A Cellular Model To Monitor Proteasome Dysfunction by α-Synuclein

Impairment of the ubiquitin-proteasome degradation system has recently been suggested to be related to the onset of neurodegenerative disorders such as Alzheimer′s disease and Parkinson's disease. In this study, we investigated whether intracellular α-synuclein affects proteasome activity in SH-SY5Y cells. To monitor intracellular proteasome activity, we used a reporter consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), which is known to be degraded by proteasome. The level of intact GFP-CL1 was dramatically increased by coexpression of GFP-CL1 and α-synuclein, as judged by confocal microscopic and immunoblot analyses. Expression of two pathogenic mutants of α-synuclein, A30P and A53T, and phosphomimetic S129D mutant increased the intensities of GFP more effectively than did wild-type α-synuclein. GFP fluorescence in cells transfected with Δ73-83 mutant or β-synuclein, which does not assemble into filaments <i>in vitro</i>, was not changed as compared with that in cells expressing GFP-CL1 alone. Thus, the ability of α-synuclein to inhibit proteasome activity is related to its propensity to assemble into filaments. Furthermore, we observed that some compounds inhibiting α-synuclein filament formation <i>in vitro</i> prevented the α-synuclein-mediated proteasome dysfunction in cells transfected with both GFP-CL1 and α-synuclein. The cellular model expressing both GFP-CL1 and α-synuclein may be a useful tool to screen compounds protecting neurons from α-synuclein-mediated proteasome dysfunction

Additional file 2: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Table S1. α-Syn concentrations in sarkosyl-insoluble fractions extracted from patients’ brains, The α-syn concentrations of sarkosyl-insoluble fractions extracted from patients’ brains used for experiments in the cultured cell model (A) and mouse model (B) are shown. (PDF 40 kb

Additional file 3: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S2. Seeding activities of serial dilutions of sarkosyl-insoluble fractions from brains of α-synucleinopathy patients, Sarkosyl-insoluble fractions extracted from brains with synucleinopathy patients were diluted and introduced into SH-SY5Y cells transiently expressing human α-syn. Immunoblot analyses of sarkosyl-insoluble fractions (ppt) and sarkosyl-soluble fractions (sup) extracted from cells transfected with serial dilutions of MSA-1(Cb), MSA-2 (FC), MSA-2 (Pu), MSA-3 (FC) and DLB-4 (FC) are shown. Phosphorylated α-syn was detected with anti-phosphorylated α-syn PSer129 antibody. α-Syn was detected with anti-syn 131–140 antibody. Cb: cerebellum, FC: frontal cortex, Pu: putamen. (PDF 283 kb

Additional file 4: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S3. Determination of protein concentration of α-syn in C57BL/6 mouse brain, Standard curve of mouse α-syn was generated by immunoblotting of serial dilutions of recombinant mouse α-syn protein. Protein concentrations of endogenous α-syn in mouse brains were determined by interpolation on a standard curve. A68 buffer-soluble fractions were extracted from C57BL/6 mouse brains (n = 3). Bands of recombinant proteins and A68 buffer-soluble fractions were detected with anti-mouse α-syn antibody. (PDF 78 kb

Additional file 1: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S1. Determination of protein concentration of phosphorylated α-syn in patients’ brains. A, Sarkosyl-insoluble fractions prepared from patients’ brains used in this study were analyzed by immunoblotting with anti-phosphorylated α-syn PSer129 antibody (upper) and anti-tau T46 antibody (lower). B, Standard curve of phosphorylated α-syn, generated by immunoblotting of phosphorylated monomer α-syn. Concentrations of phosphorylated α-syn were determined using this standard curve. Protein concentrations of sarkosyl-insoluble fractions extracted from patients’ brains are shown in Table S2. (PDF 139 kb

Additional file 5: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Table S2. Numbers of mice used in experiments. (PDF 52 kb

Immunoblotting analysis of abnormal tau in the sarkosyl-insoluble fraction.

<p>(A) Immunoblotting analysis was visualized using AT8 antibody for the sarkosyl-insoluble fraction. The numbers indicate individual mice: 1–15, MB 1 mg/kg/day group; 16–30, MB 0.3 mg/kg/day group; and 31–44, water only group. Molecular weight markers are shown on the right (kDa). P, positive control (P301L tau transgenic mouse, 20 month-old female). (B) A comparison of relative AT8 expression levels of the MB-treated groups and the water only group. The data were compared with the AT8 band intensity. (C) A comparison of relative phosphorylated tau (AT8)/total tau (HT7) levels of the MB-treated groups and the water only group. The data were compared with the AT8 band intensity, which was normalized with the total tau (HT7) band intensity. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. P<0.01 was considered to represent a statistically significant difference. a.u., arbitrary unit. N.S., no significant difference.</p

Immunohistochemical staining with a conformational antibody that recognizes aggregated tau.

<p>MC-1-positive neurons and cellular processes were seen in the motor cortex (A and B), prepotic area (C and D), posterior hypothalamus (E and F) and pons (G and H). A, C, E, G, mouse with a low AT8/HT7 ratio; and B, D, F, H, mouse with a high AT8/HT7 ratio. The calibration bar in H applies to all photomicrographs (50 µm).</p

Additional file 6: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S4. Seeding activities of serial dilutions of treated synthetic α-syn fibrils in SH-SY5Y cells, Serial dilutions of synthetic α-syn fibrils exposed to various inactivation treatments were introduced into SH-SY5Y cells. Immunoblot analysis of sarkosyl-insoluble fractions (ppt) and sarkosyl-soluble fractions (sup) extracted from cells transfected with serial dilutions of synthetic α-syn fibrils treated with 1% SDS for 1 h at room temperature, boiling, or autoclaving at 134 °C with or without 1% SDS are shown. Phosphorylated α-syn was detected with anti-phosphorylated α-syn PSer129 antibody. α-Syn was detected with anti-syn 131–140 antibody. (PDF 248 kb

Immunoblotting analysis of total tau in the Tris-soluble fraction.

<p>(A) Immunoblotting analysis was visualized using HT7 antibody for the Tris-soluble fraction. The numbers indicate individual mice: 1–15, MB 1 mg/kg/day group; 16–30, MB 0.3 mg/kg/day group; and 31–44, water only group. Molecular weight markers are shown on the right (kDa). For quantitative measure of band intensity, α-tubulin was used as an internal control for protein concentration. (B) A comparison of the relative total tau (HT7) expression levels of the MB-treated groups and the water only group. The data were compared with the HT7 band intensity, which was normalized with α-tubulin. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. a.u., arbitrary unit. N.S., no significant difference.</p