RESEARCH ARTICLE The small aromatic compound SynuClean-D inhibits the aggregation and seeded polymerization of multiple α-synuclein strains Received for publication, January 12, 2022, and in revised form, March 25, 2022 Published, Papers in Press, April 4, 2022, https://doi.org/10.1016/j.jbc.2022.101902 Samuel Peña-Díaz1,2,‡, Jordi Pujols1,2,‡, Eftychia Vasili3,4 , Francisca Pinheiro1,2, Jaime Santos1,2 , Zoe Manglano-Artuñedo1,2, Tiago F. Outeiro3,4,5,6, and Salvador Ventura1,2,7, * From the 1Institut de Biotecnologia i Biomedicina. Universitat Autonoma de Barcelona, Bellaterra, Spain; 2Departament de Bioquimica i Biologia Molecular. Universitat Autonoma de Barcelona, Bellaterra, Spain; 3Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany; 4Max Planck Institute for Experimental Medicine, Göttingen, Germany; 5Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle Upon Tyne, Newcastle, United Kingdom; 6 Scientific Employee With a Honorary Contract at Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Göttingen, Germany; 7ICREA, Passeig Lluis Companys 23, Barcelona, Spain Edited by Wolfgang Peti Parkinson’s disease is a neurodegenerative disorder charac- and processes, known as Lewy bodies and Lewy neurites, terized by the loss of dopaminergic neurons in the substantia respectively (4), whose primary protein component consists of nigra, as well as the accumulation of intraneuronal proteina- aggregated forms of α-synuclein (α-Syn) (5, 6). This intrinsi- ceous inclusions known as Lewy bodies and Lewy neurites. The cally disordered protein is a soluble monomer with diverse major protein component of Lewy inclusions is the intrinsically functions, like regulating synaptic vesicle release and traf- disordered protein α-synuclein (α-Syn), which can adopt ficking, fatty acid binding, and neuronal survival (7). However, diverse amyloid structures. Different conformational strains of under pathological conditions, α-Syn misfolds and readily ag- α-Syn have been proposed to be related to the onset of distinct gregates. The presence of altered α-Syn in the brain is a synucleinopathies; however, how specific amyloid fibrils cause common feature of the synucleinopathies that, aside from PD, distinctive pathological traits is not clear. Here, we generated include PD with dementia, multiple system atrophy (MSA), three different α-Syn amyloid conformations at different pH and dementia with Lewy bodies (8). and salt concentrations and analyzed the activity of SynuClean- The synucleinopathies differ in their symptomatology, pro- D (SC-D), a small aromatic molecule, on these strains. We show gression, and affected cellular and anatomical compartments. that incubation of α-Syn with SC-D reduced the formation of It has been hypothesized that these differences might ulti- aggregates and the seeded polymerization of α-Syn in all cases. mately rely on the conformational heterogeneity of α-Syn Moreover, we found that SC-D exhibited a general fibril amyloid structures (9–11), in analogy with the human prion disaggregation activity. Finally, we demonstrate that treatment protein, where distinct polymorphs, or strains, cause different with SC-D also reduced strain-specific intracellular accumula- histopathological lesion profiles, with divergent brain region tion of phosphorylated α-Syn inclusions. Taken together, we distributions and clinical manifestations (12). This view is conclude that SC-D may be a promising hit compound to supported by a recent cryo-EM study showing that the inhibit polymorphic α-Syn aggregation. structure of α-Syn amyloid filaments from the brains of in- dividuals with MSA differs from those of individuals with dementia with Lewy bodies (10). The therapeutic implications Parkinson’s disease (PD) is the second most prevalent of this conformational diversity are huge since molecules that neurodegenerative disorder associated with protein misfolding efficiently block the aggregation of one α-Syn strain may not and aggregation after Alzheimer’s disease and affects 0.5 to 1% necessarily recognize another polymorph. of the population aged between 65 and 69 years, increasing to Recombinant soluble α-Syn forms amyloid fibrils in vitro 1 to 3% of people aged over 80 years (1, 2). PD is characterized that, despite differing at the atomic level from those observed by the loss of dopaminergic neurons in the substantia nigra in patients’ brains (10), can seed the aggregation of endoge- pars compacta, resulting in motor symptoms such as rigidity, nous α-Syn in cultured neuronal cells, primary cultured neu- tremor, and bradykinesia and nonmotor symptoms such as rons, and animal brains (13–15). Moreover, synthetic α-Syn dementia (3). A major histopathological hallmark of PD is the fibrils formed under different solution conditions exhibit formation of proteinaceous assemblies in the neuronal body different conformation, seeding activity, and neurotoxicity in cells and when inoculated in rat brains (11, 16–22). Surpris- ‡ ingly, despite compounds like anle138b (23, 24), squalamine These authors contributed equally to this work. * For correspondence: Salvador Ventura, salvador.ventura@uab.es. (25), trodusquemine (26), BIOD303 (27), or fasudil (28) have J. Biol. Chem. (2022) 298(5) 101902 1 © 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). SC-D inhibits α-synuclein strains aggregation been shown to modulate α-Syn aggregation in vitro effectively, Low-magnification transmission electron microscopy studies addressing the activity of small molecules on distinct α- (TEM) images of negatively stained samples evidenced that Syn strains are still scarce (29). fibrillar sheet-like structures with many parallel protofilaments In the present study, we prepared three assemblies from α- and rod-like assemblies coexisted with large and abundant, Syn monomer under different conditions. After confirming apparently amorphous, aggregates in buffer A. In contrast, that they differ in their conformation and in their capacity to isolated fibrils were apparent in buffer B, whereas fibrils tend convert endogenous α-Syn into phosphorylated aggregates in a to cluster into a mesh in buffer C (Fig. S2). High-magnification cellular model, we characterized how SynuClean-D (SC-D), a images revealed that the fibrillar material in the three solutions potent inhibitor of α-Syn aggregation (30), performs on top of displayed different morphology, with fibrils in buffer A being the three polymorphs. We demonstrate that SC-D reduces mostly sheet-like, those in buffer B and C having a more ca- amyloid formation in all the experimental conditions and nonical fibrillar appearance, those in buffer B appearing as disrupts the different fibrils. In addition, it reduces the seeding straight, and the ones in buffer C twisted (Fig. 1, D–F). The capacity of preformed aggregates in vitro and in cells. Alto- width of the fibrils formed in buffers A, B, and C were 8.6 ± gether, the data converge to indicate that SC-D is active 1.9 nm, 14.6 ± 2.7 nm, and 11.9 ± 1.8 nm, respectively. against different α-Syn fibrillar conformations, thus becoming The secondary structure content of the samples was a promising hit compound for developing a generic molecule analyzed using attenuated total reflection Fourier-transform to treat the synucleinopathies. infrared (ATR-FTIR) spectroscopy (Fig. 1,G–I and Table S1). In all cases, the spectra were dominated by a band at 1624 to 1628 cm−1 attributed to the presence of intermolecular β-sheet Results structure. When comparing only the structures formed at α-Syn amyloid fibrils characterization physiological pH, buffer B favors a higher content of disorder SC-D, (5-nitro-6-(3-nitrophenyl)-2-oxo-4-(trifluoromethyl)- (28%) than buffer C (23%), which might account for a higher 1H-pyridine-3-carbonitrile) (Fig. S1A), has been previously sensitivity to proteolysis. Indeed, when we compared the described as an inhibitor of α-Syn aggregation, which can resistance to chemical denaturation with 4 M urea of the two reduce protein misfolding cyclic amplification–induced ag- species in a kinetic assay, we observed that the fibrils formed in gregates propagation and disrupt fibrils, displaying anti- buffer C were clearly more stable and could not be denatured aggregation and neuroprotective activity in animal models of entirely in this harsh condition (Fig. S3). Overall, these results PD (30). However, whether the activity of this small molecule indicated that we had successfully prepared three distinct types is restricted to the assemblies formed in the conditions in of α-Syn fibrils from the same monomer, named here strains which we performed the screening or it can target alternative A, B, and C. α-Syn aggregated conformations was unknown. To decipher this, we generated three distinct α-Syn assemblies in condi- tions that differ from those of previous assays, where we used SC-D inhibits the aggregation of α-Syn into different strains phosphate-buffered saline (PBS). Both pH and salt concen- Once the generation of different conformations was tration have been shown to influence α-Syn fibril formation confirmed, we examined the impact of SC-D (100 μM) in their (16, 31); therefore, we incubated recombinant α-Syn (70 μM) aggregation processes. First, we confirmed that the SC-D does in buffer A (50 mM sodium acetate pH 5.0) and buffer B not exhibit absorbance in buffers A, B, and C at the excitation (50 mM Tris-HCl pH 7.5) to assess the effect of the pH and in (450 nm) and emission (480 nm) wavelengths used to monitor buffer B and buffer C (50 mM Tris-HCl pH 7.5150 mM KCl) Th-T fluorescence in our assays (Fig. S1B). Kinetic analysis to test the impact of physiological salt concentration. indicated that SC-D exhibited a moderate impact on the for- Kinetic analysis indicated that in the three conditions, α-Syn mation of Th-T-positive species of strain A, reducing the assembled into thioflavin-T (Th-T)-positive structures. fluorescence at the endpoint of the reaction by 32% (Fig. 2A). Noticeably, α-Syn exhibited a high Th-T binding in buffer C, In contrast, the impact in light scattering was large, with 45% whereas the lowest levels of Th-T fluorescence corresponded reduction at 340 nm (Fig. 2A inset). Thus, SC-D effectively to those attained in buffer A (Fig. 1A). Thus, both the pH and inhibits the aggregation of α-Syn in buffer A. Its reduced effect the salt of the solution significantly conditioned the kinetics of on Th-T fluorescence can be explained by the low dye binding α-Syn amyloid formation. In contrast, the highest levels of light of the final solution, likely because of the presence of amor- scattering were observed in buffer A, with buffers B and C phous aggregates coexisting with fibrils. TEM images rendering much lower and almost identical signals (Fig. 1B). confirmed a reduction in the number and size of the aggre- This indicated the formation of aggregated species with poor gates in the presence of SC-D (Fig. 2G) when compared with Th-T staining in buffer A. the control samples (Fig. 2D). Proteinase K (PK) digestion of the aggregates indicated SC-D was very effective at inhibiting the formation of α-Syn differential susceptibility to proteolysis. The species formed in amyloid fibrils at physiological pH, both in the absence buffer B were completely cut into two smaller fragments, (Fig. 2B) and presence of salt (Fig. 2C), resulting in a 73% and whereas those in buffers A and C were significantly more 72% reduction of the Th-T fluorescence signal at the end of resistant, while exhibiting different degradation profiles the reaction for strains B and C, respectively. In addition, light (Fig. 1C). scattering measurements indicated that SC-D effectively 2 J. Biol. Chem. (2022) 298(5) 101902 SC-D inhibits α-synuclein strains aggregation Figure 1. Strain characterization. A, aggregation kinetics of strain A (blue), strain B (red), and strain C (green). Intensity of Th-T fluorescence is plotted as normalized means in function of time and error bars as standard errors of mean. B, normalized light scattering measurements of final point aggregates of strain A (blue), strain B (red), and strain C (green). Error bars are shown as standard errors of mean values. C, Tricine–SDS-PAGE gels of soluble α-Syn and aggregated strains A, B, and C before (−) and after (+) PK digestion. D–F, representative TEM images of strain A (D), strain B (E), and C (F) end point aggregates. Scale bars correspond to 200 nm. G–I, ATR-FTIR absorbance spectra in the amide I region of final point aggregates of strain A (G), B (H), and C (I). α-Syn, α-synuclein; ATR, attenuated total reflectance; PK, Proteinase K; TEM, transmission electron microscopy; Th-T; thioflavin-T. reduced the amount of aggregated material for both strains, puzzling and might indicate that despite the two processes are with a decrease in the signals at 340 nm of 44% in buffer B kinetically similar, they might involve different species for (Fig. 2B inset) and 45% in buffer C (Fig. 2C inset). Not sur- which SC-D has a distinct affinity. prisingly, TEM images corroborated that, in both cases, the Strain B seeds were very effective, abrogating the lag phase population of amyloid fibrils was significantly reduced upon of the aggregation process and inducing a faster reaction. SC-D SC-D treatment (Fig. 2, H and I), relative to untreated control did restore the sigmoidal shape of the unseeded reaction but reactions (Fig. 2, E and F). Overall, SC-D appears to act as a significantly reduced its speed and the final amount of general inhibitor of spontaneous α-Syn aggregation. Th-T-positive species by 40% (Fig. 3B). In addition, light scattering in the presence of the molecule was reduced in 38% at 340 nm (Fig. 3B inset) and thus to a lower extent than in the SC-D hampers α-Syn seeded polymerization spontaneous reaction. Preformed amyloid structures, or seeds, have been shown to Strain C fibrils effectively seeded the soluble protein induce and accelerate the aggregation of the soluble protein aggregation, shortening the lag phase and promoting a counterpart (32). We studied the seeding capacity of the three significantly faster reaction, which still displayed a sigmoidal polymorphs by adding 1% (v/v) of preformed and sonicated α- shape. SC-D is exceptionally active in this condition, abro- Syn fibrils formed in the same conditions followed for the gating almost entirely α-Syn aggregation, with an 86% reduc- correspondent assay. tion in the final Th-T fluorescence (Fig. 3C). Consistently, the No seeding capacity was observed for fibrils formed in impact in light scattering in the seeded reaction was also very buffer A. Nonetheless, SC-D still reduced Th-T fluorescence in high, with a reduction of 63% at 340 nm (Fig. 3C inset). 66% (Fig. 3A) and light scattering in 45% at 340 nm, in the These data indicate that SC-D is a very effective inhibitor of presence of seeds (Fig. 3A inset). The highest activity of SC-D α-Syn seeded polymerization for all the strains and in all in the seeded reaction, relative to the spontaneous one, is assayed conditions. The high activity on the fibrils formed in J. Biol. Chem. (2022) 298(5) 101902 3 SC-D inhibits α-synuclein strains aggregation Figure 2. SynuClean-D (SC-D) effects on strain A, strain B, and strain C aggregation. A–C, aggregation kinetics and final point light scattering mea- surements (insets) of α-Syn in the presence (blue) or absence (black) of SC-D for strain A (A), strain B (B), and strain C (C). Th-T fluorescence and light scattering are plotted as normalized means. Error bars are shown as standard errors of mean values. **p < 0.01 and ***p < 0.001. D–I, representative TEM images of α-Syn aggregates of strain A (D and G), strain B (E and H), and strain C (F and I), obtained in the presence (G–I) or absence (D–F) of SC-D. Scale bars correspond to 2 μm and 200 nm for main images and insets, respectively. TEM, transmission electron microscopy; Th-T; thioflavin-T. buffer C might be related to the fact that it is the buffer that disassemble the preformed mature fibrils of the different resembles more in ionic strength to the one in which we strains formed after 48 h of incubation (Fig. 4). Strain A performed the initial screening, and SC-D was shown to bind exhibited the highest levels of Th-T reduction after 24 h of preferentially to fibrillar structures in this condition. incubation with 100 μM of SC-D (Fig. 4A), with a decrease of 71%. Light scattering was also reduced in 53% at 340 nm (Fig. 4B). This indicated that SC-D was active against both the SC-D disrupts preformed aggregates of different strains fibrillar and nonfibrillar assemblies formed at acidic pH, which In a previous study, SC-D evidenced fibril disaggregation was corroborated by TEM images of SC-D-incubated samples capacity (30). Importantly, the compound could partially (Fig. 4, C and D). Strain C Th-T signal decreased by 57% upon Figure 3. Seeding assays with three different strains. A–C, aggregation kinetics and final point light scattering measurements (insets) of α-Syn in buffer A (A), buffer B (B), or buffer C (C) in the absence of compounds and seeds (gray dotted line), in the presence of 1% (v/v) of preformed seeds at the specific condition (black), or in the presence of seeds and 100 μM of SC-D (blue). Th-T fluorescence is plotted as normalized means. Normalized light scattering of treated (blue) and untreated (white) seeded samples at final point for each strain is shown as inset. Error bars are shown as standard errors of mean values; **p < 0.01 and ***p < 0.001. α-Syn, α-synuclein; SC-D, SynuClean-D. 4 J. Biol. Chem. (2022) 298(5) 101902 SC-D inhibits α-synuclein strains aggregation Figure 4. Disaggregational effect of SynuClean-D (SC-D). A, E, and I, Th-T-derived fluorescence assays before (black) and after (blue) the addition of SC-D to strain A (A), strain B (E), and strain C (I) preformed α-Syn fibrils. B, F, and J, final point light-scattering measurements in absence (black) and after (blue) the addition of SC-D to strain A (B), strain B (F), and strain C (J) mature α-Syn fibrils. Th-T fluorescence and light scattering are plotted as normalized means. Error bars are shown as standard errors of mean values. C–D, G–H, and K–L, representative TEM images of strain A (C–D), B (G–H), and C (K–L) aggregates in the absence (C, G, and K) or presence of SC-D (D, H, and L). Scale bars correspond to 2 μm and 200 nm for main images and insets, respectively; **p < 0.01 and ***p < 0.001. α-Syn, α-synuclein; TEM, transmission electron microscopy; Th-T; thioflavin-T. incubation with the molecule (Fig. 4I), whereas the impact on expresses α-Syn fused with enhanced green fluorescence strain B was more moderate with a 27% reduction (Fig. 4E). protein (EGFP), called HEK293-α-Syn-EGFP (33, 34). In this Overall, the disrupting activity of SC-D on top of the different cellular model, endogenous aggregation of α-Syn can be strains seems to correlate with its ability to interfere with the induced by the administration of exogenous seeds. In addition, formation of Th-T-positive species in seeded polymerization endogenous α-Syn aggregation is accompanied by an increase reactions. in phosphorylation at serine 129 (S129), recapitulating what is For both strains formed at pH 7.5, the reduction in light observed in animal models and in the brain of patients with PD scattering upon SC-D addition was similar, ranging between (35–38). Here, we incubated HEK293-α-Syn-EGFP cells with 30 and 40% (Fig. 4, F and J). Consistently, with Th-T and light the different aggregated α-Syn strains, A, B, and C (100 nM), scattering data, TEM analysis of strains B and C demonstrated and performed immunocytochemistry (ICC) experiments with a significant reduction in the number of fibrils and fibril an antibody against phosphorylated α-Syn at S129 (pS129) to clusters for samples incubated with the molecule (Fig. 4, H and identify seed-promoted endogenous intracellular inclusions. L), relative to the respective controls (Fig. 4, G and K). Overall, The α-Syn strains were pelleted and washed with Milli Q water SC-D demonstrated significant and strain-dependent fibril four times under sterile conditions; this process did not alter dismantling activity. their physicochemical and morphological properties (Fig. S4). Then, they were diluted in PBS, sonicated, and diluted in the cell culture media and further incubated for 4 days. The same α-Syn aggregates formed in the presence of SC-D show volume of PBS, buffers A, B, and C, was added to control cells. reduced seeding activity in cell culture models The percentage of cells with intracellular accumulation of α- We next investigated the effect of the different α-Syn fibril Syn-EGFP was first determined. Importantly, we did not strains on the aggregation of endogenous α-Syn, using a stable observe any α-Syn-EGFP or pS129-positive inclusions after human embryonic kidney cells 293 (HEK293) cell line that treatment only with PBS and buffers A, B, and C (Fig. S5). In J. Biol. Chem. (2022) 298(5) 101902 5 SC-D inhibits α-synuclein strains aggregation contrast, we found that treatment with seeds of the strain B led animal models of PD (41). Another class of compounds target to a statistically significant increase of the α-Syn-EGFP in- monomeric α-Syn, such as fasudil and BIOD303, which sta- clusions compared to the cells treated with seeds of the strains bilize the native protein conformation and reduce aggregation A and C (Fig. 5A). Likewise, quantification of the pS129- in cellular and animal models of PD (27, 28). Finally, molecules positive inclusions revealed a significant increase upon treat- like squalamine and trodusquemine act on destabilizing α-Syn- ment with strain B, whereas strains A and C did not affect the membrane contacts and interact with the protein’s negatively percentage of cells with pS129 inclusions (Fig. 5A). charged C-terminal domain (25, 26), displacing oligomeric These data indicate that strain B is the only one able to species from cellular membranes and thus reducing their propagate effectively in this cell model. When the α-Syn ag- toxicity (42, 43). Furthermore, as SC-D, trodusquemine can gregation reaction in buffer B was done in the presence of SC- prevent seeding-mediated aggregation of α-Syn and has shown D and the sample sonicated and added to the cell culture, the to be effective in the same Caenorhabditis elegans model of PD number of α-Syn-EGFP aggregates and especially of pS129- (26). Thus, different conformations of α-Syn can now be tar- positive inclusions decreased significantly (Fig. 5, A and B), geted effectively, but we lack information on how the activity with respect to untreated fibrils, indicating that the molecule of these molecules depends on the aggregation conditions. In interfered with the exogenous seeding of intracellular α-Syn. this study, we analyzed the different properties, aggregation propensities, and seeding capacity of three alternative poly- morphs of α-Syn in vitro and in a cellular model to decipher Discussion how the activity of SC-D responds in front of different α-Syn Prion-like proteins can aggregate into multiple fibrillar amyloid formation scenarios. structures. This conformational heterogeneity might explain Our study confirms a clear pH and salt dependence of the why a single amyloidogenic protein is behind the development in vitro aggregation of α-Syn, indicating a critical role of the of disorders with diverse phenotypic traits (19, 39). As for what chemical conditions of the microenvironment surrounding refers to α-Syn, evidence on structural fibrillar plasticity and its the nascent aggregate. In agreement with previous data, ag- linkage to diverse synucleinopathies is progressively accumu- gregation at acidic pH is fast (44–47). This comes at the lating (13–15, 17–22). The administration of brain extracts expense of the formation of abundant aggregates of lower from patients with PD or MSA into mice brains resulted in a amyloid content than at neutral pH, as deduced from Th-T, different lesion pattern and symptomatology (40). In addition, scattering, and FTIR analysis (Fig. 1 and Table S1). TEM im- when different recombinant α-Syn polymorphs are adminis- ages confirmed that short amyloid fibrils coexist with large tered to cells and animals, the damage caused and the area of amorphous aggregates (Figs. 1 and S2). These aggregates are the brain that is affected vary; the different conformations, significantly resistant to proteolysis but display low seeding aggregation, and transmission propensity of these multiple capability in vitro (Fig. 3), likely due to the low proportion of fibrillar structures are thought to be the ultimate cause of such ordered fibrils that can act as fibrillization nuclei. Accordingly, differential effect (9, 20). This has important implications for when exogenously applied to cells, strain A has a negligible treating synucleinopathies since each of these disorders might impact on the formation of endogenous aggregated and need specific drugs. phosphorylated α-Syn (Fig. 5). The C-terminal region of α-Syn Different small molecules have been described to modulate (residues 96−140) is very acidic (10 Glu + 5 Asp), and it reg- α-Syn amyloidogenesis. These include compounds that, as SC- ulates aggregation since C-terminally truncated α-Syn aggre- D, interact with aggregated forms of α-Syn like anle138b, a gates faster than the full-length protein (48, 49). It is likely the molecule that targets oligomeric but not monomeric α-Syn impact of acidic pH in the protonation state of this region that (23, 24), reducing aggregation and eliciting neuroprotection in accounts for the aggregation properties of strain A (47). SC-D Figure 5. Effect of α-Syn strains in aggregation of endogenously expressed α-Syn-EGFP. A, quantification of the percentage of cells with inclusions shows that α-Syn-EGFP (green) and pS129 positive (red) inclusions are increased under treatment with strain B. Treatment α-Syn strain B with SC-D leads to a decrease in phosphorylated α-Syn inclusions (red) and the total level of inclusions (green). B, representative images from strain B −/+ SC-D buffer. Treatment with the α-Syn strain B-SC-D at the concentration of 100 nM for 4 days revealed the formation of α-Syn inclusions phosphorylated in S129. Representative images of cells under the treatment with B strain −/+ SC-D buffer (Scale bar 30 μm). DAPI was used for nuclear staining. Data are shown as mean ± SD (>100 cells counted per condition, four independent experiments); *p < 0.05 and **p < 0.01. α-Syn, α-synuclein; DAPI, 40 60 -diamidino-2-phenylindol; EGFP, enhanced GFP; pS129, phosphorylated S129; SC-D, SynuClean-D. 6 J. Biol. Chem. (2022) 298(5) 101902 SC-D inhibits α-synuclein strains aggregation can inhibit the aggregation of α-Syn into strain A amyloid-like that, SC-D blocks the in cell seeding potential of strain B, structures, as measured by Th-T, but it is especially active in decreasing the number of intracellular aggregates and, espe- inhibiting the formation of the predominant, amorphous, β- cially, of phosphorylated α-Syn inclusions (Fig. 5). sheet-containing α-Syn aggregates formed at low pH as re- Overall, the results confirm the sensitivity of α-Syn aggre- ported by light scattering and TEM data (Fig. 2). It also gation to the solution conditions and converge to indicate that effectively disaggregates the mature ordered and nonordered SC-D acts as a pan-inhibitor of the spontaneous and seeded α-Syn aggregates formed in this condition (Fig. 4). Surpris- formation of different α-Syn amyloid polymorphs, being able ingly, SC-D is very good at inhibiting the seeded aggregation of to partially disentangle them and eventually prevent the in- strain A, which suggests that the species formed at pH 5.0 in duction of intracellular aggregates by exogenous seeds. This the absence and presence of seeds differ conformationally and generic property might be related to the fact that, according to that SC-D recognizes the later ones preferentially (Fig. 3). previous molecular dynamic simulations (30), SC-D prefer- In contrast to pH 5.0, α-Syn aggregation at pH 7.5 results in entially targets residues within (Glu61, Thr72, and Gly73) or the formation of regular amyloid fibrils with significantly close to (Ala53, Val55, and Thr59) the non-amyloid compo- higher Th-T binding and lower scattering relative to aggre- nent domain (Fig. S6), which are expected to be relevant for α- gates formed under acidic conditions (Fig. 1). However, the Syn aggregation in all the assayed conditions. However, it is properties of these assemblies are strongly dependent on the also clear that the activity of this molecule differs between salt content of the solution. Fibrils formed in the presence of strains, suggesting the establishment of conformation- physiological salt concentrations (strain C) display a higher β- dependent interactions with the different intermediate spe- sheet content and bind Th-T with higher affinity than those cies. It is worth remarking that the highest SC-D activity oc- formed in its absence (strain B) (Fig. 1 and Table S1). curs in solution conditions that resemble most those of the Accordingly, strain C is significantly more resistant to prote- initial screening study, which suggests that, in principle, one olysis and chemical denaturation than strain B (Figs. 1 and S3). can set up drug discovery assays to identify molecules that Previous studies suggested that the presence of salt induces target preferentially a specific, biologically relevant, α-Syn high-level compaction of mature fibrils by hiding the C-ter- polymorph. Meanwhile, the generic activity of SC-D positions minal acidic domain of α-Syn (31, 50), which might account this aromatic molecule as a hit compound from which evolve for the observed conformational and stability differences be- potentially active drugs for the different synucleinopathies. tween strains B and C. The fibrils formed in both conditions seeded the in vitro aggregation of soluble α-Syn, but the Experimental procedures seeding effect is more substantial for strain B, where the seeds completely abrogate the lag phase of the polymerization re- Protein expression and purification action (Fig. 3). This differential seeding capability is translated The expression and purification of α-Syn was carried out in to the cellular model, with strain B significantly stimulating the Escherichia coli BL21 DE3 strain as previously described (52). aggregation of endogenous α-Syn and the accumulation of Briefly, E. coli BL21 (DE3) containing a pET21a plasmid pS129 α-Syn (Fig. 5). In contrast, strain C had a lower effect on encoding for the α-Syn complementary DNA were grown in aggregation and a negligible impact on pS129 inclusion for- LB medium containing 100 μM/ml ampicillin and induced mation (Fig. 5). These properties fairly correlated with those with 1 mM IPTG for 4 h at an absorbance of 600 nm of 0.6. observed when injecting α-Syn fibrils formed under the same Cells were recovered by centrifugation and washed up by solution conditions as our study into the striatum of mice, with resuspension and centrifugation in PBS, pH 7.4. Pellets were the fibrils formed in the absence of KCl inducing abnormal stored at −80  C until used, when pellets were resuspended in phosphorylated α-Syn deposits through the mouse brain, 50 ml per culture liter in lysis buffer (50 mM Tris pH 8, whereas few pS129-positive deposits were induced by the fi- 150 mM NaCl, 1 μg/ml pepstatin, 20 μg/ml aprotinin, 1 mM brils formed in the presence of KCl (31). Salt-induced benzamidine, 1 mM PMSF, 1 mM EDTA, and 0.25 mg/ml compaction and masking of the C-terminal region in strain lysozyme) and sonicated using a LABSONICU sonicator (B. C might explain these differences since recent studies indicate Braun Biotech International). Then, sonicated samples were that cell surface receptors that bind α-Syn in the amyloid state boiled for 10 min at 95  C and centrifuged at 20,000g at 4  C to initiate cell-to-cell transmission recognize this acidic region for 40 min. The soluble fraction was treated with 136 μl/ml of when it is exposed at the surface of the fibrils, as it would be in 10% w/v streptomycin sulfate and 228 μl/ml of pure acetic acid strain B due to uncompensated electrostatic repulsion in the and centrifuged at 20,000g at 4  C for 10 min. Upon centri- absence of salts (51). SC-D is very effective at inhibiting fugation, soluble extracts were fractionated by adding 1:1 of the aggregation of strains B and C both in the absence and the saturated ammonium sulfate. Samples were centrifuged for presence of seeds (Figs. 2 and 3) and acts as a fibril- 10 min at 20,000g and 4  C, and the resultant pellets resus- disaggregating molecule for both polymorphs (Fig. 4). How- pended with 50% ammonium sulfate. After centrifugation (at ever, as a general trend, SC-D performs better with strain C, 20,000g and 4  C for 10 min), the pellet was resuspended in especially in seeded aggregation (Fig. 3), likely because the 100 mM, pH 8, ammonium acetate (5 ml per culture liter) and conditions in which these assemblies form resemble more in pure EtOH 1:1 (v/v) and harvested by centrifugation for terms of salt content than those in which we performed the 10 min at 20,000g at 4  C. The insoluble fraction was resus- assay in which SC-D was initially identified. Irrespective of pended in Tris 20 mM pH 8, filtered with a 0.22-μm filter, and J. Biol. Chem. (2022) 298(5) 101902 7 SC-D inhibits α-synuclein strains aggregation loaded into an anion exchange column HiTrap Q HP (GE aggregation was performed in a 96-well sealed plate, contain- Healthcare) coupled to an ÄKTA purifier high-performance ing in each well 70 μM α-Syn (in buffer A, B, or C), 40 μM Th- liquid chromatography system (GE Healthcare). Tris T, a 1/8” diameter Teflon polyball, and SC-D at 100 μM in a 20 mM, pH 8, and Tris 20 mM, pH 8, and NaCl 1 M were total volume of 150 μl (2.5% DMSO final concentration). used as buffer A and buffer B. αS was eluted using a using a Control samples contained the corresponding amount of step gradient: step 1, 0 to 20% buffer B, 5 cv; step 2, 20 to 45% DMSO (2.5% final concentration). The plate was fixed into an buffer B, 11 cv; step 3, 100% buffer B, 5 cv. Purified αS was orbital Max-Q 4000 (ThermoScientific) and constantly dialyzed against 5 L of ammonium acetate 50 mM in two steps: agitated at 100 rpm and 37  C, and Th-T fluorescence 4 h and overnight. The obtained protein was lyophilized and measured every 2 h as previously indicated. stored at −80  C until used in the experiments. In the disaggregation assays, 70 μM α-Syn was incubated for 48 h at 100 rpm and 37  C in a 96-well sealed plate, which also Sample preparation contains in each well 40 μM Th-T and a 1/8” diameter Teflon polyball. After 48 h, aggregated samples were exposed to Lyophilized α-Syn was resuspended in PBS 1X to a final 100 μM of SC-D or 2.5% of DMSO in controls. The plate was concentration of 210 μM and dialyzed in a 1:1000 (v/v) ratio in incubated for an additional period of 24 h at 100 rpm and the presence of buffer A (50 mM sodium acetate pH 5.0), 37  C in an orbital Max-Q 4000, and Th-T fluorescence buffer B (50 mM Tris-HCl pH 7.5), or buffer C (50 mM Tris- measured at final point as before. HCl pH 7.5 supplemented with 150 mM KCl) for 24 h. Protein was filtered through a 22-μm membrane to eliminate small TEM aggregates of α-Syn, and sample concentration was measured at 280 nm in a spectrophotometer Cary100 (Agilent) Aggregated samples were diluted 1:10 in PBS 1X and softly using the extinction coefficient 5960 M−1 cm−1. SC-D (5- sonicated for 5 min at intensity 2 with a Ultrasonic Cleaner nitro-6-(3-nitrophenyl)-2-oxo-4-(trifluoromethyl)-1H-pyridine- sonicator (VWR International) to avoid large accumulations of 3-carbonitrile) was resuspended in 100% dimethyl sulfoxide fibrils. Immediately, 5 μl of the samples was placed on a (DMSO) to a final concentration of 50 mM as preservation carbon-coated copper grid. Grids were carefully dried 5 min stock and kept stored at −20  C. Working solutions of 4 mM later with a filter paper to remove the excess of sample and SC-D in 100% were prepared for the different experiments just washed in Mili-Q water twice. Finally, 5 μl of 2% (w/v) uranyl before use. acetate was added on the top of the grid for 2 min and then removed with a filter paper. Grids were left to air-dry for 10 min. Representative images were obtained screening a Protein aggregation assays minimum of 30 fields per sample and using a Transmission The aggregation was performed on a 96-well sealed plate, Electron Microscopy Jeol 1400 (Peabody) operating at an containing in each well 70 μM α-Syn (in buffer A, B, or C), accelerating voltage of 120 kV. 40 μM Th-T, a 1/8” diameter Teflon polyball (Polysciences Europe GmbH), and SC-D at 100 μM. Briefly, 3.75 μl of 4 mM Light scattering SC-D stock was added to each well; 3.75 μl of 100% DMSO In all the assays, final-point–aggregated protein samples was added instead to control samples. The final volume was were carefully resuspended and recovered to measure the total 150 μl (2.5% DMSO in the assay solution). The plate was fixed aggregate formation. To a quartz cuvette, 80 μl of aggregated into an orbital Max-Q 4000 (ThermoScientific) and constantly samples were added and excited at 340 nm to collect 90 agitated at 100 rpm and 37  C. Th-T fluorescence was scattering from 320 to 360 nm in a Cary Eclipse Fluorescence measured every 2 h by exciting through a 430- to 450-nm filter Spectrophotometer (Agilent). and collecting the emission signal with a 480- to 510-nm filter, using a Victor3.0 Multilabel Reader (PerkinElmer). All the Secondary structure determination assays were done in triplicate. Data were normalized and represented as mean and SEM and fitted with GraphPad Prism ATR-FTIR spectroscopy analysis of amyloid fibrils was 6.0 software (GraphPad Software Inc) using the following performed using a Bruker Tensor 27 FTIR Spectrometer equation: (Bruker Optics Inc) with a Golden Gate MKII ATR accessory. Each spectrum consists of 16 independent scans, measured at 1 a spectral resolution of 4 cm−1, within the 1800- to 1500-cm−1 / ¼ 1− range. Second derivatives of the spectra were used to deter- kb ðekb t − 1Þ þ 1 mine the frequencies at which the different spectral compo- accounting kb and ka for the homogeneous nucleation rate nents were located. Fourier-deconvolution and determination constant and the secondary rate constant (fibril elongation and of band position of the original amide I band were performed secondary nucleation), respectively (53). using PeakFit software (Systat Software). For the seeding assays, 1% (v/v) of preformed α-Syn fibrils were added to their corresponding condition after a sonication PK digestion process of 5 min at intensity 9 with a Ultrasonic Cleaner Eighteen microliters of aggregated α-Syn at final point sonicator (VWR International). As previously mentioned, was mixed with 6 μl of PK, rendering a final concentration of 8 J. Biol. Chem. (2022) 298(5) 101902 SC-D inhibits α-synuclein strains aggregation 5 μg/ml, and incubated for 5 min at 37  C. Then, 8 μl of washed first with PBS and then with diluted 1:10 trypsin, fixed loading buffer containing 1% β-mercaptoethanol was added with 4% paraformaldehyde for 20 min at room temperature and the enzyme thermally inactivated for 10 min at 95  C in a (RT), permeabilized with 0.5% Triton X-100 (Sigma–Aldrich) ThermoCell cooling & heating block (BIOER). Finally, 7 μl of for 20 min at RT, and blocked in 1.5% bovine serum albumin/ the samples was loaded into a 12% SDS-PAGE, later stained Dulbecco’s PBS for 1 h. Cells were incubated with primary with Blue Safe. antibody overnight (1:1000; anti-pS129-α-syn Rabbit Ab51253 Abcam) and secondary antibody Alexa Fluor 555 goat anti- Mature fibril denaturation rabbit immunoglobulin G (Life Technologies-Invitrogen) for 2 h at RT, followed by nuclei staining with 40 60 -diamidino-2- For denaturation assays of strains B and C, 70 μM α-Syn was phenylindol (Sigma–Aldrich, D8417) (1:5000 in Dulbecco’s incubated for 48 h in the presence of DMSO and Th-T as PBS) for 10 min. After a final wash, coverslips were mounted described previously. The resultant aggregates were carefully by using Mowiol (Sigma–Aldrich) and subjected to fluores- resuspended and recovered. For chemical denaturation, 500 μl cence microscopy. of aggregated α-Syn fibrils was mixed with 4 M urea (final The proportion of cells with α-Syn inclusions within the concentration) in a quartz cuvette (1 ml final volume) and population was then determined by counting. Each experiment incubated for 2000 s at 37  C under constant agitation was reproduced at least three independent times. Data were (150 rpm). Meanwhile, Th-T fluorescence was recorded in a analyzed using a t test. For quantification of aggregation, at Cary Eclipse Fluorescence Spectrophotometer (Agilent) with a least 100 cells were counted per variant and per experiment. data pitch of 1 s using an excitation wavelength of 445 nm and Images were acquired using a 63× objective lens and analyzed recorded at 485 nm with an excitation and emission band- using LAS AF, version 2.2.1 (Leica Microsystems) software. width of 2.5 and 5 nm, respectively (54). Denaturing curves were fitted to a one-step reaction using GraphPad Prism 5 Statistical analysis (GraphPad Software). All graphs were generated with GraphPad Prism 6.0 soft- ware (GraphPad Software Inc). Data were analyzed by two-way Cell culture, cell treatment, and ICC ANOVA and Tukey’s Honestly Significant Difference test us- HEK293 stably expressing WT α-Syn fused to EGFP ing SPSS software, version 20.0 (IBM Analytics). All data are (HEK293- α-Syn-EGFP), under the cytomegalovirus promoter, shown as means and SEM. p < 0.05 was considered statisti- was used for the seeding experiments (34). Cells were main- cally significant. In the graphs, *, **, and *** indicate p < 0.05, tained in Dulbecco’s modified Eagle’s medium supplemented p < 0.01, and p < 0.001, respectively. For cellular assays, with 10% fetal bovine serum Gold (PAA) and 1% penicillin– statistical analysis was done using the Student’s t test for in- streptomycin (PAN). Cells were grown at 37  C in an atmo- dependent variables and the data were presented as mean ± sphere of 5% CO2. For the seeding experiments, cells were standard deviations and represent results from at least four plated on 13-mm glass coverslips in 24-well plates and incu- independent experiments. bated in 5% fetal bovine serum media. The following day, α-Syn strains were diluted in PBS, fragmented by sonication Data availability (55) and then added to cells at a final concentration of 100 nM. All data are contained within the manuscript. The fibrils were prepared in an aggregation reaction starting from 70 μM of monomeric α-Syn, in the absence or presence of 100 μM of SC-D and the corresponding proportion of Supporting information—This article contains supporting informa- DMSO for 48 h at 100 rpm and 37  C. Preformed fibrils (PFFs) tion (30). were then recovered and centrifuged at 14,000 rpm for 30 min. The supernatant was discarded, and the pellet resuspended in Acknowledgments—We thank the Servei de Microscòpia at Uni- Milli-Q water under sterile conditions. PFFs were then versitat Autònoma de Barcelona for their help with TEM. centrifuged again at 14,000 rpm for 30 min, and the pellet was Author contributions—S. P.-D., J. P., and S. V. conceptualization; resuspended in Milli-Q water under sterile conditions. This S. P.-D., J. P., E. V., F. P., J. S., and Z. M.-A. methodology; S. P.-D., step was repeated three additional times. We performed the J. P., E. V., S. V., and T. F. O. validation; S. P.-D. and S. V., formal imaging in 24-well dishes (with volume media 0.5 ml/well) and analysis; S. P.-D., J. P., E. V., F. P., J. S., and Z. M.-A. investigation; calculated to add 50 μl of PBS or PFFs (dilution 1:10). We S. P.-D. and S. V., writing–original draft; Z. M.-A. and S. V. prepared seeds by adding PFFs to sterile PBS to a final con- supervision; T. F. O. and S. V. project administration; T. F. O. and centration of 1 μM. The minimal volume that can be used for S. V. funding acquisition. sonication is 200 μl. We diluted 50 μl of sonicated PFFs or an Funding and additional information—S. V. was supported by the equivalent volume of PBS as a control, into prewarmed cell Spanish Ministry of Science and Innovation (PID2019–105017RB- media (1:10 dilution). We checked our PFF preparations, and I00), ICREA (ICREA-Academia 2015 and 2020), and the Fundación we did not detect amorphous aggregates during the prepara- La Marató de TV3 (Ref. 20144330). T. F. O. was supported by the tion. Control cells were exposed to vehicle only (PBS). Cells Deutsche Forschungsgemeinschaft (DFG, German Research Foun- were further incubated for 4 days. ICC was carried out using dation) under Germany’s Excellence Strategy (EXC 2067/ standard protocols. Briefly, after treatment, the cells were 1–390729940) and by SFB1286 (Project B8). J. Biol. Chem. 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