CMT-3 targets different α-synuclein aggregates mitigating their toxic and inflammogenic effects

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder for which only symptomatic treatments are available. Repurposing drugs that target α-synuclein aggregation, considered one of the main drivers of PD progression, could accelerate the development of disease-modifying therapies. In this work, we focused on chemically modified tetracycline 3 (CMT-3), a derivative with reduced antibiotic activity that crosses the blood-brain barrier and is pharmacologically safe. We found that CMT-3 inhibited α-synuclein amyloid aggregation and led to the formation of non-toxic molecular species, unlike minocycline. Furthermore, CMT-3 disassembled preformed α-synuclein amyloid fibrils into smaller fragments that were unable to seed in subsequent aggregation reactions. Most interestingly, disaggregated species were non-toxic and less inflammogenic on brain microglial cells. Finally, we modelled the interactions between CMT-3 and α-synuclein aggregates by molecular simulations. In this way, we propose a mechanism for fibril disassembly. Our results place CMT-3 as a potential disease modifier for PD and possibly other synucleinopathies.

Figure 1

Comparative effect of CMT-3, DOX and MINO on α-synuclein amyloid aggregation. (a) Chemical structures of CMT-3, DOX, and MINO. Atom numbering and ring labels of the tetracycline skeleton are provided on the chemical structure of CMT-3. Note that the main difference in these structures resides in the presence or not of DMA groups designated by grey shaded circles. (b) Dose–response curves from endpoint ThT intensities of α-synuclein aggregation assay in the presence of different concentrations of CMT-3, DOX or MINO. IC₅₀ values are mean values of three independent determinations. The data fitting is described in “Methods” section. (c) TEM of α-synuclein samples incubated at 37 °C for 120 h under continuous orbital agitation in the absence (αS) or in presence of CMT-3 (αS:CMT-3), DOX (αS:DOX), or MINO (αS:MINO). The scale bar corresponds to 1 μm, at ×26,000 magnification.

Figure 2

CMT-3 inhibits α-synuclein amyloid aggregation by binding to aggregated species. (a) Turbidimetry assay of 70-μM α-synuclein samples incubated in the absence (empty circles) or presence of 100 μM CMT-3 (full circles) and monitored at 500 nm. (b) ThT fluorescence of 70 μM α-synuclein samples incubated in the absence (empty circles) or presence of 100 μM CMT-3 (full circles). (c) Binding of CMT-3 to α-synuclein aggregated species monitored by fluorescence anisotropy. Changes in fluorescence anisotropy of CMT-3 were measured at λ_em 520 nm upon the addition of aliquot samples harvested at different time-points of an α-synuclein aggregation reaction. Data values represent the mean ± S.E. of 6 independent experiments.

Figure 3

CMT-3 reshapes α-synuclein early aggregates into non-toxic species. (a) Analysis of α-synuclein Amide I' band after the curve fitting procedure (see “Methods”) showing the component bands: αS in the absence (top) or in the presence of CMT-3 (bottom) after 16 h incubation. (b) TEM and (c) SEM of α-synuclein samples incubated at 37 °C under orbital agitation in the absence (top) or in the presence of CMT-3 (bottom), and harvested after 16 h for observation of oligomers. The white bar corresponds to 500 nm. (d) LDH cytotoxicity assay in SH-SY5Y cells after the addition of α-synuclein oligomers formed after 16 h of incubation at 37 °C under orbital agitation in the absence (αS_oli) or presence of CMT-3 (αS:CMT-3_oli). Cytotoxicity values were normalized by the signal observed after addition of Triton X-100, which induced complete disruption of the cells. Data represents the mean ± S.E.M (n = 11). One-way ANOVA followed by Holm-Sidak’s multiple comparisons test. ****p < 0.0001 vs NT.

Figure 4

CMT-3, unlike DOX, disassembles preformed α-synuclein amyloid fibrils (αS_PFF). (a) In ThT fluorescence assays, 70 μM α-synuclein was either incubated alone (blue line), or with 100 μM of CMT-3 (purple line) or DOX (orange line) added 44 h after the initiation of the aggregation process (arrow). (b) TEM of α-synuclein samples incubated at 37 °C under orbital agitation during 44 h to obtain fibrillar species (αS_PFF). Samples were further incubated for another 52 h either alone or with the addition of 100 μM CMT-3 or DOX. Note the dramatic reduction in αS_PFF with the addition of CMT-3, but not with DOX. The scale bar corresponds to 1 μm (×26,000). (c, d) αS_PFF and αS_PFF:CMT-3 species from (b) were further submitted to SEM and AFM. SEM scale bar corresponds to 500 nm (×50,000) and 200 nm (×200,000). AFM scale bar corresponds to 1 μm. The pseudo color scale bar represents the information of the z axis from 0 to 100 nm. The z range was chosen to allow the observation of fibrils while the aggregated fibrillar clusters are out of range since they have a height greater than 100 nm.

Figure 5

Disassembled α-synuclein PFF (αS_PFF:CMT-3) are non-toxic and unable to either disrupt membrane integrity or promote seeding. (a) In seeding assays, αS_PFF served as efficient seeds to accelerate aggregation of α-synuclein monomers (light blue squares), while αS_PFF:CMT-3 were not only unable to seed monomers (purple triangles), but also blocked the amyloid aggregation reaction of the monomers. The unseeded aggregation kinetics of αS_m (no seeds) is also shown (black circles). For this assay, solutions were incubated at 37 °C under continuous orbital agitation and aggregation was measured by ThT fluorescence emission. (b) Changes in liposomal membrane permeability upon the addition of α-synuclein monomers (αS_m), α-synuclein oligomers harvested after 16 h of incubation (αS_oli), αS_PFF or αS_PFF:CMT-3. Results show that new species produced by CMT-3-driven disassembly of αS_PFF were unable to disrupt membrane permeability. The fluorescence signal was normalized to values obtained after addition of Triton X-100, which induced complete disruption of liposomal vesicles. (c) LDH cytotoxicity assay in SH-SY5Y cells after the addition of αS_m, αS_oli, αS_PFF and αS_PFF:CMT-3. The cytotoxicity signal was normalized to values obtained after addition of Triton X-100. Data represents the mean ± S.E.M (n = 11). One-way ANOVA followed by Holm-Sidak’s multiple comparisons test. ****p < 0.0001 vs NT.

Figure 6

Disassembled α-synuclein PFF (αS_PFF:CMT-3) are less inflammogenic for microglial cells. (a) TNF-α release in microglial cells treated for 24 h with αS_PFF, αS_PFF:CMT-3 (70 μg/ml), or intact αS_PFF in wells pre-incubated with CMT-3 (CMT-3_preinc + αS_PFF). (b) Quantification of glutamate release after exposure of microglial cultures to the same treatments as in (a). Data represents the mean ± S.E.M (n = 11). One-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001 vs NT. #p < 0.05, #### p < 0.0001 vs αS_PFF._.

Figure 7

Binding mode of CMT-3 to α-synuclein fibrils and subsequent disassembly mechanism (a) Side and close-up views of the putative complex formed between CMT-3 and α-synuclein fibrils. The protein backbone is represented in cartoon while the ligand is depicted in ball-and-stick representation. The hydrogen bond network between the protein and the ligand is represented as a discontinued blue line in the enlarged side view. (b) Contact frequency between CMT-3 and α-synuclein amino acid residues. (c) Schematic representation of the hydrogen bonds between CMT-3 and atoms from amide and carbonyl groups in the main chain of α-synuclein. Contact map between the residues at the edge (E) or the core (C) of the α-synuclein fiber in the absence (d) or presence of CMT-3 (e). The color bar scale represents the frequency contact along the simulation time.