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

Unique Transcriptome Patterns of the White and Grey Matter Corroborate Structural and Functional Heterogeneity in the Human Frontal Lobe

<div><p>The human frontal lobe has undergone accelerated evolution, leading to the development of unique human features such as language and self-reflection. Cortical grey matter and underlying white matter reflect distinct cellular compositions in the frontal lobe. Surprisingly little is known about the transcriptomal landscape of these distinct regions. Here, for the first time, we report a detailed transcriptomal profile of the frontal grey (GM) and white matter (WM) with resolution to alternatively spliced isoforms obtained using the RNA-Seq approach. We observed more vigorous transcriptome activity in GM compared to WM, presumably because of the presence of cellular bodies of neurons in the GM and RNA associated with the nucleus and perinuclear space. Among the top differentially expressed genes, we also identified a number of long intergenic non-coding RNAs (lincRNAs), specifically expressed in white matter, such as LINC00162. Furthermore, along with confirmation of expression of known markers for neurons and oligodendrocytes, we identified a number of genes and splicing isoforms that are exclusively expressed in GM or WM with examples of <i>GABRB2</i> and <i>PAK2</i> transcripts, respectively. Pathway analysis identified distinct physiological and biochemical processes specific to grey and white matter samples with a prevalence of synaptic processes in GM and myelination regulation and axonogenesis in the WM. Our study also revealed that expression of many genes, for example, the <i>GPR123</i>, is characterized by isoform switching, depending in which structure the gene is expressed. Our report clearly shows that GM and WM have perhaps surprisingly divergent transcriptome profiles, reflecting distinct roles in brain physiology. Further, this study provides the first reference data set for a normal human frontal lobe, which will be useful in comparative transcriptome studies of cerebral disorders, in particular, neurodegenerative diseases. </p> </div