Additional file 1: Figure S1. of Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

Effects of Ca2+ on Ser129-phosphorylation of α-syn in rat primary cortical neurons. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 concentrations on Ser129-phosphorylation. Primary cortical neurons were treated with A23187 at the indicated concentrations for 8 h. b, c Effect of extracellular Ca2+ chelator EGTA (b) or intracellular Ca2+ chelator BAPTA-AM (B-AM) (c) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 0.25 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 8 h. d Effect of CaM inhibitor W-7 on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 0.25 μM A23187 with the indicated concentrations of W-7 for 8 h. Representative blots are shown. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Graphs show relative ratios to vehicle control cells. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P < 0.05; **, P < 0.01). (TIFF 2872 kb

Semiquantification of the optical densities of striatal dopamine nerve terminals in rats treated with vehicle or ZNS.

<p>Rats were unilaterally injected with rAAV-A53T α-synuclein (<b>A</b>, <b>C</b>) or rAAV-GFP (<b>B</b>, <b>D</b>). The sections were immunostained with antibody for TH (<b>A</b>, <b>B</b>) or DAT (<b>C</b>, <b>D</b>). In rats injected with rAAV-A53T α-synuclein (<b>A</b>, <b>C</b>), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 2 weeks after injection (left panels), and those of the injected sides at 4 weeks (middle panels) and 8 weeks (right panels) after injection. In rats injected with rAAV-GFP (<b>B</b>, <b>D</b>), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 4 weeks (left panels) and 8 weeks (right panels) after injection. Scale bars, 1.0 mm. (<b>A–D</b>) Graphs show semiquantitative analysis of striatal TH or DAT-positive fibers. As compared with the vehicle group, ZNS significantly suppressed loss of striatal TH-positive fibers at 4 weeks (<i>P</i><0.001) and 8 weeks (<i>P</i> = 0.004) after rAAV-A53T α-synuclein injection. Decreased densities of DAT-positive fibers were significantly suppressed in the ZNS group at 4 weeks (<i>P</i><0.001) and 8 weeks (<i>P</i><0.001) after viral injection. The open bars illustrate the vehicle group and the black bars illustrate the ZNS group. Data represent the mean ± SD and <i>P</i>-values were estimated by two-way ANOVA, followed by a Bonferroni’s <i>post hoc</i> test (*<i>P</i><0.05).</p

rAAV-mediated expression of A53T α-synuclein in the rat nigrostriatal system.

<p>(<b>A</b>) rAAV-A53T α-synuclein was unilaterally injected in the substantia nigra pars compacta. Immunohistochemical images of the rat substantia nigra (SN) and striatum at 2 weeks after viral injection. Upper panels show sections immunostained with LB509 antibody that recognizes total human α-synuclein, including phosphorylated and nonphosphorylated forms. Middle and bottom panels show sections immunostained with antibodies for tyrosine hydroxylase (TH) and dopamine transporter (DAT), respectively. Left and right panels show sections of rats treated with vehicle and zonisamide (ZNS), respectively. Scale bars, 1 mm. (<b>B</b>) Left panels shows western blotting of the rat ventral midbrains at 2 weeks after viral injection. 1% Triton X-100-soluble extracts (5 µg/lane) were analyzed by western blotting with Syn-1 (total α-synuclein), LB509 (human total α-synuclein), and EP1536Y (phosphorylated α-synuclein) antibodies. For loading control, the same amounts of samples were blotted with β-actin antibody. Right graphs show the ratios of the total α-synuclein and phosphorylated α-synuclein expression levels in the ZNS group relative to those in the vehicle group. Band intensities were normalized to β-actin to correct loading variations between lanes. Data represent the means ± SD.</p

Biochemical analysis of the expression levels of TH and DAT in rats unilaterally injected with rAAV-A53T α-synuclein.

<p>The striatum tissues were collected at 4 weeks after viral injection, and divided into the injected and uninjected sides. (<b>A</b>) 1% Triton X-100-soluble extracts (10 µg/lane) were analyzed by western blotting with TH, DAT, and LB509 (human total α-synuclein) antibodies. Representative blot data of the vehicle and ZNS groups are shown. Human α-synuclein is observed only in the rAAV-injected sides. Although the DAT blot of vehicle-treated samples is separated from the blot of ZNS-treated ones, these blots are originally derived from the same blot. (<b>B</b>) Graphs show the ratios of TH and DAT expression levels in the ZNS group relative to those in the vehicle group. As compared with vehicle treatment, ZNS significantly retained the expression levels of TH (n = 4 for each group, <i>P</i> = 0.013) and DAT (n = 4 for each group, <i>P</i><0.001). Data represent the means ± SD. <i>P</i>-values were estimated by unpaired <i>t</i> test (*<i>P</i><0.05).</p

Effects of late treatment with ZNS on A53T α-synuclein-induced loss of nigrostriatal neurons.

<p>ZNS administration was started 7 days after rAAV-A53T α-synuclein injection, and the treatment was performed until 4 weeks after viral injection. Sections were immunostained with antibody for TH. Graphs show the quantitative analysis of the number of TH-positive nigral neurons (<b>A</b>) and the semiquantitative analysis of the optical density of TH-positive striatal fibers (<b>B</b>). As compared with vehicle treatment, ZNS significantly suppressed loss of TH-positive nigral neurons (<i>P</i> = 0.012). Decreased densities of TH-positive nigral fibers were significantly suppressed in the ZNS group (<i>P</i> = 0.020). Data represent the mean ± SD. <i>P</i>-values were estimated by unpaired <i>t</i> test (*<i>P</i><0.05).</p

Stereological assessments of the number of nigral dopamine neurons in rats treated with vehicle or ZNS.

<p>Rats were unilaterally injected with rAAV-A53T α-synuclein (<b>A</b>, <b>C</b>) or rAAV-GFP (<b>B</b>, <b>D</b>). The sections were immunostained with antibody for TH (<b>A</b>, <b>B</b>) or DAT (<b>C</b>, <b>D</b>). In rats injected with rAAV-A53T α-synuclein (<b>A</b>, <b>C</b>), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 2 weeks after injection (left panels), and those of the injected sides at 4 weeks (middle panels) and 8 weeks (right panels) after injection. In rats injected with rAAV-GFP (<b>B</b>, <b>D</b>), photomicrographs show representative immunostainings of the injected and uninjected sides of the substantia nigra at 4 weeks (left panels) and 8 weeks (right panels) after injection. Scale bars, 0.5 mm. (<b>A–D</b>) Graphs show quantitative analysis of TH or DAT-positive nigral neurons. As compared with the vehicle group, ZNS treatment significantly delayed loss of TH-positive nigral neurons at 4 weeks (<i>P</i> = 0.004) and 8 weeks (<i>P</i> = 0.010) after rAAV-A53T α-synuclein injection. Loss of DAT-positive nigral neurons in the ZNS group was also delayed at 4 weeks (<i>P</i> = 0.008) and 8 weeks (<i>P</i> = 0.040) after viral injection. The open bars illustrate the vehicle group and the black bars illustrate the ZNS group. Data represent the mean ± SD and <i>P</i>-values were estimated by two-way ANOVA, followed by unpaired <i>t</i> test (*<i>P</i><0.05).</p

Additional file 1: Table S1. of Whole-exome sequencing and digital PCR identified a novel compound heterozygous mutation in the NPHP1 gene in a case of Joubert syndrome and related disorders

Clinical symptoms of the patient with Joubert syndrome with oculorenal defects in this study. (PPTX 85 kb

Additional file 2: Figure S1. of Whole-exome sequencing and digital PCR identified a novel compound heterozygous mutation in the NPHP1 gene in a case of Joubert syndrome and related disorders

Schematic presentation of the deletion mutation predicted by a read depth-based copy number variation detection algorithm. The approximately 470 kb heterozygous deletion including the entire NPHP1 gene is shown in the patient (II-2) and her mother (I-2). The chromosomal positions are based on NCBI build 37. (PDF 84 kb

A Segmental Copy Number Loss of the <i>SFMBT1</i> Gene Is a Genetic Risk for Shunt-Responsive, Idiopathic Normal Pressure Hydrocephalus (iNPH): A Case-Control Study

<div><p>Little is known about genetic risk factors for idiopathic normal pressure hydrocephalus (iNPH). We examined whether a copy number loss in intron 2 of the <i>SFMBT1</i> gene could be a genetic risk for shunt-responsive, definite iNPH. Quantitative and digital PCR analyses revealed that 26.0% of shunt-responsive definite iNPH patients (n = 50) had such a genetic change, as compared with 4.2% of the healthy elderly (n = 191) (OR = 7.94, 95%CI: 2.82–23.79, <i>p</i> = 1.8 x 10⁻⁵) and 6.3% of patients with Parkinson’s disease (n = 32) (OR = 5.18, 95%CI: 1.1–50.8, <i>p</i> = 0.038). The present study demonstrates that a copy number loss within intron 2 of the <i>SFMBT1</i> gene may be a genetic risk factor for shunt-responsive definite iNPH.</p></div

Copy numbers of the region in intron 2 of the SFMBT1 gene in iNPH patients and controls.

<p>Copy numbers of the region in intron 2 of the SFMBT1 gene in iNPH patients and controls.</p