Summary of FpClass PPI prediction tool data.

<p>The FpClass PPI prediction tool was used to identify partner proteins for both APP and VDR. The tool predicted 1133 partners for APP and 583 partners for VDR. An analysis of the FpClass tool data indicated that 153 of these partners interacted with both APP and VDR. A total of 153 proteins were classified according to their functions in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188605#pone.0188605.s001" target="_blank">S1 Table</a>. Five of these proteins (NUMB, catenin (CTNNB1), NOTCH1, E-cadherin (CDH1), and FHL2) were membrane or membrane-related proteins. These 5 proteins were used for further analyses with the target proteins (PS1, PS2, Nicastrin, BACE1, ADAM10) and PDIA3. The software predicted 5244 partners for these proteins, and the proteins that are the most relevant to plasma membrane interactions are presented in the figure with their PPI total score.</p

The expression of VDR and PDIA3/1,25MARRS in cortical neurons.

<p><b>A-B)</b> Cell surface staining of VDR (green) in live neurons, followed by fixation and immunofluorescence labeling of MAP2 (red) as a neuronal marker, 100x. The micrographs were taken from the same areas with different levels of focus to demonstrate the different localization of VDR on the neuronal plasma membrane. <i>A)</i> VDR protein is localized on the plasma membrane of the soma; <i>B)</i> VDR protein is localized on the plasma membranes of neurites. The 3D image of staining was obtained via confocal microscopy (63x), and the video is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188605#pone.0188605.s002" target="_blank">S1 Video</a>. <b>C)</b> VDR (green) and PDIA3/1,25MARRS (red) cell surface staining with live neurons via double immunofluorescence labelling, 100x. The localization of PDIA3/1,25MARRS on the neuronal plasma membrane was very limited. <b>D)</b> Double immunofluorescence labelling of VDR and PDIA3/1,25MARRS in fixed and permeabilized neurons, 100x. PDIA3/1,25MARRS was localized in the cytoplasm and in the endoplasmic reticulum (ER). Given its strong reactivity in a certain area in the cytoplasm and its known role as an ER chaperone, the localization was considered to be in the ER. VDR is located in the nucleus, cytoplasm, ER and axon hillock. VDR and PDIA3/1,25MARRS might co-localize in the cytoplasm, especially in the ER. <b>E)</b> VDR (green) cell surface staining with live SH-SY5Y cells via immunofluorescence labelling, 100x. <b>F)</b> Immunofluorescence labelling of VDR in fixed and permeabilized SH-SY5Y cells, 100x.</p

VDR siRNA-mediated knockdown does not change the expression of LVSCC-A1D mRNA and protein.

<p><b>A) Comparison of LVSCC-A1D mRNA levels.</b> LVSCC-A1D mRNA levels from VDR-silenced neurons were not different than control groups (p>0.05), but LVSCC-A1D mRNA levels significantly decreased after vitamin D treatment in VDR-silenced neurons. * LVSCC A1D mRNA levels from vitamin D-treated VDR-silenced neurons were statistically lower than control levels (p = 0,019, p = 0,019, p = 0,030, p = 0,013, respectively) and VDR siRNA-treated groups (p = 0,031). <b>B) Detection of LVSCC-A1D protein by western blot.</b> LVSCC-A1D protein levels did not change in VDR-silenced neurons. Vitamin D treatment in VDR-silenced neurons led to a decrease in LVSCC-A1D protein levels. Beta actin was used as a loading control. <b>C) Comparison of LVSCC-A1D protein band intensities relative to beta actin.</b> Western blot results were consistent with mRNA results. The absolute intensities were measured using Image J software, and the relative intensities were calculated from the ratio of LVSCC-A1D to beta actin absolute intensities. * LVSCC-A1D protein levels from vitamin D-treated VDR-silenced neurons were statistically lower compared to other groups (p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, respectively). <i>Control</i>: Untreated control group; <i>Vehicle</i>: Only transfection reagent-treated control group; <i>Non target siRNA:</i> Non-target siRNA-treated negative control group; <i>Cyc B siRNA</i>: Cyclophilin B siRNA-treated positive control group; <i>VDR siRNA</i>: VDR siRNA-treated group and <i>VDR siRNA+Vitamin D</i>: Following 12 hours of VDR siRNA treatment, groups were treated with vitamin D. Data are presented as a mean SD.</p

siRNA-mediated knockdown of VDR induces expression of LVSCC-A1C mRNA and protein. A) Comparison of LVSCC-A1C mRNA levels.

<p>VDR suppression resulted in increased LVSCC-A1C mRNA expression, but the effects of VDR suppression on LVSCC-A1C were normalized after vitamin D treatment.* LVSCC-A1C mRNA levels from VDR-silenced neurons were statistically higher than in other groups (p = 0,015, p = 0,034, p = 0,002, p = 0,024, respectively). ** LVSCC-A1C mRNA levels were statistically lower than in VDR siRNA-treated group (p = 0,013). <b>B) Detection of LVSCC-A1C protein by western blot.</b> Although LVSCC-A1C protein increased in VDR-silenced neurons, vitamin D treatment decreased LVSCC-A1C expression to control levels. Beta actin was used as loading control. <b>C) Comparison of LVSCC-A1C protein band intensities relative to Beta actin.</b> Western blot results were consistent with mRNA results. The absolute intensities were measured using Image J software, and the relative intensities were calculated from the ratio of LVSCC-A1C to Beta actin absolute intensities. * LVSCC-A1C protein levels from VDR-silenced neurons were statistically higher compared to control groups (p<0.01, p<0.01, p<0.05, respectively). ** LVSCC-A1C protein levels from vitamin D-treated VDR-silenced neurons were statistically lower compared to the VDR siRNA-treated group (p<0.001). <i>Control</i>: Untreated control group; <i>Vehicle</i>: Transfection reagent-treated control group; <i>Non target siRNA:</i> Non-target siRNA-treated negative control group; <i>Cyc B siRNA</i>: Cyclophilin B siRNA-treated positive control group; <i>VDR siRNA</i>: VDR siRNA-treated group and <i>VDR siRNA+Vitamin D</i>: Following 12 hours of VDR siRNA treatment, groups were treated with vitamin D. Data are presented as a mean SD.</p

Seven-day-old primary cortical neurons were used to determine the neuron/glia ratio.

<p>The glia ratio was 10% in cultured cells. Neurons, green (FITC tagged PAN neuronal marker antibody); Glia, red (Texas Red (TR) tagged GFAP antibody). 40X magnification.</p

siRNA-mediated knockdown of VDR leads to a significant reduction in NGF release.

<p>* VDR-silenced neurons had significantly lower NGF release compared to control, vehicle, non-target siRNA and cycB siRNA-treated groups (p<0.05, p<0.01, p<0.01, p<0.01, respectively). Data are presented as a mean SD.</p

siRNA-mediated knockdown of VDR.

<p><b>A) Comparison of VDR mRNA levels.</b> VDR siRNA treatment suppressed VDR mRNA expression. After 12 hours of vitamin D treatment (1×10⁻⁷ M) applied to VDR-silenced neurons, VDR mRNA levels increased. These results indicate that vitamin D increases VDR expression in cortical neurons * VDR mRNA levels from VDR-silenced neurons were statistically lower than in the control groups (p<0.001, p<0.001, p<0.001, p<0.001, respectively). ** VDR mRNA levels from Vitamin D-treated VDR-silenced neurons were statistically higher than in the VDR siRNA-treated group (p<0.001). <b>B) Western blot detecting VDR protein level.</b> VDR siRNA treatment suppressed VDR protein expression. After 12 hours of vitamin D treatment (1×10⁻⁷ M) in VDR-silenced neurons, VDR protein levels increased. Beta actin was used as the loading control. <b>C) Comparison of VDR protein band intensities relative to Beta actin.</b> The absolute intensities of VDR and Beta actin protein bands were measured using Image J software, and the relative intensities were calculated from the ratio of VDR to Beta actin absolute intensities. * VDR protein levels from VDR-silenced neurons were statistically lower than in control groups (p<0.001, p<0.001, p<0.001, p<0.001, respectively). ** VDR protein levels from vitamin D-treated VDR-silenced neurons were statistically higher than in the VDR siRNA-treated group (p<0.05). <i>Control</i>: Untreated control group; <i>Vehicle</i>: Transfection reagent-treated control group; <i>Non target siRNA:</i> Non-target siRNA-treated negative control group; <i>Cyc B siRNA</i>: Cyclophilin B siRNA-treated positive control group; <i>VDR siRNA</i>: VDR siRNA-treated group and <i>VDR siRNA+Vitamin D</i>: Following 12 hours of VDR siRNA treatment, groups were treated with vitamin D. Data are presented as a mean SD.</p

Vitamin D Receptor Regulates Amyloid Beta 1–42 Production with Protein Disulfide Isomerase A3

The challenge of understanding the biology of neuronal amyloid processing could provide a basis for understanding the amyloid pathology in Alzheimer’s disease (AD). Based on our previous studies, we have suggested that AD might be the consequence of a hormonal imbalance in which the critical hormone is vitamin D. The present study primarily focused on the creation of a condition that prevents the genomic or nongenomic action of vitamin D by disrupting vitamin D receptors (VDR or PDIA3/1,25MARRS); the effects of these disruptions on the series of proteins involved in secretases that play a crucial role in amyloid pathology and on amyloid beta (Aβ) production in primary cortical neurons were observed. VDR and PDIA3/1,25MARRS genes were silenced separately or simultaneously in E16 primary rat cortical neurons. The expression of target genes involved in APP processing, including Presenilin1, Presenilin2, Nicastrin, BACE1, ADAM10, and APP, was investigated with qRT-PCR and Western blot in this model. 1,25-Dihydroxyvitamin D₃ treatments were used to verify any transcriptional regulation data gathered from siRNA treatments by determining the mRNA expression of the target genes. Immunofluorescence labeling was used for the verification of silencing experiments and intracellular Aβ1–42 production. Extracellular Aβ1–42 level was assessed with ELISA. mRNA and protein expression results showed that 1,25-dihydroxyvitamin D₃ might affect the transcriptional regulation of the genes involved in APP processing. The intracellular and extracellular Aβ1–42 measurements in our study support this suggestion. Consequently, we suggest that 1,25-dihydroxyvitamin D₃ and its receptors are important parts of the amyloid processing pathway in neurons