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Vitamin D receptor regulates amyloid beta 1-42 production with protein disulfide isomerase A3 Duygu Gezen-Ak, Irem L Atasoy, Esin Candas, Merve Alaylioglu, Selma Yilmazer, and Erdinc Dursun ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00245 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 15, 2017
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ACS Chemical Neuroscience
Vitamin D receptor regulates amyloid beta 1-42 production with protein disulfide isomerase A3 Duygu Gezen-Ak1, Irem L. Atasoy1, Esin Candaş1, Merve Alaylıoğlu1, Selma Yılmazer1, Erdinç Dursun1* 1
Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey
Short title: VDR and PDIA3 regulate amyloid beta production
*Correspondence to: Erdinç Dursun Address: Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, 34098, Istanbul, Turkey. Phone: +90 212 414 30 00/21627 +90 533 339 98 82 Fax:
+90 212 414 30 42
e-mail:
[email protected] [email protected] 1 ACS Paragon Plus Environment
ACS Chemical Neuroscience
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ABSTRACT 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 non-genomic 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.
Key words: Alzheimer’s disease / amyloid / protein disulfide isomerase A3 (PDIA3) / Vitamin D receptor (VDR) / vitamin D /
INTRODUCTION
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ACS Chemical Neuroscience
The basic obstacle that obstructs the understanding of the action of vitamin D in neurodegeneration is its misnomer. Vitamin D is not a vitamin but a secosteroid hormone that regulates more than 1000 genes in different conditions, at different developmental stages, in different cell types. VDR ChIP-Seq datasets and the results of the ENCODE project indicate that there are 1000-10,000 genomic VDR binding sites per cell 1. These studies demonstrated that VDR transcriptomes are surprisingly numerous and diverse; they are heterogeneous depending on cell type and time 2. The basic properties of vitamin D, its evolution from a membrane component to a transcription factor, and the non-skeletal action of vitamin D and its receptors have been meticulously reviewed by valuable researchers in the field
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. Considering that vitamin D deficiency is a lifelong process and that
neurodegeneration, especially the process of Alzheimer’s disease (AD) pathology, takes at least two decades, we have suggested that AD might be a consequence of a hormonal imbalance in which the critical hormone is 1,25-Dihydroxyvitamin D3 11, and this suggestion is supported by our in vivo and in vitro studies
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and by other publications in the field. Assuming that this theory is valid, the basic
challenge would be to evaluate AD pathology as a result of vitamin D deficiency or the inefficient utilization of vitamin D. In light of this suggestion, the present study primarily focused on the creation of a condition that prevents the genomic or non-genomic action of vitamin D by disrupting/silencing vitamin D receptors (VDR or protein disulfide isomerase A3 -PDIA3/1,25MARRS-) and 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 1-42 (Aβ 1-42) production in primary cortical neurons were observed. The second focus became the cellular distribution of amyloid beta during different cell culture conditions. RESULTS Cytotoxicity Assay and Apoptotic index The cytotoxicity assay and apoptotic index were used to determine the overall effect of short-term VDR or PDIA3/1,25MARRS silencing and double silencing on neuronal survival. There was no significant difference in lactate dehydrogenase (LDH) release between siRNA-treated groups and control groups in cortical neurons after 24 hours of treatment, whereas LDH release was increased in siRNA-treated groups after 48 hours of treatment (p
