Abstract
Alpha-synuclein (αS) fibrils are toxic to cells and contribute to the pathogenesis and progression of Parkinson’s disease and other synucleinopathies. β-Synuclein (βS), which co-localizes with αS, has been shown to provide a neuroprotective effect, but the molecular mechanism by which this occurs remains elusive. Here we show that αS fibrils formed in the presence of βS are less cytotoxic, exhibit reduced cell seeding capacity and are more resistant to fibril shedding compared to αS fibrils alone. Using solid-state NMR, we found that the overall structure of the core of αS fibrils when co-incubated with βS is minimally perturbed, however, the dynamics of Lys and Thr residues, located primarily in the imperfect KTKEGV repeats of the αS N-terminus, are increased. Our results suggest that amyloid fibril dynamics may play a key role in modulating toxicity and seeding. Thus, enhancing the dynamics of amyloid fibrils may be a strategy for future therapeutic targeting of neurodegenerative diseases.
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Introduction
Parkinson’s disease (PD) is a progressively debilitating neurodegenerative disorder that is estimated to affect 1% of the world’s population over the age of 601. Amyloid fibril deposits of the protein alpha-synuclein (αS) are found in Lewy bodies (LB) and Lewy neurites (LN)2,3 in the substantia nigra and other brain regions of PD patients. Myriad evidence shows that αS fibrils are toxic to cells4,5,6,7, yet the precise role of αS in the pathology of PD and other synucleinopathies is still unclear.
Several hypotheses have been proposed to explain the experimentally observed cellular toxicity of the fibrils. Among these, seeding-propagation is a proposed mechanism to explain the observed cytotoxicity of the fibrils and the progressive nature of the disease. This process involves the release of mature αS amyloid seeds from the cell8,9,10 that can then be taken up by a neighboring cell10,11; these seeds then template the further misfolding and aggregation of endogenous monomeric αS in the recipient cell7,12. Fibril polymorphism and protofilament packing have been shown to play an important role in seeding capacity and toxicity6,13,14,15,16, while the ability of the fibrils to “shed” oligomer and protofibril species may also contribute to cellular toxicity and propagation17,18. Previous studies have identified some of the cellular-level details of the internalization, seeding, and propagation of αS fibrils10,11,19,20,21,22,23,24,25,26,27. However, these studies lack the information needed to understand the molecular details of how fibrils can template further aggregation, and critically, the mechanisms by which αS fibril seeding of endogenous αS is affected by inhibitors of αS aggregation.
Beta-synuclein (βS), a homologous protein which is co-localized with αS and is expressed at variable levels relative to αS in different synucleinopathies28,29, has been recognized as a natural inhibitor of αS aggregation30. A transgenic mouse model that simultaneously expresses both human αS and βS had fewer inclusions and less neurodegeneration compared with only αS-expressing transgenic mice30. Interestingly, no detectable amount of βS has been found in LB31,32 even though βS can be over-expressed in certain parts of the PD brain28, begging the question of how exactly βS interacts with αS to provide neuroprotection and influence αS fibril-induced cellular toxicity.
We have previously investigated the sequence and domain level interactions that mediate the influence of βS on the aggregation and fibril formation of αS33,34. We have found that head-to-tail transient complexes between βS and αS35, mediated by multi-pronged N- and C-terminal interactions33, provide enough of a kinetic trap at the earliest stages of αS aggregation to slow down the assembly of αS into fibrils. However, even though βS slows down αS aggregation and reduces the overall αS fibril load in a concentration dependent manner35,36, it does not fully abolish αS fibril formation. Therefore, a detailed understanding of the conformational properties and cytotoxicity of αS fibrils formed in the presence of βS will provide us with a deeper understanding of the mechanisms underlying fibril toxicity.
Here, we show that co-incubation of the monomeric intrinsically disordered αS with the monomeric intrinsically disordered βS results in unique “co-incubated αS/βS” fibrils that show a significant reduction in cellular toxicity, a reduction in seeding capacity, and are more resistant to fibril shedding. Solid-state NMR experiments revealed that while the overall structure of the core of αS/βS fibrils is minimally perturbed, the imperfect KTKEGV consensus motif repeats of αS in the preNAC N-terminus become dynamic and more water accessible. Our results offer insight into the mechanism of amyloid fibril toxicity and highlight that increased dynamics of co-incubated αS/βS fibrils may interfere with their templating ability, thereby reducing their seeding capacity. Targeting amyloid fibrils by enhancing their dynamics may be a new strategy in designing therapeutics against neurodegenerative diseases.
Results
Co-incubation with βS induces subtle differences in αS fibril morphology
We studied the differences in the morphology of αS fibrils formed from the incubation of monomeric N-terminally acetylated αS, and αS fibrils formed by co-incubation of monomeric N-terminally acetylated αS with monomeric N-terminally acetylated βS at a 1:3 ratio (called αS/βS co-incubated fibrils). We have used the N-terminally acetylated forms of αS and βS, since this post-translational modification is constitutively present in the native forms of these intrinsically disordered proteins37,38. Consistent with our previous work33,35, βS delays αS fibril formation in the Thioflavin T (ThT) aggregation assay (Fig. S1), whereby the co-incubation of αS with βS results in a longer lag time and slower growth kinetics compared with αS by itself. Fibrils formed as the end products of these two monomer aggregation assays display differences in their polymorph composition. Atomic force microscopy (AFM) images show that αS by itself (Fig. 1a) forms long straight or twisted fibril polymorphs, similar to previous reports13,39,40, while co-incubated αS/βS forms fibril polymorphs that are shorter and straight (Fig. 1b), with no discernable twisting pattern. On average, the height of αS fibrils (6.0 ± 1.1 nm) tends to be shorter than αS/βS fibrils (7.9 ± 1.7 nm) (Fig. 1c), while the length of αS/βS fibrils (0.3 ± 0.2 μm) tend to be shorter than αS fibrils (0.5 ± 0.4 μm) (Fig. 1d).
Morphological differences between αS and co-incubated αS/βS fibrils. (a,b) Amplitude modulated (AM) AFM images of fibrils formed from (a) 70 μM monomeric αS and (b) 70 μM monomeric αS co-incubated with 210 μM monomeric βS. The x-y axes length scale bar is 500 nm. Assessment of the height (c) and length (d) of αS (black) versus co-incubated αS/βS (red) fibrils. Histograms of height and length data are presented as normalized probability densities, and the best-fit probability density function is overlaid to better visualize the distributions.
We quantified the monomer composition of the co-incubated αS/βS fibrils to try to ascertain whether these morphological changes could be induced by incorporation of βS into the protofilaments that make up the fibril. Mature fibrils were solubilized in 4 M guanidine hydrochloride and analyzed by ESI-MS. Surprisingly, the co-incubated αS/βS fibrils are composed of less than 6% βS (Fig. S1).
αS/βS co-incubated fibril core structure is maintained while the N-terminal dynamics are increased
We investigated the conformational and dynamics properties of the αS fibril when co-incubated with βS utilizing solid-state NMR (ssNMR) spectroscopy. We first assessed the secondary structure of our fibrils from 2D 13C-13C and 15N-13C correlation spectra. Figure 2a shows the 2D 13C-13C 100 ms DARR spectra and Fig. S5 the 2D 15N-13C correlation spectra of αS fibrils in black overlaid with co-incubated αS/βS fibrils in red. This experiment utilizes a cross-polarization period to transfer 1H to 13C magnetization, which preferentially detects the rigid residues that make up the core of the fibril and does not detect the dynamic or disordered residues that make up the bulk of the N- and C-terminal regions. The 13C chemical shifts, 13C-13C cross peaks, and 15N-13C cross peaks of the spectra do not show marked differences between αS and αS/βS fibrils, indicating that the core structure of the fibril does not change even when formed in the presence of a stoichiometric excess of βS (Fig. 2a). To ascertain whether our fibrils maintain the common Greek-key motif core structure previously identified13,39,40,41, without conducting a full structure determination by ssNMR, we compared our spectra with the 13C chemicals shifts of αS fibrils deposited into the Biological Magnetic Resonance Data Bank42 (BMRB). Out of 15 total entries in the BMRB of αS fibril chemical shifts (8 human WT, 6 human mutants, 1 mouse WT) only one is associated with a high-resolution 3D structural model of human WT αS fibrils (BMRB 25518, PDB 2N0A)41. Using the 13C chemical shifts from BMRB 25518 we created a 13C-13C chemical shift correlation map, shown in green in Fig. 2c, and overlaid it with our αS fibril 13C-13C spectrum; we observe relatively good agreement between the published chemical shifts and our αS spectrum. In addition, we created a 13C-13C chemical shift correlation map using an average of the 13C chemical shifts from the core residues (44–96) in the 8 human WT entries in the BMRB (Entries: 1693943, 1749844, 1791045, 1824346, 1886044, 2551841, 2553547, 2689048) (Fig. 2d). When overlaid with our αS fibril spectrum, we again find relatively good agreement between the two spectra. This comparison between the previously published 13C chemical shifts, in particular the 13C chemical shifts from PDB 2N0A, and our own 13C spectra suggests that the secondary structure of our αS fibril core is consistent with a Greek-key topology. A full structure determination and peak assignment is currently underway to confirm this assessment.
Characterization of the Fibril Core. (a) Overlay of αS fibril (black) and co-incubated αS/βS fibril (red) 2D 13C-13C correlation spectra (100 ms DARR), showing that the conformation of the core does not differ significantly between these two fibrils. (b) Expansion of select regions of the 2D 13C-13C spectra in (a) that show the major cross peak intensity differences, which are the Lys, Thr and a tentatively assigned Asn residues. (c,d) Overlays of the αS 2D 13C-13C correlation spectrum in (a) with chemical shift correlation maps derived from (c) the published solid-state NMR structure of αS fibrils (green, PDB: 2N0A, BMRB: 2551841) and (d) an average of the 13C chemical shifts of αS fibrils deposited into the BMRB (BRMB: 1693943, 1749844, 1791045, 1824346, 1886044, 2551841, 2553547, 2689048).
While the core of our αS and co-incubated αS/βS fibrils are unchanged, there are some subtle differences in peak intensities between αS and αS/βS fibrils. The Cβ-Cα, Cβ-Cγ and Cβ-CO cross-peaks of the threonine residues show marked intensity decreases in the co-incubated fibril (Fig. 2b). We also observed intensity decreases in the cross peaks of lysine Cε-γ and Cβ-γ of N65 of the co-incubated fibril (Fig. 2b); the N65 Cβ-γ assignment is tentative, and could also plausibly be from I88 Cβ-CO or F94 Cβ-CO. The loss of intensity of these peaks could be caused by increased dynamics of these residues in the co-incubated fibril. To further investigate this point we measured the 13C T1ρ relaxation time49, which reports on μs timescale dynamics, of the threonine Cβ, since these peaks are well resolved from chemical shift overlap of any other residue in the region from ~65–70 ppm (Fig. 2a and Fig. S4a,b). We found that the 13C T1ρ relaxation time of the Thr Cβ’s from co-incubated αS/βS fibrils decreased relative to the Thr Cβ’s from αS fibrils (Fig. S4c), indicating an increase in dynamics of these residues.
To further characterize any differences between αS and co-incubated αS/βS fibrils, we probed the changes in water accessibility and hydration between the two fibrils. Figure 3 shows the water-edited 2D 13C-13C correlation spectra50,51 of 13C,15N-labeled αS fibrils (Fig. 3a) and co-incubated αS/βS fibrils where only αS is uniformly labeled with 13C and 15N (Fig. 3b). The basic premise of this experiment is to observe how the transfer of water 1H magnetization varies across the fibril. The long water 1H spin-diffusion (SD) mixing time (100 ms, black) spectra represent a state where the water 1H magnetization has fully equilibrated across each fibril, while the short water 1H SD mixing time (3 ms, red) spectra illustrate the fibril residues that are in closest proximity to water. The relative proximity or accessibility of a residue to water is then most easily compared by taking the ratio between these two intensities (Int3ms/Int100ms). Due to sample sensitivity and time constraints, in lieu of obtaining residue specific full SD build up curves, the reason for using the ratio between the short and long mixing times is to provide normalization of the intensities measured in the short mixing time experiment and allow for a relative comparison of the initial spin-diffusion buildup rates between different samples. For example, residues that are far from water or are located in the center of the fibril core will have smaller water-accessibility ratios, while residues that are on the surface of the fibril will have larger water-accessibility ratios. This approach has been used previously to probe the differential hydration environments of amyloid fibrils and other biomolecules51,52,53,54,55.
Changes in fibril water accessibility and fibril degradation. (a–c) Water-edited solid-state NMR 13C-13C correlation spectra of (a) αS and (b) co-incubated αS/βS fibrils. Magnetization was equilibrated at long water spin-diffusion times (100 ms, black) compared with the initial water-protein magnetization transfer at short spin-diffusion times (3 ms, red). (c) 1D slices taken at the blue dashed lines in (a) and (b) of αS fibrils (left side) and αS/βS fibrils (right side), showing the intensities of cross-peaks to lysine (top) or valine (bottom) side chains. The ratio of the cross-peak intensities at long and short spin diffusion times (Int3ms/Int100ms) indicates the relative proximity of water on a residue-specific basis. (d) Digestion of αS and αS/βS fibrils at various concentrations of proteinase K. Full-length gels are presented in Supplementary Fig. 6. (e) Map of the residues that show the largest degree of change in water accessibility between αS and αS/βS fibrils, lysine (blue) and threonine (green), on the core-residues (44–96) of PDB structure 2N0A.
Slices from the water edited 2D 13C-13C spectra show decreases in the relative intensities of several of the threonine and lysine cross-peaks of the αS/βS fibrils compared to αS fibrils, while those lysine cross-peaks that remain in the αS/βS fibril spectra have increased water spin-diffusion (i.e. larger Int3ms/Int100ms ratios) relative to αS fibrils (Fig. 3c). The increase in water spin-diffusion of the αS/βS fibril lysine peaks indicates that these residues are more water accessible. Conversely, the water spin diffusion ratios of the hydrophobic alanine and valine residues do not change between αS and co-incubated αS/βS fibrils (Fig. 3c), indicating that the hydration environment of these residues does not significantly change between the two fibrils.
Co-incubated fibrils are more sensitive to proteinase K digestion
Proteasomal impairment has been implicated in several neurodegenerative diseases56, including PD57, and as proteasome activity decreases with age cells become more vulnerable to deleterious protein aggregation58. Therefore, an understanding of how synuclein fibrils and aggregates undergo protease degradation and clearance may shed critical light on PD progression. In order to understand the differences in protease degradation and fibril stability between αS fibrils and co-incubated αS/βS fibrils, we carried out a series of digestion assays with increasing concentrations of proteinase K (Fig. 3c). We observed that co-incubated αS/βS fibrils are more sensitive to proteinase K digestion at a concentration of 5 μg/ml. The co-incubated fibrils display an enhanced propensity to be degraded to low molecular weight species (i.e. intense band at ~5 kDa only) compared to αS fibrils, which have a larger proportion of high molecular species (i.e. intense bands at ~10 and ~15 kDa).
Co-incubated αS/βS fibrils are less toxic and exhibit reduced seeding and proliferation capacity compared to αS fibrils in neuroblastoma cells
Having now characterized the conformational and polymorphic differences between αS and co-incubated αS/βS fibrils, we investigated how these changes in protofilament packing and dynamics affects the cytotoxicity of the fibrils. It is common practice to sonicate amyloid fibrils directly before use in cell toxicity assays, in order to obtain a more uniform distribution of fibril lengths and size. However the sonication process also produces smaller aggregates and oligomers59, with an unknown distribution of shapes and sizes, that show different toxicities, fibril seeding abilities, and cell incorporation capacities19. In order to avoid forming these additional oligomers, we have elected to use un-sonicated fibrils in our assays of cell toxicity and seeding, with polymorph compositions (Fig. 1a,b) and size distributions (Fig. 1c,d) illustrated in our AFM images. As a consequence of using long un-sonicated fibrils, the concentration of fibrils used in our assays (~1 μM) is higher than the nM concentrations used previously for smaller sonicated fibrils, since these smaller sonicated species are more easily incorporated into cells60,61. However, our fibril preparations are internalized similarly to sonicated fibrils, showing the characteristic fluorescent punctate-like structures (Fig. S7).
Fibrils of αS or αS/βS were added to cultures of human SH-SY5Y neuroblastoma cells and incubated for 24 hours, at which time cellular viability was assessed by the ability of the cells to reduce 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Compared with untreated cells, or cells treated with monomeric αS, fibrils of αS induced a 30% reduction in cell viability (***p < 0.001), whereas αS/βS fibrils had no significant impact on cell viability (Fig. 4a).
Cellular toxicity of αS and co-incubated αS/βS fibrils and their seeding potential. (a) Viability of SH-SY5Y cells assessed by MTS assay after treatment with αS or αS/βS fibrils (1.3 μM monomer equivalents), or monomeric αS (1.3 μM) as control, for 24 hours. Data shown are means and standard errors of the mean (SEM) of 3 independent experiments run in triplicates. ***ANOVA p < 0.001. (b) Confocal fluorescence microscopy images of SH-SY5Y cells treated with 1.3 μM αS fibril (top), αS/βS fibril (middle), or αS monomer (bottom) for 24 hours before fixing and staining. Separate channels are presented showing the presence of all αS species (anti-αS antibody, red), all amyloid species (ThioS, green), and cell nuclei (DAPI, blue), along with the three channels overlaid (merge). The scale bar is 40 μm.
We also investigated the ability of αS and αS/βS fibrils to seed aggregation in vitro using a quiescent thioflavin-T (ThT) fluorescence assay. Figure S3 shows that while preformed seeds of both αS and αS/βS fibrils have the ability to induce fibril formation in the presence of αS monomers, the time for the ThT fluorescence curve to plateau takes longer with αS/βS fibril seeds. This indicates that αS/βS fibrils have a reduced capacity to seed further αS aggregation. We confirmed these observations of αS and αS/βS fibril seeding capacity in cell, by assessing the ability of these fibrils to seed aggregation of endogenous αS in SH-SY5Y cells through the analysis of the fluorescence intensities of dyes that specifically bind to αS and amyloid structures (Fig. 4b). Cells were treated with monomeric αS, αS fibrils or αS/βS fibrils for 24 hours before being fixed and stained with purified mouse anti-αS (anti-α-synuclein) antibody, thioflavin S (ThioS), and 4’,6-diamidino-2-phenylindole (DAPI). Cells were then imaged by confocal fluorescence microscopy, where the anti-αS antibody fluoresces red and indicates the presence of any synuclein species present, ThioS fluoresces green and indicates the formation of amyloid species, and DAPI stains the cell nucleus blue (Fig. 4b). Compared with cells treated with monomeric αS (Fig. 4b, bottom row), cells treated with αS fibrils showed an increase in anti-αS antibody fluorescence of 7.3× (Fig. 4b, top row), while cells treated with αS/βS fibrils showed a smaller increase of 4.4× (Fig. 4b, middle row). ThioS staining indicating amyloid formation showed a similar trend with a 4.8x increase with αS fibrils vs a 3.4x increase with αS/βS fibrils.
Oligomers shed from αS or αS/βS fibrils have different morphologies, toxicities and seeding capacities
It has been hypothesized that as the endpoint of misfolding and aggregation of several neurodegenerative disease associated proteins, amyloid fibrils might act as a “sink” to sequester misfolded toxic species62. However, amyloid fibrils do not represent a completely stable species in solution, rather they exist in a dynamic equilibrium between fibril and oligomer forms. Indeed, toxic oligomers have even been observed to shed from mature αS fibrils over time18. To understand the effect of βS on the stability and equilibrium of αS fibrils, we sought to determine the morphology, toxicity and cell seeding capacities of the oligomers that are shed from αS fibrils and αS/βS fibrils.
We first measured the thermostability of the two fibrils using far-UV circular dichroism (CD) spectroscopy. The CD spectra show that both αS and αS/βS fibrils have the characteristic spectral minimum at 218 nm, indicating the presence of β-sheet structure (Fig. S2). We monitored the change in ellipticity of the 218 nm signal as a function of temperature, and found that change in ellipticity of co-incubated αS/βS fibrils is less than that of αS fibrils as temperature increased, indicating that αS/βS fibrils are more thermostable than αS fibrils (Fig. S2). AFM images show that the oligomers that are shed from αS fibrils (Fig. 5a) primarily adopt small globular morphologies, while oligomers shed from αS/βS fibrils tend to adopt short proto-fibril morphologies with some larger globular species also present (Fig. 5b). We next measured the toxicity of the shed oligomers in SH-SY5Y cells. After a 48 hour period of incubation with shed oligomers from either αS or αS/βS fibrils, we found that oligomers shed from αS reduced cell viability by 17% compared to the untreated cells and cells treated with monomeric αS, whereas oligomers shed from αS/βS did not (Fig. 5c). We also assessed the ability of shed oligomers to seed further aggregation in cells, using confocal fluorescence microscopy. Compared with cells treated with monomeric αS (Fig. 5d, bottom row), cells treated with oligomers shed from αS fibrils showed an increase in anti-synuclein antibody fluorescence of 1.6× (Fig. 5d, top row), while cells treated with oligomers shed from αS/βS fibrils showed an increase of 1.3× (Fig. 5d, middle row). ThioS staining indicates that amyloid formation increased by 1.6× in cells treated with oligomers shed from αS fibrils and by 1.3× in cells treated with oligomers shed from αS/βS fibrils.
Morphology and toxicity of oligomeric species that are shed from mature fibrils. (a,b) AM-AFM images of the oligomeric species that are shed from mature αS fibrils (a) and mature αS/βS fibrils(b). The length scale bar is 500 nm. (c) Viability of SH-SY5Y cells assessed by MTS assay after treatment for 48 hours with the shed oligomers from αS fibrils or shed oligomers from αS/βS fibrils (0.7 μM monomer equivalents), or with monomeric αS (0.7 μM) as a control. Data shown are means ± SEM of 3 independent experiments run in triplicates. **ANOVA p < 0.01. (d) Confocal fluorescence microscopy images of SH-SY5Y cells treated with oligomers shed from αS fibrils (top), oligomers shed from αS/βS fibrils (middle), or with αS monomer (bottom) for 48 hours before fixing and staining. Separate channels are presented showing the presence of all αS species (anti-αS antibody, red), all amyloid species (ThioS, green), and cell nuclei (DAPI, blue), along with the three channels overlaid (merge). The scale bar is 16 μm.
Discussion
Amyloid fibrils of αS are key pathologic features of PD and have been recognized as contributing to the progression of the disease. These fibrils are thought to contribute to cellular toxicity through their ability to seed further aggregation of endogenous αS, and the ability of the fibrils to “shed” oligomer and protofibril species that may be toxic. The αS fibrils studied in this work should be distinguished from fibrils that are contained within aggresome-like LBs. Fibrils that are formed as the end product of the aggregation pathway of αS (either in vivo or in vitro), and are not yet collected into LBs, exist in a dynamic equilibrium with oligomers, as evidenced by the ability of fibrils to “shed” smaller molecular species18,63. Here we have demonstrated that αS fibrils formed in the presence of the natural inhibitor βS, while maintaining similar core structures as αS fibrils alone, exhibit reduced toxicity to neuroblastoma cells, reduced seeding properties, and are in dynamic equilibrium with oligomers that also share reduced toxicity and seeding (Fig. 6).
αS and αS/βS fibril toxicities and seeding potentials. αS aggregates and misfolds along a nucleation-dependent fibril formation pathway, generating various oligomeric species before finally adopting a characteristic repeating cross-beta amyloid fibril structure. When αS aggregates on its own (left pathway), the resulting fibrils are toxic to cultured human neuroblastoma cells, and the oligomeric species that shed from these fibrils are also toxic to cells. However, if αS is co-incubated with βS and allowed to aggregate (right pathway), then the resulting fibrils are no longer toxic to cells, and oligomer species that shed from these fibrils are also non-toxic. The αS and αS/βS fibrils also display differential seeding capacities (confocal images, bottom). αS fibrils are able to efficiently seed amyloid formation, while co-incubated αS/βS fibrils have reduced propensity to seed further aggregation, as evidenced by the difference in green intensity in confocal images.
We have utilized ssNMR and the changes in 13C chemical shifts to probe how the core residues and dynamics of αS fibrils and co-incubated αS/βS fibrils differ from one another. While we have not yet completed a full assignment and structure determination of our fibrils, 13C chemical shifts are very sensitive reporters of amino acid type and secondary structure64. The cross-polarization based ssNMR experiments used in this work preferentially detect molecules and domains that are rigid, and dynamic and disordered domains are not detected. The dynamic regions of αS fibrils are the very N-terminal (residues ~1–43) and C-terminal (residues ~97–140) domains; what we detect in our spectra are the relatively rigid core of the amyloid fibrils (residues ~44–96). The 13C resonances observed in our spectra show characteristic β-sheet chemical shifts. Comparison of our spectra with the chemical shift lists (BMRB Entry 25518) and spectra reported by Rienstra and coworkers41, who have previously determined the core fibril structure of full-length αS by ssNMR, show relatively good agreement (Fig. 2c). Since we have prepared our fibrils in a similar manner to those used for the published αS fibril structure41, we can reasonably assume that our fibrils adopt a similar core structure. αS has a 140 amino acid primary sequence generally described by 3 domains: a 60 residue polyampholyte N-terminal domain, a 35 residue hydrophobic NAC domain, and a 45 residue highly negatively-charged polyelectrolyte C-terminal domain. Lysine and threonine (Fig. 3e) are almost exclusively located in the N-terminal and NAC regions of the αS sequence, where they are clustered into imperfect KTKEGV repeats. These two regions form the “Greek-key” motif of the αS fibril core structures, roughly spanning residues 44–96. Of particular note is the preNAC domain (44–60), which makes up the packing interface between two αS protofilaments13,39,40, and contains two full KTK repeats.
While the overall fold of the core residues does not change between αS and αS/βS fibrils, and is similar to previously determined αS structures, based on the assignments of the Thr and Lys cross-peaks in our 2D 13C-13C spectra of the co-incubated αS/βS fibril we do find changes to these residues that make up the preNAC domain’s final two KTKEGV repeats. First, from the loss of intensity in our 2D spectra and measurements of T1ρ relaxation, we have found an increase in the local dynamics of the Thr and Lys residues of co-incubated αS/βS fibrils relative to αS fibrils alone. Second, we observed that the water accessibility of the Lys residues increases in the co-incubated αS/βS fibrils relative to αS fibrils. Our ssNMR measurements of the fibril core structure (residues 44–96), and residue dynamics and water-accessibility highlight that βS does not perturb the core structure of αS fibrils, but instead may have an impact on the residues at the protofilament packing interface.
Recent structures of αS fibrils determined by cryo-electron microscopy have begun to show the importance of protofilament packing to the observed differences in fibril polymorphism, rather than a change in the conformation of the protofilament core13,39,40. Our AFM images indicate that the primary distinctions between αS and αS/βS fibrils lie in the average height and length, although these parameters are widely distributed (Fig. 1c,d). The more subtle distinction lies with the change in polymorph composition of the fibrils: αS fibrils show a mixture of twisted and straight polymorphs (Fig. 1a) while αS/βS fibrils appear to only have straight polymorphs (Fig. 1b). These observations suggest that βS modulates the packing of the protofilaments in the mature fibril, and is less likely to be incorporated into the cross-beta structure of the individual protofilament. A change in protofilament packing is also supported by the proteinase K digestion profiles, which indicate that αS/βS fibrils are more easily accessible to cleavage by proteinase K, suggesting that the co-incubated αS/βS fibrils might be more susceptible to degradation in vivo (Fig. 3d). These profiles resemble those from previous work by Miake and coworkers, who established that proteinase K digestion of αS preferentially cleaves the N- and C-terminal portions of αS and leaves the fibril core from residues 31–109 intact65. In addition, our results demonstrate that while αS fibrils are indeed toxic to neuroblastoma cells (Fig. 4a), co-incubated αS/βS fibrils are not (Fig. 4a) and have a lesser tendency to cause the formation of synuclein aggregates and amyloid species compared with αS fibrils (Fig. 4b). Therefore, co-incubation of βS with αS results in fibrils that are not toxic to cells and have reduced ability to seed further aggregation in a cellular environment. The reduced seeding ability also suggests that βS interferes with the ability of αS fibrils to catalyze secondary nucleation processes on the fibril surface, proposed previously by Knowles and coworkers66. Taken together with our earlier results, these results again suggest that βS is associated with the N-terminal domain and protofilament packing interface along αS fibrils, inducing more dynamic flexibility in the N-terminal portions of the αS fibrils, and may suggest that N-terminal domain dynamics and packing may play a role in this seeding process.
We have also found that αS fibrils are less thermostable (Fig. S2) and shed primarily small globular and amorphous oligomers (Fig. 5a), while co-incubated αS/βS fibrils are more thermostable (Fig. S1) and shed primarily short proto-fibril aggregates (Fig. 5b). The proto-fibril species shed from αS/βS fibrils also show reduced seeding propensity compared to the small globular and amorphous αS oligomer species (Fig. 5d). This finding suggests that the dynamic equilibrium is shifted away from the formation of small toxic oligomers towards less toxic proto-fibrils in the presence of βS (Fig. 5c). Taken together in the context of our previous observations, the protofilament-packing of co-incubated αS/βS fibrils appears to be more stable than αS fibrils, while increasing the local dynamics of the N-terminal domain. This results in a reduced capacity for seeding and shedding of toxic oligomeric species.
βS has previously been identified in studies of transgenic mice as a natural anti-Parkinsonian factor which has the ability to reduce αS inclusion formation30. Yet, even though it reduces αS positive inclusions, it does not completely abolish the formation of αS fibrils. We propose that the role of βS as an inhibitor is multifaceted, influencing αS aggregation at multiple points along its fibril-formation pathway. In the earliest stages of αS aggregation, βS can stabilize αS in αS-βS heterodimers35, which help to slow down the conversion of αS into higher order aggregates. As αS continues to aggregate, βS has been found to stabilize and eliminate the formation of toxic oligomers67,68. In this work, we have now shown that in the last stage of αS aggregation, co-incubation with βS minimizes the toxicity and seeding ability of αS fibrils, and furthermore alters the fibril-oligomer equilibrium. Our findings demonstrate that βS can reduce the effects of toxic αS fibrils in cells without changing the core structure of αS fibrils, and provide insight into how the dynamics and the surface of these fibrils may directly contribute to their toxicity and seeding ability. The multi-pronged targeting of αS by βS highlights the potential of βS as a lead for the future design of inhibitors that provide therapeutic intervention in synucleinopathies at multiple stages of αS aggregation.
The misfolding and aggregation of endogenous αS monomers due to seeding by fibrils is believed to be critical to the progression of synucleinopathies. The mechanism by which mature αS fibrils seed further aggregation is believed to proceed by surface-mediated secondary nucleation69,70,71, where the surface properties of αS fibrils govern their interaction with endogenous αS monomers and template further aggregation. The exact details of how additional αS monomers undergo templated conversion are not yet known, but the present work provides some clues. The recent cryo-EM structures of αS fibrils show that a steric-zipper motif in the N-terminal domain mediates the interface between two protofilaments and stabilizes the mature fibril morphology13,40. Our results show that co-incubated αS/βS fibrils have increased dynamics and water accessibility of residues in the N-terminal domain, particularly in the KTKEVG repeats in the preNAC region, and allow for enhanced protease degradation of the fibril, suggesting that the protofilament interface may be altered and more dynamic. These observations also highlight the importance of dynamics in mediating the seeding ability of αS fibrils: increased dynamics of the N-terminal domain may lead to reduced seeding, as secondary nucleation may necessitate a rigid N-terminal domain for proper templating of αS aggregation. Our results suggest that enhancing amyloid fibril dynamics at templating domains may be an approach for future therapeutic intervention for neurodegenerative diseases.
Materials and Methods
Protein expression and purification
Expression of N-terminally acetylated human αS and βS proteins was performed via co-expression with pNatB plasmid (Addgene #53613) in E. coli BL21(DE3) cells, and protein purification was performed as described previously72. Uniformly 13C, 15N isotopically labeled αS for ssNMR experiments was expressed in M9 minimal media supplemented with 13C-glucose and 15N-ammonium chloride as the sole carbon and nitrogen sources, respectively. Protein molecular weight and purity were assessed by ESI-MS, and stored at −20 °C as a lyophilized powder until use.
Fibril sample preparation
Lyophilized acetylated αS or βS was dissolved in 10 mM PBS (pH 7.4), and large aggregates were removed by centrifuge filtration (50 kDa MWCO, Millipore Sigma, St. Louis, MO). The dissolved protein was concentrated in 3 kDa centrifuge units (Millipore Sigma, St. Louis, MO) to 1 mg/mL (αS) or 3 mg/mL (βS). To create fibrils, 100 uL of each sample mixture was loaded into 96-well clear bottom plates (Corning, Corning, NY) with a single Teflon bead (3 mm, Saint-Gobain N.A., Malvern PA). The plates were sealed with Axygen sealing tape (Corning, Corning, NY) and shaken at 600 rpm and 37 °C in a POLARstar Omega fluorimeter (BMG Labtech, Cary, NC). Fibrils were allowed to form for at least 72 hours. Samples used for AFM, ESI-MS, ssNMR, and cell toxicity and shedding experiments were collected by centrifugation at 14k rpm for 2 hours, and washed through multiple rounds of re-suspension in 10 mM PBS (pH 7.4) and centrifugation at 14k rpm for 2 hours in order to remove residual soluble and non-fibrillar components.
Preparation of oligomer species shed from fibrils
Fibril samples were re-suspended in 1 mL of 10 mM PBS (pH 7.4) and incubated at 37 °C for 72 h, followed by removal of mature fibrils by using 0.22 µm filter (Millipore Sigma, St. Louis, MO). Samples were concentrated with 3 kDa centrifuge units (Millipore Sigma, St. Louis, MO), and protein concentration was measured using a bicinchoninic acid (BCA) assay (Thermo Scientific, Waltham, MA).
Proteinase K digestion
Fibrils at a concentration of 1 mg/mL were incubated with various concentrations (0.1, 0.5, 1.0, 2.0, 5.0 μg/mL) of proteinase K (Sigma Aldrich, St. Louis, MO) in 10 mM PBS (pH 7.4) at 37 °C for 1 h. The digestion reaction was quenched by the addition of a 1200:1 molar excess of phenylmethane sulfonyl fluoride (Sigma Aldrich, St. Louis, MO) followed by the addition of 2 M guanidine thiocyanate (Sigma Aldrich, St. Louis, MO) and incubation at room temperature for 4 h. The results of the degradation reaction were mixed with 4x SDS-PAGE loading buffer (Invitrogen, Carlsbad, CA), loaded onto precast gels (Bio-Rad, Hercules, CA), and run at 120 V for 50 min.
Atomic Force Microscopy (AFM)
Samples (20 μL) were placed onto freshly cleaved mica (Ted Pella Inc., Redding, CA) and incubated for 15 min at room temperature, followed by 3 washes of 200 μL each deionized water as described previously34. All images were collected on a NX-10 instrument (Park Systems, Suwon, South Korea) using non-contact mode tips (PPP-NHCR, 42 N/m, 330 kHz; Nanosensors, Neuchatel, Switzerland). Image processing and analysis were carried out in the Gwyddion software package73.
Solid-State Nuclear Magnetic Resonance Experiments
All MAS ssNMR experiments were carried out on an Avance III HD 600 MHz (14 T) spectrometer (Bruker BioSpin, Billerica, MA) using a 1.6 mm triple resonance MAS probe (Phoenix NMR, Loveland, CO) tuned to 1H/13C/15N frequencies. Typical radiofrequency (rf) field strengths were 118 kHz for 13C, 74 kHz for 15N, and 100–145 kHz for 1H. 13C chemical shifts were referenced to the 13CH2 signal of adamantane at 38.48 ppm on the tetramethylsilane (TMS) scale, and 15N chemical shifts were referenced to the 15N signal of N-acetylvaline at 122.0 ppm on the liquid ammonia scale. All experiments utilized a MAS rate of 13.333 kHz, and sample temperature was controlled to 25 °C, unless otherwise noted. One-dimensional (1D) 13C MAS spectra were recorded using a conventional cross-polarization (CP) sequence. Two-dimensional (2D) 13C-13C dipolar-assisted rotational-resonance (DARR) experiments74 utilized a mixing period of 100 ms. A 2D water-edited DARR50 experiment, with a DARR mixing period of 100 ms, a T2-filter of 6 ms, and a 1H spin-diffusion period of either 3 ms or 100 ms, was used to measure the water-protein 1H spin diffusion differences between the two fibril samples. 2D 15N-13C correlation spectra were measured using a REDOR-based pulse sequence75, utilizing a REDOR period of 1.35 ms to observe long range correlations. A standard Bruker CP based pulse sequence was used to measure the 13C T1ρ relaxation of the Thr Cβ49. A 13C ω1 field of 2.25ωr was applied on resonance with the Thr Cβ (70 ppm) during the spin-lock period.
Analysis of fibril composition by ESI-MS
Mature fibril samples were dissolved in 4 M guanidine hydrochloride overnight, then buffer exchanged with 50 mM ammonium acetate with 0.1% formic acid. Samples were concentrated to 10 µM for ESI-MS analysis.
Neuroblastoma cell culture
Human SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA) were cultured in DMEM/F12 (GE Healthcare, Boston, MA) with 10% fetal bovine serum (Gibco Co., Dublin, Ireland) and kept in a 37 °C, 5% CO2 humidified atmosphere. Before cell viability assays or immunocytochemistry, cells were plated into 96-well (Corning, Corning, NY) or 12-well plates (Cellvis, Mountain View, CA), and allowed to grow for 24 h.
Cell viability MTS reduction assay
SH-SY5Y cells were treated with 1.3 μM fibril (24 h), 0.7 μM shed species (48 h), or an equivalent concentration of monomer as a control (24 h or 48 h). Cell viability was assessed by adding 20 μL MTS per 100 μL cell culture (Promega, USA) and incubating for 2.5 h at 37 °C, before measuring absorbance at 490 nm.
Immunocytochemistry
SH-SY5Y cells were treated with 1.3 μM fibril (24 h), 0.7 μM shed species (48 h), or an equivalent concentration of monomer as a control (24 h or 48 h). Cells were fixed with 10% formalin (Sigma Aldrich, St. Louis, MO) and permeabilized with 0.5% Triton in PBS (Sigma Aldrich, St. Louis, MO). Cells were then blocked by incubation with 5% Donkey Serum solution (Sigma Aldrich, St. Louis, MO) for 30 min at 37 °C. Cells were incubated with 0.01% thioflavin S (Acros Organics, Waltham, MA) for 10 min at 37 °C and then washed with PBS, followed by incubation with purified mouse anti-α-synuclein primary antibody (BD Biosciences, Franklin Lakes, NJ; Cat.# 610786, RRID: AB_398107) at 4 °C overnight in the dark. Cells were washed with PBS 3 times and incubated with fluorophore-conjugated secondary antibody TRITC (Sigma Aldrich, St. Louis, MO; Cat.# T5393, RRID: AB_261699) for 1 h, then washed again 3 times with PBS. Cells were incubated with DAPI for 1.5 min at room temperature and then washed with PBS, to visual cell nuclei. All samples were imaged using a Zeiss LSM 780 confocal laser scanning microscope with 20x objective (Zeiss, Oberkochen, Germany), and images were processed using the Fiji distribution of ImageJ76. To analyze the anti-αS-antibody and ThioS stained fluorescence images in a more quantitative manner, Fiji was used to filter and threshold each channel based on the DAPI-stained nuclei to extract the raw intensity per cell for each channel. These intensities were then normalized by the intensity per cell of the monomer treated cells, to give the relative increase in intensity reported in the results section. DIC overlays of fluorescence images presented in Figs. 4b and 5d are shown in the SI (Fig. S9).
Preparation of ATTO-550 labelled fibrils
Fibrils were labeled with the fluorescent ATTO550-NHS-ester (ATTO-TEC GmbH) per the manufacturer’s procedure. In brief, fibrils formed as described above were incubated with a 2 M excess of ATTO550-NHS-ester in labeling buffer (pH 8.3 PBS/sodium bicarbonate solution) for 1 hour at room temperature. Conjugated ATTO550-fibrils were then separated from unreacted fluorophore by centrifugation at 16k rpm for 30 min and resuspension of the ATTO550-fibril pellet in pH 7.4 PBS; this centrifugation/resuspension wash was repeated twice.
Statistical analysis
All cell viability experiments were performed in triplicate, and each assay was repeated at least three times. Statistical significance was determined by one-way ANOVA with post-hoc Bonferonni analysis (GraphPad Prism).
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Acknowledgements
This work was supported by National Institutes of Health (NIH) Grant GM110577 (J.B.). M.M.M. is the William Dow Lovett Professor of Neurology and is funded by NIH grants NS096032, AT006868, NS073994, and NS101134, the Michael J. Fox Foundation for Parkinson’s Research, and the American Parkinson Disease Association.
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J.B. and M.M.M. conceived the study. X.Y. expressed and purified protein, and prepared fibril and oligomer samples for all experiments. X.Y. performed the proteinase K assay, AFM and ESI-MS experiments. X.Y. and R.Y. designed and carried out the SH-SY5Y cell culture, MTS assay, and immunocytochemistry experiments. J.K.W. designed and performed the solid-state NMR experiments. X.Y., J.K.W., M.M.M. and J.B. analyzed the data and wrote the manuscript. All authors reviewed, edited and approved the manuscript.
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Yang, X., Williams, J.K., Yan, R. et al. Increased Dynamics of α-Synuclein Fibrils by β-Synuclein Leads to Reduced Seeding and Cytotoxicity. Sci Rep 9, 17579 (2019). https://doi.org/10.1038/s41598-019-54063-8
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DOI: https://doi.org/10.1038/s41598-019-54063-8
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