Single point mutations at the S129 residue of α-synuclein and their effect on structure, aggregation, and neurotoxicity

Abstract

Parkinson's disease is an age-related neurological disorder, and the pathology of the disease is linked to different types of aggregates of α-synuclein or alpha-synuclein (aS), which is an intrinsically disordered protein. The C-terminal domain (residues 96-140) of the protein is highly fluctuating and possesses random/disordered coil conformation. Thus, the region plays a significant role in the protein's solubility and stability by an interaction with other parts of the protein. In the current investigation, we examined the structure and aggregation behavior of two artificial single point mutations at a C-terminal residue at position 129 that represent a serine residue in the wild-type human aS (wt aS). Circular Dichroism (CD) and Raman spectroscopy were performed to analyse the secondary structure of the mutated proteins and compare it to the wt aS. Thioflavin T assay and atomic force microscopy imaging helped in understanding the aggregation kinetics and type of aggregates formed. Finally, the cytotoxicity assay gave an idea about the toxicity of the aggregates formed at different stages of incubation due to mutations. Compared to wt aS, the mutants S129A and S129W imparted structural stability and showed enhanced propensity toward the α-helical secondary structure. CD analysis showed proclivity of the mutant proteins toward α-helical conformation. The enhancement of α-helical propensity lengthened the lag phase of fibril formation. The growth rate of β-sheet-rich fibrillation was also reduced. Cytotoxicity tests on SH-SY5Y neuronal cell lines established that the S129A and S129W mutants and their aggregates were potentially less toxic than wt aS. The average survivability rate was ∼40% for cells treated with oligomers (presumably formed after 24 h of incubation of the freshly prepared monomeric protein solution) produced from wt aS and ∼80% for cells treated with oligomers obtained from mutant proteins. The relative structural stability with α-helical propensity of the mutants could be a plausible reason for their slow rate of oligomerization and fibrillation, and this was also the possible reason for reduced toxicity to neuronal cells.

Keywords: Raman; alpha-synuclein; amyloid; fibrillization; neurotoxicity; secondary structure.

FIGURE 1

Circular dichroism spectra of wt and mutant alpha synucleins and spectral deconvolution. One Gaussian at 198 nm is fitted for random coil and one at 218 nm for beta sheets. Two Gaussians at 208 and 222 nm are fitted for alpha helix, and the sum of the two is shown as “Fit alpha.” Summation of all the fitted Gaussians and the residuals are also shown. (A) CD spectra of wild-type aSn protein at different time points of incubation. (B) Circular dichroism spectra of S129A α-synuclein at different time points of incubation. (C) Circular dichroism spectra of S129W α-synuclein at different time points of incubation. For 72 h, a positive peak is observed at 195 nm, which is a signature of the beta-sheet. One Gaussian is additionally fitted for the beta-sheet in this spectrum. Summation of all the fitted Gaussians and the residuals are also shown. (D) Changes in the secondary structural composition over the period of incubation for wt and the mutants.

FIGURE 2

Time-dependent ThT fluorescence assay for determining the fibril formation growth kinetics of wild-type α-synuclein (wt aS) and the mutant variants (S129A and S129W). For each incubated sample, at different time points of incubation, 20 µL of the incubated solution was mixed with 500 µL of ThT solution (∼20 µM) and the ThT fluorescence was measured, and the sample was excited at 440 nm. The magenta line is the fit of Eq. 1 to the data points obtained for wt aS. Similarly, the olive line is the fit of Eq. 1 to the data points obtained for S129A, and the blue line is a fit of Eq. 1 to the data points obtained for S129W.

FIGURE 3

Different elongation kinetics of wt, S129A, and S129W aS proteins as obtained from AmyloFit and surface morphology of α-synuclein aggregates formed at different time points, as observed by atomic force microscopy. (A) Nucleation elongation kinetics for wild-type aS and AFM images obtained at 24 and 72 h. (B) Secondary nucleation-based elongation kinetics for S129A and AFM images obtained at 24 and 72 h. (C) Fragmentation-dominated elongation kinetics for S129W and AFM images obtained at 24 and 72 h. All AFM images are of dimension 2.5 µm × 2.5 µm.

FIGURE 4

Raman spectra of wild and two mutants of alpha synuclein, their monomer, and fibril in the frequency range of 1200–1800 cm⁻¹ by 532-nm laser. Both monomer (A) and fibril (B) solution were prepared in 20 mM sodium phosphate buffer pH 7.4 and dropped cast on a glass coverslip, and spectra were recorded at room temperature (25 °C) after being air dried. Laser power at the source was 50 mW at the sample.

FIGURE 5

Cytotoxicity evaluation of wild-type and mutant aS proteins using MTT assay. Wild-type and mutant protein oligomers were incubated for 0, 24, 48, and 72 h, and then, their effects were checked for viability of SH-SY5Y neuroblastoma cells. (A) Representative microscopic images of SH-SY5Y cells after 24 h treatment of different variants of wild-type and mutant proteins. Cells were treated with 15 µM concentration for imaging purposes. Arrows indicate dead cells. (B) Cells were treated with two different concentrations of oligomer variants (10 μM and 15 µM) and incubated for 24 h, followed by MTT assay. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated cell (control) as mean ± SEM in a bar diagram where *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to control. NaP, sodium phosphate buffer; W0, W24, W48, and W72, wild-type alpha synuclein incubated for 0, 24, 48, and 72 h, respectively; SW0, SW24, SW48, and SW72 mutant S129W alpha-synuclein incubated for 0, 24, 48, and 72 h, respectively; SA0, SA24, SA48, and SA72 mutant S129A alpha-synuclein incubated for 0, 24, 48, and 72 h, respectively.

FIGURE 6

AFM topographic images of SH-SY5Y cells in presence of fibrils. The figure displays topographic images acquired through atomic force microscopy (AFM) of SH-SY5Y cells. SH-SY5Y cells were incubated for 24 h using 72 h incubated aS. Then, the cells were processed for AFM, which (A) depicts control cells without any alpha-synuclein (aS) fibrils, while (B) shows SH-SY5Y cells in the presence of wild-type aS fibrils. (C) shows SH-SY5Y cells exposed to the S129A aS fibril, and (D) displays SH-SY5Y cells exposed to the S129W aS fibril. In all panels, the presence of fibrils is indicated with a white arrow. Scale bar = 10 µm.