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 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

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 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

Data_Sheet_1_Distinct eye movement patterns to complex scenes in Alzheimer’s disease and Lewy body disease.docx

BackgroundAlzheimer’s disease (AD) and Lewy body disease (LBD), the two most common causes of neurodegenerative dementia with similar clinical manifestations, both show impaired visual attention and altered eye movements. However, prior studies have used structured tasks or restricted stimuli, limiting the insights into how eye movements alter and differ between AD and LBD in daily life.ObjectiveWe aimed to comprehensively characterize eye movements of AD and LBD patients on naturalistic complex scenes with broad categories of objects, which would provide a context closer to real-world free viewing, and to identify disease-specific patterns of altered eye movements.MethodsWe collected spontaneous viewing behaviors to 200 naturalistic complex scenes from patients with AD or LBD at the prodromal or dementia stage, as well as matched control participants. We then investigated eye movement patterns using a computational visual attention model with high-level image features of object properties and semantic information.ResultsCompared with matched controls, we identified two disease-specific altered patterns of eye movements: diminished visual exploration, which differentially correlates with cognitive impairment in AD and with motor impairment in LBD; and reduced gaze allocation to objects, attributed to a weaker attention bias toward high-level image features in AD and attributed to a greater image-center bias in LBD.ConclusionOur findings may help differentiate AD and LBD patients and comprehend their real-world visual behaviors to mitigate the widespread impact of impaired visual attention on daily activities.</p

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

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

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

<p>(A) Immunoblot analysis was visualized using HT7 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; 31–44, water only group. Molecular weight markers are shown on the right (kDa). (B) A comparison of relative total tau (HT7) expression levels in the sarkosyl-insoluble fraction of the MB-treated groups and the water only group. The data were compared with the HT7 band intensity. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. a.u., arbitrary unit.</p