Data_Sheet_1_Elevation of inositol pyrophosphate IP7 in the mammalian spinal cord of amyotrophic lateral sclerosis.pdf

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder associated with progressive impairment of spinal motor neurons. Continuous research endeavor is underway to fully understand the molecular mechanisms associating with this disorder. Although several studies have implied the involvement of inositol pyrophosphate IP7 in ALS, there is no direct experimental evidence proving this notion. In this study, we analyzed inositol pyrophosphate IP7 and its precursor IP6 in the mouse and human ALS biological samples to directly assess whether IP7 level and/or its metabolism are altered in ALS disease state.MethodsWe used a liquid chromatography-mass spectrometry (LC-MS) protocol originally-designed for mammalian IP6 and IP7 analysis. We measured the abundance of these molecules in the central nervous system (CNS) of ALS mouse model SOD1(G93A) transgenic (TG) mice as well as postmortem spinal cord of ALS patients. Cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) from ALS patients were also analyzed to assess if IP7 status in these biofluids is associated with ALS disease state.ResultsSOD1(G93A) TG mice showed significant increase of IP7 level in the spinal cord compared with control mice at the late stage of disease progression, while its level in cerebrum and cerebellum remains constant. We also observed significantly elevated IP7 level and its product-to-precursor ratio (IP7/IP6) in the postmortem spinal cord of ALS patients, suggesting enhanced enzymatic activity of IP7-synthesizing kinases in the human ALS spinal cord. In contrast, human CSF did not contain detectable level of IP6 and IP7, and neither the IP7 level nor the IP7/IP6 ratio in human PBMCs differentiated ALS patients from age-matched healthy individuals.ConclusionBy directly analyzing IP7 in the CNS of ALS mice and humans, the findings of this study provide direct evidence that IP7 level and/or the enzymatic activity of IP7-generating kinases IP6Ks are elevated in ALS spinal cord. On the other hand, this study also showed that IP7 is not suitable for biofluid-based ALS diagnosis. Further investigation is required to elucidate a role of IP7 in ALS pathology and utilize IP7 metabolism on the diagnostic application of ALS.</p

Additional file 1 of Polygenic effects on the risk of Alzheimer’s disease in the Japanese population

Additional file 1: Figure S1. The excluded region around the APOE gene. We removed the APOE region, consisting of ±500 kb, from around the top-hit SNP rs1160985 (chr19:45403412) in our data. Each data point indicates GWAS p values from Jansen et al. [32] used as SNP weights in the PRS calculation. Figure S2. Associations between the PRS and covariates. Age at baseline examination and years of education were examined by Spearman correlation. Sex and doses of APOE ε4 and ε2 alleles were analysed by t tests or ANOVAs. CN = cognitively normal; MCI = mild cognitive impairment; ADD = Alzheimer's disease dementia. Figure S3. Associations between the PRS.adjLD and covariates. Age at examination and years of education were examined by Spearman correlations. Sex and dose of APOE ε4 and ε2 alleles were analysed by t tests or ANOVAs. Figure S4. Comparison of AD conversion between the APOE ε4 carriers and the APOE ε4 noncarriers with high PRS. Kaplan–Meier survival curves for conversion rates of MCI to AD in the APOE ε4 carriers and the APOE ε4 noncarriers with high PRS values. p-values were calculated by log-rank test. Figure S5. Age differences between the low- and high-PRS groups and between the nonconverters and converters. Baseline ages were compared between groups using the Wilcoxon rank-sum test. Each violin plot includes the kernel probability density of the data at different values and the box plots with the median value and the interquartile range

Alteration of <em>POLDIP3</em> Splicing Associated with Loss of Function of TDP-43 in Tissues Affected with ALS

<div><p>Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by selective loss of motor neurons. In the ALS motor neurons, TAR DNA-binding protein of 43 kDa (TDP-43) is dislocated from the nucleus to cytoplasm and forms inclusions, suggesting that loss of a nuclear function of TDP-43 may underlie the pathogenesis of ALS. TDP-43 functions in RNA metabolism include regulation of transcription, mRNA stability, and alternative splicing of pre-mRNA. However, a function of TDP-43 in tissue affected with ALS has not been elucidated. We sought to identify the molecular indicators reflecting on a TDP-43 function. Using exon array analysis, we observed a remarkable alteration of splicing in the polymerase delta interacting protein 3 (<em>POLDIP3</em>) as a result of the depletion of TDP-43 expression in two types of cultured cells. In the cells treated with TDP-43 siRNA, wild-type <em>POLDIP3</em> (variant-1) decreased and <em>POLDIP3</em> lacking exon 3 (variant-2) increased. The RNA binding ability of TDP-43 was necessary for inclusion of <em>POLDIP3</em> exon 3. Moreover, we found an increment of <em>POLDIP3</em> variant-2 mRNA in motor cortex, spinal cord and spinal motor neurons collected by laser capture microdissection with ALS. Our results suggest a loss of TDP-43 function in tissues affected with ALS, supporting the hypothesis that a loss of function of TDP-43 underlies the pathogenesis of ALS.</p> </div

Additional file 1 of A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

Additional file 1. Supplementary methods, results (including Scheme S1), figures (S1–S7), and a table (S1)

Reduction of TDP-43 expression makes cells smaller.

<p>(<b>A</b>) A flow cytometer was used to obtain forward scatter (FSC) histograms of SH-SY5Y cells transfected with siRNA targeting POLDIP3 (blue) or TDP-43 (red) or non-targeting control (black). The FSC histogram is one representative transfection from which 10,000 cells were counted. (<b>B</b>) Data represent the mean with standard error of forward scatter of the siRNA-transfected cells from three independent experiments. Note that down-regulation of TDP-43 leads to a reduction in cell size as well as POLDIP3 depression. Asterisk indicates significant difference (*<i>P</i><0.01, Tukey multiple comparison test). (<b>C and D</b>) Cell lysates from the SHSY5Y cells stably expressed GFP-tagged POLDIP3 variants were subjected to immunoblotting for POLDIP3 (<b>C</b>) or TDP-43 (<b>D</b>). Anti-actin immunoblotting served as a loading control. (<b>E</b>) Recovery rate of the cell size in the TDP-43-depleted SH-SY5Y with or without expression of GFP-tagged POLDIP3 variant. Data represent the mean with standard error from three independent experiments. Note that the cell size recovery rate is significantly increased in the cells expressing POLDIP3 variant-1 compared to in the cells expressing variant-2. Asterisk indicates significant difference (*<i>P</i><0.01, Student <i>t</i> test).</p

TDP-43 RRM1 domain is necessary for including <i>POLDIP3</i> exon 3.

<p>(<b>A</b>) TDP-43 binds to <i>POLDIP3</i> mRNA. Whole cell lysate from HeLa cells was immunoprecipitated with anti-TDP-43 or control rabbit IgG, subjected to the isolation of RNA and RT-PCR analysis using primers located in exon 5 of <i>POLDIP3</i>. (<b>B–F</b>) Analysis of <i>POLDIP3</i> splicing by TDP-43 with mutated in RRM domains. (<b>B</b>) Schematic diagram of the constructs for wild-type (WT) and mutated TDP-43 expression plasmid (mRRM1, mRRM2, and mRRM1/2). Phe (F) to Leu (L) alterations were introduced in RRM1, RRM2, or both motifs of the TDP-43. Boxes indicate RRM motif. Numbers indicate the location of substituted amino acids. (<b>C</b>) TDP-43 depletion caused by siRNA targeting 3′-UTR of <i>TDP-43</i> (TDP-43 3′-UTR siRNA) was rescued by ectopic expression of WT and mutated TDP-43 in HEK293T cells. Mutated TDP-43s were similarly expressed. HEK293T cells were transfected with control (lane 1) or TDP-43 siRNA targeting for 3′-UTR region (lanes 2–6), followed by transfection with empty vector (EV) or various TDP-43 expression plasmids. TDP-43 expression was assessed by probing with anti-TDP-43 antibody. Anti-actin immunoblotting served as a loading control. (<b>D</b>–<b>F</b>) <i>POLDIP3</i> mRNA levels in culture cells transfected with <i>TDP-43</i> 3′-UTR siRNA as a fold change relative to those of cells transfected with control siRNA. The amounts of variant-1 (<b>D</b>), variant-2 (<b>E</b>) or total <i>POLDIP3</i> mRNA (<b>F</b>) were quantified by real-time qRT-PCR using the primers indicated with arrows in Fig. 2A. In this experiment, <i>RPLP1</i> and <i>RPS18</i> were used as reference genes. Data represent the mean with standard error from three independent experiments. Asterisk indicates significant difference among <i>TDP-43</i> 3′-UTR siRNA-transfected cells (*<i>P</i><0.01, Tukey multiple comparison test).</p

Additional file 2: of Heterogeneity of cerebral TDP-43 pathology in sporadic amyotrophic lateral sclerosis: Evidence for clinico-pathologic subtypes

Contains supplementary methods regarding ISH and biochemical studies, and legends to Supplementary Figure 1. (DOC 36 kb

Genes with altered splicing by depletion of TDP-43 in culture cells.

<p>(<b>A</b>) Western blot analysis of TDP-43 in HeLa and SH-SY5Y cells with or without TDP-43 siRNA. TDP-43 and actin were shown by immunoblotting with anti-TDP-43 (top) and anti-actin (bottom) antibody, respectively. (<b>B</b>) Microarray analyses were performed in control (transfected with siControl non-targeting pool) and TDP-43 knockdown HeLa and SH-SY5Y cells in triplicate. Venn diagram shows overlapping of altered splicing genes between HeLa and SH-SY5Y cells. Only 15 genes overlapped between them. From the array data, we calculated the <i>P</i> value and the splicing index (|SI|). Both <i>P</i><0.05 and |SI|>0.5 were used as thresholds to identify the genes with altered splicing. (<b>C</b>) Exon structure diagram of the main isoform (top) and candidate variant (bottom) for <i>POLDIP3</i> and <i>HP1BP3.</i> From the exon array result, we selected the candidate splicing variant that was induced by the depletion of TDP-43. The name of each transcript is labeled on the side of the exon structure. The red box indicates the exons that are expected to be altered by TDP-43 depletion. The black bars indicate the position of the primers we used in this experiment. The gene views show the expression of exons as determined by analyzing the results of exon array in HeLa cells using Genespring GX. The expression levels are shown on a log2 scale; the error bars show standard errors of means. TDP-43 siRNA, red circle; control siRNA, blue square. (<b>D</b>) qRT-PCR analysis revealed that the splicing alteration of <i>POLDIP3</i> and <i>HP1BP3</i> gene were validated in both HeLa and SH-SY5Y cells. <i>RPLP1</i> and <i>RPS18</i> were used as reference genes. Data represent the mean with standard error from three independent experiments. Asterisk indicates significant difference (*<i>P</i><0.01, Student <i>t</i> test).</p

Morphological studies.

<p>(A) Light micrographs of K_v7.2 immunoreactivity in the sensorimotor cortex. Note the immunostained neuronal cell bodies in layers II to VI. (B) Cell count of K_v7.2-immunopositive neurons in layers II/III and V in the frontal cortices of wildtype, heterozygous, and homozygous Y284C mice. No differences were noted.</p

Primer sequences.

a<p>amplicon size: 7.5 kb.</p>b<p>expected amplicon size: 1.8 kb; non-homologously recombined DNA is not expected to amplify.</p>c<p>expected amplicon size: 0.7 kb; wildtype allele is not expected to amplify.</p>d<p>expected amplicon size: 0.35 kb; wildtype and mutant transcripts were simultaneously amplified.</p><p>All primers were synthesized by GeneNet (Fukuoka, Japan).</p