Chicago sky blue 6B inhibits α-synuclein aggregation and propagation

Abstract

Abnormal deposition of α-synuclein aggregates in Lewy bodies and Lewy neurites is the hallmark lesion in Parkinson's disease (PD). These aggregates, thought to be the culprit of disease pathogenesis, spread throughout the brain as the disease progresses. Agents that inhibit α-synuclein aggregation and/or spread of aggregates would thus be candidate disease-modifying drugs. Here, we found that Chicago sky blue 6B (CSB) may be such a drug, showing that it inhibits α-synuclein aggregation and cell-to-cell propagation in both in vitro and in vivo models of synucleinopathy. CSB inhibited the fibrillation of α-synuclein in a concentration-dependent manner through direct binding to the N-terminus of α-synuclein. Furthermore, both seeded polymerization and cell-to-cell propagation of α-synuclein were inhibited by CSB treatment. Notably, CSB alleviated behavioral deficits and neuropathological features, such as phospho-α-synuclein and astrogliosis, in A53T α-synuclein transgenic mice. These results indicate that CSB directly binds α-synuclein and inhibits its aggregation, thereby blocking α-synuclein cell-to-cell propagation.

Keywords: Aggregate propagation; Chicago sky blue 6B; Parkinson’s disease; Protein aggregation; α-synuclein.

Fig. 1

CSB inhibits α-synuclein fibrillation and reduces its toxicity. a CSB inhibits α-synuclein fibrillation in a concentration-dependent manner. CSB was incubated with recombinant human α-synuclein for 9 days, and Thio-T fluorescence assays were performed daily. b TEM analysis of α-synuclein fibrils incubated with different concentrations of CSB or vehicle (DMSO) control. Scale bars: 1 μm (low magnification, top line) and 200 nm (high magnification, bottom line). c Circular dichroism spectra of α-synuclein protein incubated with CSB (0.1 μM, 1 μM and 10 μM) or DMSO (control). d CSB (0.1, 1 and 10 μM) significantly improved the viability of α-synuclein–overexpressing cells. Data are presented as means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Dunnett’s post hoc test)

Fig. 2

CSB inhibits seeded fibrillation of α-synuclein and reduces its cell-to-cell propagation. a Inhibition of α-synuclein fibrillation by CSB in the presence of pre-formed fibril seeds (5%, w/w). b, c Reduction of α-synuclein aggregate propagation by CSB in the dual-cell BiFC model. Propagated α-synuclein is indicated by white arrows. Scale bar: 20 μm. Data are presented as means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

CSB directly binds N-terminal and NAC regions of α-synuclein. a Superposition of ¹H-¹⁵N HSQC spectra of α-synuclein (80 μM) alone (blue) and in the presence of a fivefold molar excess of CSB (red). Strong signal attenuation together with chemical shift changes were observed for select residues. b Residue-specific changes in the intensities of ¹H-¹⁵N HSQC cross-peaks of α-synuclein induced at increasing concentrations of CSB. I₀ and I are the intensities of ¹H-¹⁵N HSQC cross-peaks in the absence and presence of CSB, respectively. c Intensity changes (I–I₀) of non-overlapping residues at the N-terminus of α-synuclein reflecting exchange behavior show prominent slowing on the NMR chemical shift scale in the presence of increasing concentrations of CSB. Lines represent global fits to the experimental data for all selected residues based on a reversible 1-to-1 binding model

Fig. 4

CSB improves motor functions in a transgenic mouse model of synucleinopathy. a Schematic representation of the experimental timeline. Six-month-old A53T Tg and WT littermates were intraperitoneally injected weekly with CSB or PBS for 3 months. b Open field test. c Forelimb grip strength test. d–f Balance beam test. Data are presented as means ± SEM (*p < 0.05, **p < 0.01, ****p < 0.0001; ns, non-significant difference)

Fig. 5

CSB reduces S129-phosporylated α-synuclein levels and astrogliosis in a transgenic mouse model of synucleinopathy. a, b Representative images showing immunohistochemical staining for S129-phosphoryated α-synuclein (pS129) (a) and GFAP (b). HP, hippocampus; Pfcx, prefrontal cortex. c, d Quantification of pS129 levels in the HP (c) and Pfcx (d). e, f Quantification of GFAP levels in the HP (e) and Pfcx (f). Scale bar: 50 μm. Data are presented as means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, non-significant difference.)