Additional file 1: Table S1. of Distribution of Îą-synuclein in the spinal cord and dorsal root ganglia in an autopsy cohort of elderly persons

Clinicopathological characteristics of 265 subjects with LBAS. (XLSX 12 kb

Additional file 2: Figure S1. of Distribution of Îą-synuclein in the spinal cord and dorsal root ganglia in an autopsy cohort of elderly persons

Intraneuronal cytoplasmic bodies in the large motor neurons. (PDF 93 kb

Additional file 1: Figure S1. of Serum microRNA miR-501-3p as a potential biomarker related to the progression of Alzheimer’s disease

This study’s definitions of patients with Alzheimer’s disease (AD) and controls on the basis of Braak staging in the ROW discovery set. (PDF 186 kb

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

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

Age-dependent neuritic abnormalities in Tau- or MAP2-expressing worms.

<p>(<b>A–E</b>) CLSM images of neurites in the posterior part of the worm are shown. (<b>A</b>) Mock/DsRed-transgenic (Tg) worm, young. (<b>B</b>) Mock/DsRed-Tg worm, aged. (<b>C</b>) Tau(0N3R)/DsRed-Tg worm, young. (<b>D</b>) Tau(0N3R)/DsRed-Tg worm, aged. (<b>E</b>) Tau(0N4R)/DsRed-Tg worm, young. (<b>F</b>) Tau(0N4R)/DsRed-Tg worm, aged. (<b>G</b>) MAP2/DsRed-Tg worm, young. (<b>H</b>) MAP2/DsRed-Tg worm, aged. The scale bar is 100 µm. (<b>I</b>) Numbers of abnormal kinks (arrows) per 100 µm neurite. “Young” indicates 4–5 days after hatching, and “aged” indicates 10–11 days. The data are expressed as the mean±SEM. Asterisks indicate significant differences versus mock in each age group (one-way ANOVA followed by Bonferroni–Dunn <i>post hoc</i> test). # indicates a significant difference in the young versus aged group in the same line (P<0.05, Student's t-test). n = 21 to 23.</p

Aß assembly in the presence of the lipids extracted from SPMs isolated from autopsy brains.

<p>A, Representative original (<i>upper</i>) and binarized (<i>lower</i>) AFM images of Aß mixtures incubated on the reconstituted membranes prepared from extracted lipid samples; <i>P1</i> from the amyloid-free precuneus, <i>C1</i> from the calcarine cortex of the brain providing <i>P1</i>, <i>P2</i> from the amyloid-bearing precuneus, and <i>C2</i> from the calcarine cortex of the brain providing <i>P2</i>. The SPM sample containing same amount of proteins was used for each analysis. AFM images were taken after Aß incubation at 20 μM for 15 min. B, Area of Aß42 assembly on the membrane. C, Area of Aß42 assembly on the membrane of <i>P2</i> in the presence of 4396C or normal IgG. The mean of two independent experiments is shown in B and C. D, Western blots of insoluble (Ppt) and soluble (Sup) Aß42 following incubation with liposomes prepared from <i>P2</i> and <i>C2</i>. The insoluble Aß42 assemblies were solubilized in formic acid prior to Western blot analysis. The blot was immunostained with the anti-Aß antibody 6E10. E, TEM image of Aß42 mixtures incubated with liposomes prepared from <i>P2</i>. Typical amyloid fibrils with 6–10 nm diameter were observed (arrowheads). Asterisks indicate liposomes. Scale: 500 nm (A), 100 nm (E).</p

Lipid analyses of cholesterol and sphingomyelin of SPMs.

<p>A, Cholesterol level of SPMs. A, The cholesterol level of SPMs was determined by the cholesterol oxidase method using an Amplex-Red cholesterol assay kit. B, Composition of sphingomyelins of SPMs. Extracted sphingomyelins from SPMs were analyzed by reverse-phase LC-MS using a C30 column. The proportion of each sphingomyelin species containing diverse ceramide structures in four major sphingomyelins (containing d18 or d20 as the sphingoid base and C18:0 in the fatty acid chain) is expressed as mean ± SEM. <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. The SPM sample containing same amount of proteins was used for each analysis.</p

MAP2 is not involved in the growth process of NFTs in the AD brain.

<p>(<b>A</b>) Diagram of three site-specific anti-MAP2 antibodies and anti-Tau antibodies. (<b>B</b>) Human temporal cortex tissues from three AD patients and three normal controls were homogenized sequentially in detergent-containing buffers. Sarkosyl-insoluble, SDS-soluble fractions were prepared and subjected to SDS-PAGE followed by western blotting using anti-Tau antibodies (AT8 and PHF1) and three newly raised site-specific anti-MAP2 antibodies (MAP2-#39 and #40 are not shown). Total protein was used as the loading control. The staining intensity of Tau was increased markedly in the Sarkosyl-insoluble, SDS-soluble fractions from AD brains compared with normal brains. By contrast, the MAP2 antibodies failed to detect any increased patterns in AD brains compared with normal brains. Phosphatase treatment was performed to avoid effects from MAP2 phosphorylation. NC, normal brains; AD, Alzheimer's disease brains. Information about the cases is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089796#pone.0089796.s004" target="_blank">Table S1</a>. (<b>C</b>) Double immunofluorescence staining of the homologous carboxyl-terminal sequences of Tau and MAP2 in the AD brain. AD brain paraffin-embedded sections were double-labeled by anti-Tau antibody (PHF1) and anti-MAP2 antibody (MAP2-#41). Tau but not MAP2 localized in NFTs as well as in NTs. Representative Tau-positive-only neurons (arrowhead), MAP2-positive-only neurons (arrow) and Tau/MAP2-double-positive neurons (star) are indicated. Scale bars = 25 µm. (<b>D</b>) Average number of the three neuron types was counted per 640 µm². The data are presented as the mean±SD.</p