monomer band intensities in denaturing aggregation profile of soluble fractions.

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Effect of antioxidant and reducing conditions on NA-mediated α-syn oligomerization. Formation of SDS-resistant oligomers of α-syn suggests covalent modification at lysines by NA like DA (37). It could thus be hypothesised that the oxidative intermediates of NA might induce oligomerisation of αsyn as in the case of DA where its oxidised form has been found to modify the fibrillation kinetics of α-syn (40). To investigate this possibility, we tested the effect of an antioxidant and a reducing agent on the aggregation of α-syn in the presence NA. α-syn was incubated alone and with 200 μM NA in the presence of ascorbic acid, an antioxidant or DTT (Figure 6). Whereas NA was observed to suppress the aggregation significantly in the absence of reducing agents, there was no noticeable effect seen when samples were incubated with ascorbic acid and DTT. Interestingly, at 100 µM and 1 mM concentration of ascorbic acid, no suppression of α-syn fibrillation was observed. The results were further confirmed by incubating the samples with 10 mM DTT which was also observed to prevent the effect of NA on α-syn fibrillation as it was not able to oxidise in the reducing conditions suggesting that oxidation of NA is essential for the formation of α-syn oligomers. The results are consistent with the findings reported by Pham et al (41) where no DA mediated oligomerisation of αsyn was observed in reducing conditions. This also supports the study by Bisaglia et al (42) where they have demonstrated the potential reactivity of indole-5,6-quinone towards α-syn. Binding of NA with different species of α-syn formed in the fibrillation pathway and TRFS measurements. For a quantitative assessment of the thermodynamics of NA interaction with different fibrillation stage species, we employed ITC as shown in Figure 7 (A-E). NA was taken up in the syringe of the ITC instrument. Aliquots were titrated into the calorimeter cell containing the 0h, 4h, 9h, 12h, and 48h α-syn fibrillation samples. The thermograms obtained for every reaction, showed almost similar binding interaction pattern that was calculated to be in millimolar range with an exothermic heat of binding. The data acquired for each reaction showed best fit for the one-site binding model and possessed one kind of binding site for each type of species with the values of Ka equivalent to (1.18, 1.62, 1.28, 1.2, 1.09) x 104 M-1 respectively, whereas stoichiometry was found to be increased approximately from 1 to 3 which showed that the binding became non-specific as the fibrillation proceeded. ∆G˚ of binding calculated for all the reactions was found to be negative that demonstrated the spontaneity of each reaction (SI Table 2).Further, time resolved fluorescence using time correlated single photon counting (TCSPC) was used to study tyr emission decays. With excitation at 280 nm, the emission decay of α-syn control showed the average lifetime (τavg) of 1.13 ns (SI Table 3) which was insignificantly decreased to 1.02 ns upon addition of NA up to 5-fold molar excess (SI Figure 7). This indicated that the quenching of fluorescence was mediated predominantly by a static quenching mechanism suggesting the formation of a complex as formation of static ground state complexes do not decrease the decay time of the uncomplexed fluorophores because only the unquenched fluorophores are observed (43). Cytotoxicity of NA-induced α-syn aggregates. Further, the toxicity of the species formed with the progression of time was determined using MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5diphenyltetrazolium bromide) assay that quantifies functional mitochondrial activity. All the time6 ACS Paragon Plus Environment

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point samples of α-syn alone and those formed in the presence of NA were found to be significantly toxic to the SH-SY5Y cell line as the cell viability was reduced by approximately 40%. It was interesting to note that the toxicity caused by species formed in the presence of NA at any given point of time, was comparable to what was seen for the control species formed at 9h that are likely to correspond to oligomeric or pre-fibrillar species (Figure 8A). Hence, it could be concluded that similar kind of species were formed in the presence of NA as there was no significant difference noticed in the toxicity levels of the two. In fact, the level of cytotoxicity was found to be comparable for both control as well as NA incubated samples as the reduction in viability was significant in both the samples. This indicates that NA induces the formation of species which exhibit either equal or more cytotoxicity than the controls. Also, the species that were formed after the dissaggregation of different time point α-syn aggregates were found to be cytotoxic (Figure 8B) as the cell viability was significantly reduced to 60% as was observed in the above case. This could be due to the combined effect of decreased length of individual fibrils and increase in the number of fibrillar particles caused by fibril disaggregation which could have potential for membrane disruption as reported by Xue et al (44). The cytotoxicity data of resulting species revealed that NA stabilized the cytotoxic oligomers when bound to initial stages of α-syn and promoted the disaggregation of intermediate species to cytotoxic oligomers and short fibrils that are more toxic than full length fibrils. Reports have also shown that higher order α-syn oligomers are capable of inducing intracellular aggregation (45), supporting the observed cytotoxicity of NA-induced oligomers in our study. This could also be one of the possible reasons for the noradrenergic neuronal decay in PD. The assumption is also supported by the occurrence of α-syn-Lewy inclusions in noradrenergic neurons (46) which might imply molecular events similar to those eventually leading to dopaminergic cell death.

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NA-induced oligomers are capable of seeding the fibrillation of monomeric α-syn. We attempted to investigate whether NA-induced oligomers could function as seeds for the aggregation of monomeric α-syn. For this, 70 µM of α-syn in the presence and absence of seeds was incubated with agitation for 38 hours and fibrillation kinetics were monitored by ThT fluorescence (Figure 9). Mature α-syn fibrils were used to seed monomeric α-syn as a positive control and α-syn monomers without any seeds served as a control. In the absence of preformed seeds, monomeric α-syn assembled into ThT-positive fibrils with a lag phase of ~ 8 h. The presence of 10 % v/v fibrils reduced this lag phase to ~ 3 h. In contrast, the addition of 10% v/v of NA-generated oligomeric seeds decreased the lag phase of fibril assembly to ~ 4 h. This indicated that NA-induced oligomers seed the fibril formation. Interestingly, when α-syn was allowed to fibrillate in the presence of 10 % fibrillar seeds and 200 µM NA, ThT intensity increased at first followed by its decrease upon reaching the maximum intensity suggesting the possible sequestration of ThT binding sites in the resulting species. The kinetic measurements were then correlated with atomic force microscopy which revealed dense fibrils in the positive control sample seeded with fibrils exhibiting average height of 8-9 nm (SI Figure 8A). Elongated fibrils were imaged in unseeded control sample (SI Figure 8B). In contrast, species formed as a result of seeding by NA-induced oligomers were observed to resemble small protofibrillar structure that differed with the morphology of fully matured control fibrils (SI Figure 8C). Species formed in the presence of mature fibril seeds and NA showed distinct structures of varying size (SI Figure 8D). A significant finding was that unlike DA-induced α-syn oligomers, which have been reported to exhibit the capability of seeding the monomeric α-syn (36), species formed in the presence of NA were capable of seeding the fibrillation of α-syn monomers into fibrils with different morphology. The cytotoxicity of these species would need further confirmation.

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FRET and Molecular Docking to unravel possible NA binding sites on α-syn. To obtain additional information on the binding of NA with α-syn, we carried out fluorescence resonance energy transfer (FRET) measurements wherein mutated α-syn A90C was used and labelled with Alexa 350 fluorescence dye. For the improved dye stability, ascorbic acid (8 µM) was added in order to retard photobleaching of labelled protein (47). The spectral overlap integral value for NAAlexa350, estimated as 1.92 X 1013 (Figure 10A), suggested that the FRET pair has a good spectral overlap, which is favourable for the transfer of energy. The Fӧrster distance (R0) of FRET pair was calculated by the spectral overlap integral method. Considering random orientation of the fluorophore dipole moments, the value of R0 was found to be 18.5 Å for FRET pair NA-Alexa350. For FRET analysis, the donor (NA) was excited at 275 nm alone and in the presence of Alexa350 labelled A90C α-syn to observe the quenching of donor fluorescence and enhancement in acceptor fluorescence. The emission spectrum showed moderate quenching in the donor emission along with a relative increase in the acceptor emission, which indicates energy transfer (Figure 10B). Efficiency of energy transfer, E was found to be 0.206 and the distance (r) between the label and NA was estimated to be 23 Å, which was within a factor of 2 of R0and, hence, falling under the acceptable range of r = 0.5R0 to 2R0.

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To supplement FRET studies, NA was docked onto seven selected conformations of α-syn from its experimental structural ensembles in aqueous solution obtained by NMR combined PRE measurements (48)from pE-DB: Protein Ensemble Database (49) and also onto NMR structure of αsyn fibril, PDB id 2N0A (50). Seven conformations were selected from the ensemble based on the RMSD calculations done by comparing a randomly picked structure from the ensemble, with rest of the structures in the ensemble using ProFit server (51). RMSD cut-off of 19 Å was used for choosing the structures. As suggested by the docking scores, the interaction of NA with seven conformations of α-syn as well as with the fibril was found to be significant in each docking model with binding free energies calculated to be around ~ -4 kcal / mol. Several C-terminal residues mainly located in 94FVKKD98 (Figure 11C, SI Figure 9 A-C), along with few residues in NAC region, predominantly 80KTVEG84 (Figure 11C, SI Figure 9 D-E) and 70VVTGV74 (Figure 11B, SI Figure 9C) were observed to establish specific hydrogen bonds and hydrophobic contacts with NA (SI Table 1). Interaction of NA with fibril was also found to be in the C-terminal region; preferentially to 98DQLG101 residues (Figure 11D).

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The calculated donor to acceptor separation distance of 23 Å implied that NA is binding at the site which is 23 Å away from the A90 residue of α-syn. The assumptions correlated well with the molecular docking data where NA was observed to form stable contacts with α-syn, predominantly to residues located in C-terminal region and NAC region. Interestingly, the two binding regions in amyloid forming core of α-syn belong to the sixth and seventh imperfect 11-amino acid repeats containing the KTKEGV consensus sequence (Figure 11A). The results are comparable with the findings using DA by Dibenedetto et al (52), where DA has been shown to bind non specifically to 125YEMPS129 residues of C-terminal and to some residues in NAC region. Observed preferential binding of NA with 98DQLG101 residues in C-terminal region of the fibril structure further authenticates the potential involvement of NA in disaggregation of fibrils. Though molecular docking, which is developed for structured proteins, would have limitations for interaction studies of intrinsically disordered proteins like α-syn, such studies still give an idea about putative interacting sites of NA on α-syn. Further studies to confirm the binding site for the interaction of NA with α-syn at the residue level would be required using NMR or mass spectroscopy to strengthen the findings reported here.

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Based on our results, we hypothesise a model, illustrating the proposed mechanism of NA towards oligomerization of monomeric α-syn as well as the disaggregation of α-syn fibrils (Figure 12).The initial phase involves the interaction of NA to the C-terminal and the NAC region of α-syn monomers to form the early small intermediates. This might be followed by destabilization of the autoinhibitory structure of α-syn which is reported to be stabilized by long range interactions between the C-terminal region and hydrophobic core region (48,53). Although, C-terminal region remains significantly unstructured without participating in the assembly of amyloid forming core, it is known to play a key role in the interactions between the protofibrils necessary for the maturation of fibrils (54,55). Thus, binding of NA to these two critical regions involved in fibril formation would interfere with the assembly of mature fibrils, leading to the accumulation of structured, kinetically stabilized intermediate higher order oligomers. On the other hand, disaggregation of protofibrillar and fibrillar species might be taking place due to the destabilization of fibrillar assembly upon binding of NA, predominantly to the solvent-exposed C-terminal region of α-syn.

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CONCLUSION

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We have identified a novel, physiologically relevant interaction between NA and α-syn, key molecules likely to be implicated in the noradrenergic neuronal decay in PD. Our findings that NA can bind weakly to the initial as well as intermediate states of α-syn fibrialltion pathway leading to the development of cytotoxic species, is, to our knowledge, the first of its kind and provides potential insights to understand the pathogenesis of PD in brain regions other than substantia nigra. The incubation of NA with α-syn over time resulted in a dose dependent formation of distinct SDS-stable, structured, and higher order amyloid oligomers. It was also shown that NA binds to NAC as well as C-terminal region of the protein by FRET measurements and molecular docking. It is, therefore, possible that the decay of LC neurons which is the major noradrenergic centre of brain could possibly take place due to neurotoxic effects of such species. Our investigations into morphology, structure and neurotoxocity of NA-generated α-syn oligomers would help further in our understanding of the mechanism underlying progressive loss of the LC noradrenergic neurons and associated symptoms which could help in the development of alternative strategies to cure PD.

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

METHODS Chemicals and Reagants. NA used in this study was purchased from Sigma (St. Louis, MO). All other chemicals were purchased either from Sigma or from other companies with analytical grade quality. A plasmid containing the full length genes of the human α-syn was a generous gift from Dr. Peter T. Lansbury (Harvard Medical School, Cambridge, MA). The plasmid was further confirmed through sanger sequencing (SI figure 2). Protein expression and purification. Expression and purification of recombinant α-syn was performed as described previously (56) with slight modifications (57,58). The Protein concentration was determined by measuring the absorbance at 280 nm and using molar extinction coefficient, ε = 5960 M−1 cm−1. The purity of protein was determined by 15 % SDS-PAGE (SI Figure 1). ThTfluorescence assay. Fibril formation was monitored using Thioflavin T (ThT) (59). ThT stock solution was prepared in 20 mM sodium phosphate buffer, pH 7.4 and its concentration was calculated using molar extinction coefficient value of 35000 M−1 cm−1 at 412 nm. α-syn (70 μM) 9 ACS Paragon Plus Environment


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samples at pH 7.4 in 20 mM sodium phosphate buffer, 100 mM NaCl were shaked at 37 °C in Varioskan flash microplate reader (Thermo Scientific, USA) at 240 rpm and 10 mm diameter continuously for 48-72 hours or in a Clariostar Plus microplate reader (BMG Labtech, Germany) to assess fibril formation. ThT was excited at 445 nm and emission was recorded at 480 nm with a bandwidth of 5 nm using a 96-well corning, flat bottom plate. Measurements were done in triplicates. Seeding fibrillation assay. For generating seed fibrils, α-syn was allowed to aggregate in the absence and presence of 200 µM NA for 72 hours at 37˚C and 200 rpm. The aggregated samples were then centrifuged at 14000 rpm for 30 min. The fibrillar pellet was washed twice with phosphate buffer and resuspended in 500µl of 20 mM sodium phosphate buffer, 100 mM NaCl, pH 7.4 followed by homogenization by vortexing. 10% v/v of respective seeds were added to monomeric α-syn in order to monitor their effect on fibrillation kinetics using ThT fluorescence on Varioskan flash microplate reader as described for ThT fluorescence assay. Light scattering measurements. α-syn fibrillation in the absence and presence of NA and its fibril disaggregation by NA was monitored by static light scattering at 90° and 500 nm with excitation and emission slits of 2.5 nm each using Cary eclipse spectrofluorimeter (Varian Inc., Palo Alto, USA). Denaturing SDS-PAGE. α-syn solution in 20 mM sodium phosphate, 100 mM NaCl, pH 7.4, was agitated at 37 °C, 240 rpm and 10 mm diameter in the absence and presence of 200 µM NA. At the end of fibrillation, the samples were ultracentrifuged at 100,000g for 30 min (S52-ST rotor, Sorvall MTX 150 ultracentrifuge, Thermo Scientific, USA). Fractions were separated immediately. Supernatant was directly resolved on a 4% –15% SDS –PAGE whereas the pellet was first treated with 8 M urea and then incubated for 1h at room temperature, and resolved on the gel. Image J software was used to quantify the band intensities (60). Atomic force microscopy (AFM). To visualize the fibrils, experiments were performed by diluting the samples of 70µM fibrils to ~1:3 with 20 mM Sodium phosphate, pH 7.4, 150 mM NaCl. Monomeric sample was also prepared with same concentration. Freshly cleaved mica was loaded with 20 µl of α-syn samples and was allowed to adsorb for 20 min. The mica stubs were then rinsed four times with deionized water and allowed to dry until imaged. Imaging was done using AFM (Witec GmbH, Germany) in tapping mode, using a cantilever with a force constant of 40 N/m and a resonance frequency of ~80 KHz. The scan rate was held constant at 0.5 Hz and each image (512 X 512 data points) was flattened and analysed using Project FOUR software (Witec GmbH, Germany). Circular dichroism measurements (CD). CD spectral measurements were performed at 25°C on a J815 spectropolarimeter (Jasco, Tokyo, Japan) with a data point resolution of 0.1 nm and a scanning speed of 50 nm / min using a 0.1 mm quartz cuvette. Changes in the secondary structure of α-syn (70 µM) during fibrillation in the absence and presence of 200 µM NA were monitored in the far-UV region between 190-250 nm by withdrawing samples at different stages of fibrillation. Three consecutive scans were averaged to obtain the final scan and corrected by subtracting corresponding blanks. The data were analyzed using the Jascow32 software provided by the supplier for noise reduction and smoothing. Transmission electron microscopy (TEM). Different time point samples of fibrillation stages were placed on carbon coated 200 mesh copper grids followed by negative staining using 1% uranyl acetate and blotting with filter paper after 10 s. The samples were then allowed to dry in the air for next 30 10 ACS Paragon Plus Environment

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min and examined using a JEOL TEM 2100 microscope operating with an accelerating voltage of 200 kV.

39

I(t) = ∑𝒊𝜶𝒊𝒆𝒙𝒑 (𝝉𝒊)

40 41 42

where αi is the fractional amplitude and τi is the fluorescence lifetime decay of the ith component. The decay curves were fitted to biexponential model and the average lifetime, τavg was calculated using the formula,

43

τavg =

Isothermal titration calorimetry (ITC). ITC analysis of α-syn samples withdrawn at different stages of fibrillation was carried out with a VP-ITC system (MicroCal, Northampton, MA). The cell containing α-syn (210 µM) was equilibrated at 37 °C and titrated with 10 mM NA. In total, 30 injections of 10µl, each with duration of 20s, were carried out with a spacing time of 480s. The cell was continuously stirred at 502 rpm; the reference power was set to 15 µcal/s, and the initial delay was 120 s. For relevant control titrations, data for NA titrated into buffer and buffer titrated into protein solution were subtracted from the actual titration data. A nonlinear least squares method was used to obtain the best fitting parameters for the association constant (Ka), stoichiometry of NA binding to different time point samples of α-syn aggregation pathway (N), enthalpy of interaction (ΔH), change in the entropy (ΔS) and free energy of binding (ΔG). The integration of the calorimeter signals, base-line corrections, fitting of the data and normalization with respect to protein and NA concentration were done using Microcal ORIGIN 7.0 software. MTT cytotoxicity assay. Human neuroblastoma cells (SHSY5Y) were used for cell toxicity measurements. Briefly, 30,000 cells/well were seeded into poly-L-lysine-coated 96-well plates with growth medium as previously described (61) and allowed to attach for 24 h at 37 °C. α-Syn samples fibrillated in the absence and presence of NA and collected at different time intervals were added (final concentration 5µM) to the cells and incubated for an additional 48 h at 37 °C. Cell viability was determined using 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide (MTT) toxicity assay with the addition of 20 µl of MTT (5 mg/ml) to each well. After 3 h, cells and the formed formazan crystals were dissolved in 100% DMSO and incubated for additional 10 min shielded from light. The absorbance at 560 nm was measured and background was subtracted using a Varioskan Flash microplate reader. The statistical analysis was done using Student's t-test and values over 0.05 were considered as insignificant. Time Resolved Fluorescence Spectroscopy (TRFS). Time-resolved fluorescence measurements were carried out using the time correlated single-photon counting (TCSPC) technique with the help of time resolved fluorescence spectrometer FL 920 (Edinburgh Instruments, UK). All the fluorescence lifetime decays of 40 µM α-syn solutions at the ratio of 1:0 to 1: 5 with NA were monitored at the emission maxima (λmax) of 303 nm, using a NanoLED pulsed laser upon excitation at 280 nm. The instrument response function (IRF), was obtained by using a dilute colloidal suspension of silica (Ludox) at the excitation wavelength of 280 nm. Fluorescence lifetime decay curves were collected for 20 ns and resolved into 4096 channels, until a total of 3000 counts accumulated at maximum. The fluorescence decays were deconvoluted using FAST software. The decays were analyzed using the multiexponential model. The multiexponential decay function used was, 𝒕

𝜶𝟏𝝉𝟏 + 𝜶𝟐 𝝉𝟐 𝜶𝟏 + 𝜶𝟐

... (1)

... (2)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

where α1 ,α2 are the amplitudes and τ1, τ2 are the decay constants obtained upon fitting the data to biexponential decay model.

32

Ro6 = 8.79 ⨯ 10-25κ 2 n-4φ J (λ)

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Where κ 2 is the orientation factor, n is the refractive index of the medium, φ denotes the quantum yield of the donor. κ 2 was taken as 2/3 expecting random orientation of the fluorophores, n was typically assumed to be 1.33 as refractive index of water for protein in aqueous solution (63) and φ was considered as 0.10 as reported for tyrosine(64)and thereby for NA, which is tyrosine derived molecule J (λ) is the spectral overlap integral, which can be calculated by using the following equation:

Size Exclusion Chromatography. Fibrillation samples of α-Syn prepared in the absence and presence of 200 µM NA were collected at different time points and centrifuged at 14,000 rpm for 30 min. The supernatant was then injected into a superdex 200 10/300 GL column (void volume, 7.8 ml) (GE healthcare, USA), equilibrated with 20 mM sodium phosphate, 100 mMNaCl, 0.02% sodium azide buffer using Ӓkta Explorer FPLC instrument (GE Healthcare, USA). Fractions were collected and subjected to DLS analysis. The column was calibrated with Thyroglobulin (670 kDa), Bovine γglobulin (158 kDa), Chicken ovalbumin (44 kDa), Equine myoglobin (17 kDa) and Vitamin B12 (1.3 kDa) standards (BioRad) (SI Figure 6A). Dynamic Light Scattering (DLS). Dynamic light scattering measurements were carried out on XtalSpectrosize – 300 DLS / SLS instrument (Hamburg, Germany). For each sample, 10 scans were averaged to obtain final data. Mass histograms were collected for each sample and analyzed with Origin 8.5 Pro software. Fluorescent labelling of A90C (α-Syn). A90C mutant of α-syn was used in the studies as prepared and described earlier (26). The cysteine residue of the mutant (210 µM) was labelled with a 3-fold molar excess of maleimide modified Alexa Fluor 350 (Alexa350) dye (Invitrogen, USA) via thiol moiety as previously reported (62) in 10 mM Sodium Phosphate buffer of pH 7.4. The labeled protein was purified from free dye using PD10 desalting column (GE Healthcare, USA) with Sephadex G-25 matrix. Alexa-labelled α-syn was used for FRET measurements. The labelling efficiency (95%) was calculated using a molar extinction coefficient of 17000 M-1 cm-1 at 350 nm for Alexa350. Fӧrster Resonance Energy Transfer (FRET) Analysis. The FRET efficiency and the distance (r) between the Alexa 350 labelled at cysteine residue of A90C α-syn (acceptor) and NA (donor) was estimated from the spectral overlap of the absorption spectrum of the Alexa350 and the fluorescence emission spectrum of NA, using the equations described elsewhere (43). From the data in excitation and emission spectra, the Fӧrster radius R0 for the FRET pairs was calculated from the excitation and emission spectra using the following equation:

... (3)

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∞

∫𝑭(𝝀)𝛆(𝝀)𝝀 𝒅𝝀 𝟒

1

𝟎

2

J (λ) =

... (4) ∞

∫𝑭(𝝀)𝒅𝝀

3

𝟎

4 5 6

where F(λ) is the emission spectrum of the donor, ε (λ) is the absorption spectrum of the acceptor, both as a function of wavelength (λ). FRET efficiency was calculated by using the following equation:

7

E= 1 -

8 9

Where FDA is the fluorescence intensity of donor (NA) in the presence of acceptor (Alexa350 A90C αsyn) and FD is the fluorescence intensity of donor in the absence of acceptor.

10 11

From the calculated FRET efficiency and R0 value, distance (r) was calculated using the following equation:

12

r = 𝑹𝟎

𝑭𝑫𝑨 𝑭𝑫

𝟔

𝟏―𝑬 𝑬

... (5)

... (6)

13 14 15 16 17 18 19

Molecular Docking. The possible docking modes between α-syn and NA were studied using the AutoDockVina program (65). The molecules were prepared and bonds, bond orders, explicit hydrogens, charges, flexible torsions were assigned if they were missing for both the protein and the ligand before docking. The docking simulations were performed considering a rigid macromolecule (α-syn) and flexible ligand (NA). LigPlot+ program (66), PyMOL and Discovery Studio 4.5 were used for analysis and visualization of the interaction of docked α-syn-NA complex.

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ASSOCIATED CONTENT

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Supporting Information SDS-PAGE analysis of purified α-syn; Sanger sequencing of Recombinant Human α-syn plasmid; Far-UV CD spectra of α-syn control samples; DLS analysis of monomeric α-syn and Molecular docking studies of NA with different conformations of α-syn.

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AUTHOR INFORMATION

29

Corresponding Author

30 31 32

* School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067 India. Email: [email protected]



1 2 3

ORCIDiD Priyanka Singh: 0000-0003-4420-0664 Rajiv Bhat: 0000-0003-2298-3717

4 5

Author Contributions

6 7

P.S. and R.B. designed the research. P.S. performed the experiments. P.S and R.B. analyzed the data and wrote the manuscript.

8 9 10

Funding sources

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The research was supported by DBT, Ministry of Science and Technology, Govt. of India fellowship to P.S. DBT-BUILDER facility (BT/PR/ 5006/INF/22/153/2012) , UPE II and DST-PURSE (DST/SR/PURSE II/11) grants are also acknowledged for consumables. Notes The authors declare no conflict of interest.

16 17

ACKNOWLEDGMENT

18 19 20 21 22 23 24 25 26

The authors thank Prof. Peter Lansbury (Harvard Medical School) for providing the human α-syn clone and acknowledge Dr. Gajendra Saini, Saroj K. Jha, and Dr. Neetu Singh of AIRF, JNU for technical assistance in TEM, AFM and TRFS experiments respectively. Authors would like to acknowledge the use of A90C mutant that was prepared by Dr. Manish Jain earlier in the lab. PS acknowledges the kind help by Geetika Verma (JNU, New Delhi) in ultracentrifugation experiments and Dr. Fatima Zaidi for help in molecular docking studies. PS would like to thank Prof. Perdita Barran, Manchester Institute of Biotchnology for the use of Clariostar Plus microplate reader. The authors thank Dr. Dushyant K. Garg (JNU, New Delhi) for help in AFM and FPLC experiments. P.S. would like to thank DBT, Govt. of India for the research fellowship.

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ABBREVIATIONS

29 30 31 32 33

PD, Parkinson’s disease; NA, Noradrenaline; AFM, Atomic force microscopy; TEM, Transmission electron microscopy; FPLC, Fast protein liquid chromatography; CNS, central nervous system; ThT, Thioflavin T; α-syn, alpha synuclein; CD, Circular dichroism; DLS, Dynamic light scattering; LC, locus coeruleus; TRFS, Time resolved fluorescence spectroscopy; FRET, Fӧrster resonance energy transfer.

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Figure 1. (A)

(B)

(C)

Figure 1.Inhibition of fibril formation by NA.(A)ThT fibrillation kinetics of α-syn in the presence of varied concentrations of NA (0 μM -200 μM). (B) Light scattering analysis at the end of fibrillation. (C) TEM images of α-syn (70 μM) fibrils in the absence of NA (I) and in the presence of 10 μM (II), 50 μM (III), 100 μM (IV), 150 μM (V) and 200 μM (VI) NA. Scale bar = 100 nm.


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Figure 2. (A)

(C)

(B)

Figure 2. Effect of NA on aggregation of α-Syn upon its addition at different time points of fibrillation. (A)ThT fibrillation kinetics of α-syn (70 μM) in the presence of 200 μM NA, with NA added at 0h, 4h, 9h, 12h, 24h and 34h. (B) Light scattering analysis at the end of fibrillation. (C) TEM images of α-syn (70 μM) samples before (I-III) and after addition of NA (200 μM) (IV-VI) at 0h, 12h and 34h.



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Figure 3. (A)

(B)

(C)

Figure 3. (A) AFM images of α-syn (70 μM) samples before (I-III) and after addition of NA (200 μM) (IV-VI) at 0h, 12h and 34h along with their respective cross-sectional graphs shown on the right panel of each image. Scale bar = 1 µM; Arrow signifies the area selected for representation as cross sectional graph. (B) Far-UV CD spectra of different time point samples of α-syn (70 μM) formed in the presence of 200 μM NA.(C) Far-UV CD spectra showing β-sheet structure of α-syn species formed after the addition of 200 μM NA at different time interval of fibrillation kinetics.


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Figure 4. (A)

(B)

Figure 4. Denaturing SDS-PAGE profiles of soluble fraction (A) and insoluble fraction (B) in the absence (lanes 2 and 3) and presence (lanes 4-6) of 200 µM NA added at different time points of αsyn fibrillation pathway. Lane 1 represents the molecular weight ladder, Lane 2 represents 0h monomeric α-syn control (without incubation) and Lane 3 is the α-syn fibril sample. Samples were ultracentrifuged at 100,000 g and the pellet was treated with 8M urea prior to the gel run. Figure 5. (A)

(B)

Figure 5. SEC of α-syn samples pre-incubated in the presence of 200 µM NA and withdrawn at different time intervals (A) and upon addition of 200 µM NA at varied time points (B) using Superdex 200 10/300 GL column. The samples in panel B were subjected to SEC after incubation for 72h. The samples (both A and B panels) were ultracentrifuged at 100,000g before subjected them to SEC.



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Figure 6.

Figure 6. ThT fibrillation kinetics of α-syn (70 μM) alone and with 200 µM NA in the absence or presence of 100 µM and 1 mM ascorbic acid and 10 mM DTT incubated at 37 ˚C, 240 rpm showing the effect of oxidising and reducing conditions on the fibrillation kinetics of α-syn. Figure 7.

Figure 7. Binding of NA with α-syn species, corresponding to different stages in the fibrillation pathway. ITC analysis performed at 37°C by titration of NA (10 mM) into 20 mM sodium phosphate, 100 mMNaCl buffer (pH 7.4), containing α-syn (210 μM) withdrawn at 0h (A), 4h (B), 9h (C), 12h (D) and 48h (E) of fibrillation. Each injection was 10 μl with a total of 30 injections. (Upper) Raw data. (Lower) integrated data. The black line shows the fitted curve assuming a one-site binding model with one type of site. 24 ACS Paragon Plus Environment

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Figure 8. (A)

(B)

Figure 8. MTT cytotoxicity assay for monitoring the cell viability of SHSY5Y cells in the presence of species generated over time in the absence and presence of 200 μM NA (A) and species generated upon addition of NA at different time points of α-syn fib-rillation pathway (B). 5 μM of fibrils and other species were added to 30,000 cells / well. Values represent means ± s.d, ***P < 0.001.C1: cells only and C2: cells + NA. The statistical analysis was done using Student's t-test and values over 0.05 were considered as insignificant. Figure 9.

Figure 9. Analysis of seeded assembly reactions: ThT binding assay. 70µM α-syn incubated alone, with 10% v/v fibrillar seeds, NA-induced oligomer seeds and fibrillar seeds along with 200 μM NA.



1 2 Figure 10. 3 (A) (B) 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 10.(A) The spectral overlap between donor (NA) emission and acceptor (Alexa 350) 20 absorbance. (B) Fluorescence emission spectra of acceptor (green line), donor alone (black line) and 21 in the presence of Alexa 350 labelled A90C α-syn (dashed red line) at λex = 275 nm. The quenching 22 of donor (arrow) with rise in acceptor emission due to energy transfer can be observed. 23 24 25 26 27 Figure 11. 28 29 (A) 30 31 32 33 34 35 36 37 (B) (C) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 26 ACS Paragon Plus Environment

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(D)

Figure 11.(A) Schematic representation of α-syn, showing preferential binding sites of NA in NAC region (61-95) within sixth and seventh imperfect KTKEGV repeat and C-terminal (95-140). Molecular docking analysis of NA interaction with two conformations of α-syn monomer (B, C) and fibril (D). In the schematic diagrams, generated with LigPlot+, the atoms involved in the interaction are shown as a combination of lines and cartoon depiction. In the scheme, Hydrogen Bonds (HBs) are indicated by dashed lines (green) between the atoms involved, while Hydrophobic contacts (HCs) are represented by an arc with spokes radiating toward the ligand atoms in contact.

Figure 12.

Figure 12. A schematic model illustrating the proposed mechanism of NA induced oligomerization of monomeric α-syn and disaggregation of α-syn fibrils. 27 ACS Paragon Plus Environment


Schematic for NA-induced oligomerization of monomeric α-synuclein and disaggregation of fibrils 160x96mm (300 x 300 DPI)

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Binding of Noradrenaline to native and intermediate states during

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