Effect of D609 on APP expression.

<p>CHO-APP cells were treated with increasing concentration of D609 for 48 h and the expression of APP was measured at mRNA level by qPCR (A) and protein level by western blotting (B). The housekeeper protein β-actin shows equal protein loading. (C) Relative optical density of APP protein bands was quantified using ImageJ software. Data represent mean (n=3) <u>+</u> SE.</p

Cell viability with D609 treatment.

<p>CHO-APP cells were treated with increasing concentration of D609 for 48 h and the cell viability assessed by MTT assay (A) and morphology under a phase-contrast microscope (20×) (B). Data represent mean (n=6) <u>+</u> SE.</p

Impact of altering SGMS activity on Aβ generation.

<p>(A) Sphingomyelin and glycosphingolipid synthesis pathway. (B) CHO-APP cells were treated with the SGMS inhibitor D609 (50 μM) and sphingomyelin measured by an enzymatic assay. (C) CHO-APP cells were treated with increasing concentration of D609 for 48 h or 50 μM D609 for 0, 24 and 48 h and secreted Aβ and sAPPα were measured by western blotting. (D) Relative optical density of Aβ bands was quantified using ImageJ software. (E) Aβ42 was also measured by ELISA. Data represent mean (n=3) <u>+</u> SE, ∗<i>p</i> < 0.05, ∗∗<i>p</i> < 0.005.</p

Effect of altering GSL level on Aβ generation.

<p>(A) CHO-APP cells were treated with the GSL synthesis inhibitor NB-DNJ and GSL measured by HPLC. (B) CHO-APP cells were treated with NB-DNJ and secreted Aβ and sAPPα and cellular APP measured by western blotting. The housekeeper protein β-actin shows equal protein loading. (C) Relative optical density of Aβ bands was quantified using ImageJ software. Data represent mean (n=3) <u>+</u> SE, ∗<i>p</i> < 0.05, ∗∗<i>p</i> < 0.005.</p

Effect of <i>MAPT-AS1</i> over- and knock-down expression on <i>MAPT</i> expression in HEK293 and SK-N-MC cells.

<p>A) H1 (white columns) and H2 (black columns) haplotype <i>MAPT</i> promoter-driven luciferase activity. Luciferase activity is normalized to each control transfection levels. B) Endogenous transcript levels of either total <i>MAPT</i> (light grey columns) or 4 repeat <i>MAPT</i> transcript (dark grey columns). Transcript levels are normalized to each control transfection levels C) Endogenous haplotype-specific DNA methylation with H1 (white columns) and H2 (black columns) specific data indicated. Methylation levels from <i>MAPT-AS1</i> over- or under-expression are normalized to control transfection levels. Error bars indicate standard error of the mean from 5 independent experiments. *, p < 0.05; *** p < 0.0001.</p

Heatmap of gene expression of oligodendrocyte cell markers.

<p>Shows the expression profile of oligodendrocyte cell markers in GM and WM. There was an expression bias towards WM with the up-regulation of <i>SOX10, GJC2, MOG, MAG, MAL, GAL3ST1, UGT8</i> being statistically significant (q-value<0.05).</p

Multivariate Repeat Measure Mixed Linear Model Regression analyses for the effects of disease status and <i>MAPT</i> transcript levels.

<p>Multivariate Repeat Measure Mixed Linear Model Regression analyses for the effects of disease status and <i>MAPT</i> transcript levels.</p

Role of the Long Non-Coding RNA <i>MAPT-AS1</i> in Regulation of <i>Microtubule Associated Protein Tau (MAPT)</i> Expression in Parkinson's Disease

<div><p>Studies investigating the pathogenic role of the microtubule associated protein tau (<i>MAPT</i>) gene in Parkinson’s disease (PD) have indicated that DNA methylation of the promoter region is aberrant in disease, leading to dysregulated <i>MAPT</i> expression. We examined two potential regulators of <i>MAPT</i> gene expression in respect to PD, a promoter-associated long non-coding RNA <i>MAPT-AS1</i>, and DNA methyltransferases (DNMTs), enzymes responsible for new and maintenance of DNA methylation. We assessed the relationship between expression levels of <i>MAPT</i> and the candidate <i>MAPT-AS1</i>, <i>DNMT1</i>, <i>DNMT3A</i> and <i>DNMT3B</i> transcripts in four brain regions with varying degrees of cell loss and pathology (putamen, anterior cingulate cortex, visual cortex and cerebellum) in N = 10 PD and N = 10 controls. We found a significant decrease in <i>MAPT-AS1</i> expression in PD (p = 7.154 x 10⁻⁶). The transcript levels of both <i>MAPT-AS1</i> (p = 2.569 x 10⁻⁴) and <i>DNMT1</i> (p = 0.001) correlated with those of <i>MAPT</i> across the four brain regions, but not with each other. Overexpression of <i>MAPT-AS1</i> decreased <i>MAPT</i> promoter activity by ∼2.2 to 4.3 fold in an <i>in vitro</i> luciferase assay performed in two cell lines (p ≤ 2.678 x 10⁻⁴). Knock-down expression of <i>MAPT-AS1</i> led to a 1.3 to 6.3 fold increase in methylation of the endogenous <i>MAPT</i> promoter (p ≤ 0.011) and a 1.2 to 1.5 fold increased expression of the 4-repeat <i>MAPT</i> isoform transcript (p ≤ 0.013). In conclusion, <i>MAPT-AS1</i> and <i>DNMT1</i> have been identified as potential epigenetic regulators of <i>MAPT</i> expression in PD across four different brain regions. Our data also suggest that increased <i>MAPT</i> expression could be associated with disease state, but not with PD neuropathology severity.</p></div

Disease-specific expression levels of <i>MAPT</i>, <i>DNMT1</i>, <i>DNMT3A</i> and <i>DNMT3B</i> across four brain regions.

<p>Putamen (black circle), ACC (dark grey circle), visual cortex (light grey circle) and cerebellum (open circle). Data points are derived from estimated marginal means after adjusting for significant demographic predictors apart from disease status.</p

Comparison of demographics and <i>MAPT</i> haplotype frequency in brain tissue cohort.

<p>Comparison of demographics and <i>MAPT</i> haplotype frequency in brain tissue cohort.</p