Doxycycline inhibits α-synuclein aggregation through the formation of off-pathway oligomers

To analyze the ability of doxycycline to interfere with the fibril assembly process of α-synuclein, we incubated 70 μM α-synuclein in the absence or in the presence of 100 μM doxycycline at 37 °C under orbital agitation. Self-association kinetics of α-synuclein was monitored as an increase in fluorescence emission of Thioflavin T (ThT) at 482 nm (λ exc 450 nm) upon binding of this probe to aggregates rich in cross-β structure22. In accordance to previous reports23, the aggregation kinetic of α-synuclein in the absence of doxycycline shows a lag phase of 18 h followed by an exponential growth to finally reach a plateau at 48 h (Fig. 1a). Conversely, in the presence of doxycycline the aggregation kinetic of α-synuclein is severely affected. According to the fluorescent probe, when doxycycline is added at the beginning of the incubation, the formation of aggregates with cross-β structure is dramatically diminished. However, once the exponential growth phase has been reached, addition of doxycycline has no observable effect on the aggregation kinetic of α-synuclein (Fig. 1a).

Figure 1: Effects of doxycycline on α-synuclein aggregation and seeding. (a) Fluorescence emission intensity of 25 μM thioflavin T in a solution containing 70 μM α-synuclein alone (empty circle), or with the addition of 100 μM doxycycline at 0 h (full circles), or after 16 h of incubation (inverted triangles) (doxycycline addition is indicated by an arrow). A solution containing 100 μM doxycycline alone (asterisk) is also shown as a control. (b) Transmission electron microscopy (TEM) of α-synuclein samples incubated at 37 °C under orbital agitation in the absence (top) or in the presence of doxycycline (bottom), and harvested after 16 h (left) or 96 h (right). The white bar corresponds to 200 nm at 220000X and 70000X magnification for observation of oligomers and fibrils respectively. (c) Fresh solutions of monomeric α-synuclein were seeded with oligomers preincubated in the absence (empty square) or in the presence of doxycycline (full square). The resulting solutions were incubated at 37 °C under orbital agitation and aggregation was assayed by ThT fluorescence emission. Unseeded aggregation kinetics are shown in (empty circle). Full size image

To evaluate the impact of doxycycline on the morphology of the species present along the aggregation pathway, we performed transmission electron microscopy (TEM) studies on samples of monomeric α-synuclein incubated with or without doxycycline and harvested at different times (Fig. 1b). After 16 h of incubation, we were able to detect oligomeric species in both conditions, which were morphologically undistinguishable by TEM. Nevertheless, only in the absence of doxycycline, oligomers evolved into fibrils, which were observed after 96 h incubation. This data suggests that doxycycline halt the assembly of α-synuclein oligomers into larger aggregates since only oligomers but not fibrils are still observed at this time. In fact, the absence of fibrils in the samples that contained doxycycline is consistent with the lack of cross-β observed using the ThT fluorescent probe (Fig. 1a).

To assess whether α-synuclein oligomers formed in the presence of doxycycline are on-pathway or off-pathway intermediates in the fibril formation process, we also carried out seeding experiments. For this, seeds were produced by incubating α-synuclein solution at 37 °C under orbital agitation for 16 h in the absence or presence of doxycycline. Then, an aliquot of the solution containing α-synuclein seeds was added to fresh solutions of monomeric α-synuclein and further incubated for 72 h (Fig. 1c). The addition of seeds formed in the absence of doxycycline accelerates the aggregation process and more species with cross-β structure are self-assembled from native α-synuclein as indicated by increased ThT fluorescence. In contrast, the oligomers formed in the presence of doxycycline are not able to further convert into amyloid fibrils. This indicates that oligomers formed in the presence of doxycycline did not function as template for the conversion of unpolymerized proteins into amyloid fibrils and we will further refer to them as off-pathway oligomers throughout the paper in contrast to the on-pathway oligomers formed in the absence of doxycycline.

Inhibition of α-synuclein fibrillation is mediated by binding of doxycycline to oligomeric species

The anti-amyloid activity of doxycycline prompted us to explore details of its binding to α-synuclein by NMR spectroscopy. To analyze this interaction we used 1H-15N NMR heteronuclear multiple quantum correlation (HMQC) spectra. The 1H-15N spectrum of a sample of uniformly 15N-labeled α-synuclein, reflecting the intrinsically disorder nature of the protein, is shown in Fig. 2a. Upon titration of 15N-enriched α-synuclein with increasing concentrations of doxycycline, the 1H-15N HMQC spectra retained the excellent resolution of the uncomplexed, monomeric protein. Notably, no broadening or chemical shift perturbations could be observed, even at high ligand:α-synuclein ratios (5:1), indicating that doxycycline is unable to interact with monomeric α-synuclein.

Figure 2: Analysis of doxycycline binding to α-synuclein by NMR. (a) Overlaid contour plots of 1H-15N SOFAST-HMQC spectra of 70 μM monomeric α-synuclein in the absence (black) or presence (red) of 350 μM doxycycline. (b) 1H NMR spectra of 200 μM doxycycline alone (black line) or upon the addition of 100 μM monomeric α-synuclein (red line). (c) 1H NMR spectra of 200 μM doxycycline in the absence (black line) or presence of 100 μM α-synuclein aged for 8 h (red line), 24 h (blue line) or 48 h (green line). In panels (b) and (c), the asterisk denotes isolated doxycycline 1H signals that broaden upon binding to higher order amyloidogenic structures of α-synuclein. In panel (c), the symbol # denotes isolated monomeric α-synuclein signals vanishing as a consequence of the progression of the aggregation process. NMR spectra were acquired at 15 °C (a) and 25 °C (b) and (c). Samples were dissolved in 20 mM HEPES supplemented with 150 mM NaCl and 10% D 2 O. Full size image

To assess further the nature of the α-synuclein species involved in the interaction with doxycycline, we conducted NMR experiments aimed at detecting directly the signals belonging to the anti-amyloid compound. High-ordered oligomeric or prefibrillar α-synuclein species are invisible to the NMR approach used here24,25, although certain oligomers retaining high flexibility at the C-terminal region could be even detected26. Hence, a potential interaction of the antibiotic with those species will not become evident by using an NMR strategy based on the detection of protein backbone resonances. On the contrary, the 1D 1H NMR spectrum of doxycycline shows well-resolved resonances in the 6.0–7.0 ppm region, which constitutes an excellent probe for exploring the binding features of the antibiotic to α-synuclein. As expected, addition of 0.5 equiv. of monomeric α-synuclein to doxycycline samples caused no perturbations in the 1D 1H NMR spectrum of the small ligand, confirming the lack of interaction of the compound with the monomeric form of the protein (Figure 2b,c). By contrast, a substantial broadening of doxycycline signals was observed when aliquots of aged α-synuclein samples (24–48 h) were added, indicating that doxycycline is able to bind to the larger molecular species formed during the aggregation event (Fig. 2c). Even though changes in the signals of doxycycline upon the addition of α-synuclein aged for 8 h were not so prominent we still observed a small decrease in signal intensities, specially in the set of resonances centered at c.a. 6.95 ppm. These small perturbations are likely attributed to the small number of oligomers present in the sample at short aggregation times. In order to confirm this hypothesis, we analyzed the spectra recorded for doxycycline alone or upon the addition of fresh or aged (16 h) α-synuclein samples (Supplementary Figure S1). Although no fibrils could be detected on these samples (see Fig. 1a,d), they were centrifuged to spin down and remove minor insoluble species that could have been formed under our experimental conditions. Even after this treatment, signal attenuation in doxycycline remained unaltered (see the set of peaks centered at 6.95 ppm and 6.35 ppm), suggesting an interaction landscape formed mostly by soluble oligomers or early protofibrils. A comparative analysis between the intensities of α-synuclein resonances located at the methyl and aromatic regions (only signals showing no overlapping with doxycycline resonances were considered), shows no differences between the spectra recorded at 0 and 16 h, indicating the absence of slow-tumbling, highly-ordered structures such as amyloid fibrils or large protofibrils27, in full agreement with ThT and TEM data. Since monomeric α-synuclein is not able to interact with doxycycline (Fig. 2a,b and Supplementary Figure S1a), we assume that the attenuation observed for the signals of the compound under the experimental conditions described above are reflective of an interaction process with low-order oligomeric or protofibrillar α-synuclein species retaining some degrees of molecular flexibility. Altogether, these evidences indicate that binding of doxycycline to α-synuclein proceeds initially via interactions with aggregated species formed during the early stages of the assembly process, a molecular event that might provide a central mechanistic basis for the inhibitory process of doxycycline on α-synuclein fibrillation.

Off-pathway oligomers present a different structural arrangement

In order to gain further insights on how doxycycline impacts on α-synuclein aggregation leading to the formation of off-pathway species, three complementary techniques were employed. These were small angle X-ray scattering (SAXS), bis-ANS fluorescence, and Fourier Transformed Infrared spectroscopy (FT-IR). SAXS reports changes in protein aggregates morphology (shape and size), bis-ANS infers the amount of hydrophobic surface exposed to the solvent, while FT-IR gives deeper insight into the oligomers structural organization being a technique particularly sensitive to β-sheet structure arrangements28.

For SAXS measurements, α-synuclein was incubated up to 24 h at 37 °C under orbital agitation in the absence or in the presence of doxycycline. SAXS data were acquired from aliquots taken at different elapsed times along the aggregation process (Fig. 3a–d). Note that right after its addition, doxycycline produces no immediate impact on the scattering curve from in-solution α-synuclein (Fig. 3a). However, its influence can be clearly detected after 2 h of incubation through the increase in the scattering intensity at low q values in comparison to the scattering from doxycycline-free α-synuclein (Fig. 3b). Such a behavior is a fingerprint of the formation of larger α-synuclein aggregates in comparison to those self-assembled in the absence of doxycycline. This was systematically reproduced over time. Therefore, SAXS data demonstrated that off-pathway α-synuclein oligomers formed in the presence of doxycycline are larger than on-pathway oligomers. Interestingly, the corresponding pair distance distribution functions, p(r), show that on-pathway oligomers evolve to fibril-like aggregates along time (Fig. 3e). The maximum frequency of distances (r max ) inside the protein aggregate remains practically constant at 4 nm, while the maximum dimension, D max , elongates from 30 nm to 40 nm over 24 hours (Fig. 3e). On the other hand, off-pathway aggregates present a displacement toward larger distances in both r max (up to 22 nm), and D max (up to 55 nm) (Fig. 3f). It should be stressed that aggregates larger than 55 nm can be formed but such dimension is over the upper detection limit of SAXS under our experimental conditions. Taken together with TEM results, the changes in the p(r) function features with time indicate that doxycycline impacts on α-synuclein aggregation pathway by deviating early elongated on-pathway oligomers into the formation of larger off-pathway oligomers (Fig. 1b).

Figure 3 SAXS analysis of monomeric and oligomeric species of α-synuclein (a) SAXS curves from 175 μM α-synuclein incubated at 37 °C in the absence (red symbols) or presence (black symbols) of 250 μM doxycycline at 0 h (a), 2 h (b), 16 h (c) and 24 h (d). Corresponding distance distribution functions p(r) in the absence (e) or in the presence of doxycycline (f): 0 h (dark line); 2 h (red line), 16 h (blue line) and 24 h (magenta line). Full size image

Infrared spectra, in the amide I region (1,700–1,600 cm−1), is particularly sensitive to the backbone conformation of proteins, allowing to discriminate the relative composition of secondary structure elements29. In order to perform a comparative analysis between α-synuclein on-pathway and off-pathway oligomers, the protein was incubated in the presence or in the absence of doxycycline and aliquots were taken at regular times for infrared spectra collection. At the beginning of incubation, samples are clearly very similar and suggest that the structural content is comparable regardless the presence of doxycycline (Fig. 4a). After Fourier self-deconvolution process, the amide I shows a contour centered at 1,649 cm−1 that is typical of an unfolded protein30. However, after 2 h incubation (Fig. 4b), off-pathways oligomers reflect increased content of ordered secondary structure, as revealed by the decrease in the FTIR band at 1,641 cm−1 (Table 1).

Figure 4 FTIR spectra of the amide I region of 280 μM α-synuclein in deuterated buffer in the absence (blue line) or in the presence (red line) of 400 μM doxycycline after 0 h (a), 2 h (b) and 16 h (c) incubation at 37 °C, pD 7 under orbital agitation. Deconvolved spectra (top) was performed using a mixed Gaussian/ Lorentzian line shape of 18 cm−1 and a resolution enhancement factor of 2. Derivative spectra (bottom) were obtained with a power of three and a break-point of 0.3. Bis-ANS fluorescence signal of α-synuclein solution (70 μM) incubated at 37 °C under orbital agitation in the absence (d) or in the presence of 100 μM doxycycline (e). Aliquots of the samples were taken throughout time. All results shown are representative of at least four independent experiments. Full size image

Table 1 FTIR-based evaluation of secondary structure content during the fibrillation process for human α-synuclein with doxycycline introduced at selected time. Full size table

The half-width at half-height (HWHH) of the Amide I’ band reflects the structuration process of a protein and, as shown in Fig. 4b and c, off-pathway species seems to be more structured than than the on-pathway ones. Moreover, the amount of β-structure and the arrangement of the β-strands were also different in both species. In fact, off-pathway α-synuclein oligomers are rich in parallel β-sheet structure as shown by the presence of a band at 1,619–1,634 cm−1 and the absence of an absorption band at approximately 1,685 cm−1. On the contrary, the β-sheet structure of the on-pathway oligomers is predominantly antiparallel as indicated by a band at about 1,625 cm−1 with a high frequency component at approximately 1,685 cm−1, approximately fivefold weaker than the band at 1,625 cm−1. Remarkably, the differential organization of the β-sheet structure into parallel or antiparallel forms has been previously related to cytotoxicity28.

The intrinsic physicochemical properties of the oligomeric species, like hydrophobic solvent exposition, have also been associated to toxicity due to its relation to membrane binding and perturbation as well as cellular dysfunction. Accordingly, we measured the surface hydrophobicity of on-pathway and off-pathway α-synuclein oligomers using bis-ANS, a dye known to increase its fluorescence quantum yield upon binding to hydrophobic pockets on protein surfaces31. The emission spectra of bis-ANS when added to pre-incubated solutions of α-synuclein, reveals that there is a constant increase of hydrophobic surface exposure during aggregation (Fig. 4d), as reflected by a 65% enhancement in the quantum yield of the probe (calculated from areas under the curve). On the contrary, when doxycycline was present in the preincubated mixture, the fluorescence intensity remained unaltered up to 16 h (Fig. 4e), and reached a 10% increase in fluorescence between 24 and 72 h, indicating that off-pathway oligomers expose hydrophobic patches to a lesser extent.

Overall, these results suggest that doxycycline induces differential conformational changes in α-synuclein oligomers, resulting in the formation of off-pathway oligomers with well-distinctive features in terms of shape, size, structure and hydrophobic surface exposure.

Doxycycline reshapes α-synuclein into non-toxic oligomers

Cytotoxicity of α-synuclein has been attributed to transient prefibrillar species formed during protein aggregation5. Indeed, the pathogenicity associated to the these prefibrillar species has been linked to the conformational arrangement of the aggregate species as revealed by conformation-specific antibodies32 as well as FTIR studies28. Therefore, we assessed the impact of the structural changes induced by doxycycline on the pathogenicity of α-synuclein oligomers. First, we carried out cell viability studies on SH-SY5Y cell line using the MTT assay33, which reports viable cell number based on the mitochondrial activity. For this, on-pathway and off-pathway α-synuclein oligomers were obtained by preincubation of 140 μM α-synuclein in the absence or presence of 200 μM doxycycline, respectively for 16 h at 37 °C under orbital agitation. A 25 μl aliquot of each preparation was added to SH-SY5Y cells and further incubated at 37 °C. As previously described23, on-pathway oligomeric species were able to decrease cell viability as reflected by the 40% decrease in the turnover of MTT (Fig. 5a). When cells were treated with off-pathway oligomers we observed no significant difference from controls on MTT turnover. It should be noticed that the presence of doxycycline in the culture medium was not capable to prevent the deleterious effect that on-pathway oligomers exerted on SH-SY5Y cells.

Figure 5: Modulation of α-synuclein oligomers cytotoxicity by doxycycline. (a) Cell viability, and (b) cell death of SH-SY5Y cells 24 h after the addition of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (control), on-pathway α-synuclein oligomers, off-pathway α-synuclein oligomers, or on-pathway α-synuclein oligomers with doxycycline added to the culture medium. (c) Changes in liposomal membrane permeability upon the addition of the same species. The fluorescence signal was normalized by the signal observed after the addition of Triton X-100, which induced complete disruption of the vesicles. A monomeric solution of α-synuclein was also used as a negative control for cell viability, cytotoxicity and leakage assays. On-pathway, and off-pathway α-synuclein oligomers were prepared by incubating 140 μM α-synuclein in the absence, or in the presence of 200 μM doxycycline respectively for 16 h under orbital agitation at 37 °C. Full size image

Interestingly, one of the proposed mechanisms by which α-synuclein oligomers produce cytotoxicity is by inducing a perturbation on the cell membrane integrity34. Therefore, we measured the release of the cytosolic enzyme lactate dehydrogenase (LDH) from cells into the medium, which is commonly used as a measure of cytotoxicity by membrane impairment (Fig. 5b). Once again, on-pathway oligomers showed cytotoxicity against SH-SY5Y cells as reflected by the 40% increase in the release of LDH into the culture medium. On the contrary, in the presence of off-pathway oligomers cell integrity is well preserved. Preservation of cell integrity is not observed when doxycycline is added to the culture medium before the addition of on-pathway oligomers.

The differential capability of α-synuclein oligomers to affect membrane integrity, as showed by LDH assays, was also analyzed by content leakage assays on synthetic membranes. To do that, we monitored the release of calcein entrapped in rat brain lipid vesicles upon the addition of α-synuclein oligomers (Fig. 5c). In line with cytotoxicity assays, on-pathway α-synuclein oligomers induced the release of the fluorescent probe from liposomes, confirming the ability of these species to disrupt membrane integrity; while off-pathway oligomers showed no destabilizing effect over lipid membranes. The addition of doxycycline to liposomes did not prevent calcein release induced by on-pathway oligomers showing that the antibiotic does not stabilize the membrane by a direct interaction nor does it blocks the deleterious effect of on-pathway oligomers upon binding to their surface.

Altogether, these results suggest that the protective effect of doxycycline could be attributed to the remodeling of α-synuclein into off-pathway oligomers that are not able to impair membrane integrity and not by a direct effect of the antibiotic on the cells.