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

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 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

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

Imbalance of “<i>a</i>-series”-“<i>b</i>-series” composition in GD1(d20:1–20:0) in the brains harboring Aß deposition.

<p>Composition of GD1-gangliosides in relation to <i>a</i>- and <i>b</i>-series of gangliosides. The proportion of each ganglioside in detected gangliosides is expressed as mean with ± SEM in parentheses. <i>P1</i>, <i>C1</i>, <i>P2</i>, and <i>C2</i> indicate lipid samples extracted from SPMs of the amyloid-free precuneus, the calcarine cortex of the brain with the amyloid-free precuneus, the amyloid-bearing precuneus, and the calcarine cortex of the brain with the amyloid-bearing precuneus, respectively.</p><p>Imbalance of “<i>a</i>-series”-“<i>b</i>-series” composition in GD1(d20:1–20:0) in the brains harboring Aß deposition.</p

Increased ratio of level of (d20:1–20:0) to that of (d20:1–18:0) in GD1b-ganglioside in the amyloid-bearing precuneus.

<p>Ratios of the level of (d20:1–20:0) to that of (d20:1–18:0) in GD1a- and GD1b-gangliosides were calculated from the levels of <i>a</i>- and <i>b</i>-series of gangliosides obtained by normal-phase LC-MS using an NH₂ column. The ratio is expressed as mean ± SEM. *, <i>p</i><0.05. <i>P1</i>, <i>C1</i>, <i>P2</i> and <i>C2</i> indicate lipid samples extracted from SPMs of the amyloid-free precuneus, the calcarine cortex of the brain with the amyloid-free precuneus, the amyloid-bearing precuneus, and the calcarine cortex of the brain with the amyloid-bearing precuneus, respectively.</p

Additional file 2: Table S1. of Serum microRNA miR-501-3p as a potential biomarker related to the progression of Alzheimer’s disease

Top 20 deregulated serum miRNAs that were identified by NGS in the ROW discovery set after adjusting for age, sex, APOE genotype, and hemolysis ratio. Table S2. Significantly deregulated miRNAs that were identified by NGS in the temporal cortex of the ROW discovery set. Table S3. Significantly deregulated miRNAs that were identified by NGS in the temporal cortex of the ROW discovery set after adjusting for age, sex, APOE genotype, and RIN. Table S4. Significantly differentially expressed genes that were identified by NGS in hsa-miR-501-3p overexpression in cultured cells. Table S5. Gene Ontology enrichment analysis on the significantly downregulated genes in hsa-miR-501-3p overexpression in cultured cells. Table S6. Gene Ontology enrichment analysis on the significantly upregulated genes in hsa-miR-501-3p overexpression in cultured cells. (XLS 172 kb

Analyses of gangliosides by normal-phase LC-MS with regard to the glycan structure of gangliosides.

<p>A, Scheme of synthesis pathway of <i>a</i>- and <i>b</i>-series of gangliosides. B, Representative chromatograms of normal-phase LC-MS of gangliosides using an NH₂ column. GM1(d18:1–¹³C16:0) [<i>m/z</i> 1532.88 (shown on the left side on the chromatogram)] was used as ISD. In this system, <i>a</i>-series (GD1a) and <i>b</i>-series (GD1b) GD1-gangliosides were separately detected on the basis of the diversity of the glycan structure. The expression level of each ganglioside species was calculated by measuring each peak area above the baseline.</p