Vitamin D receptor regulates transcription of mitochondrial DNA and directly interacts with mitochondrial DNA and TFAM.

Vitamin D receptor (VDR) is an essential transcription factor (TF) synthesized in different cell types. We hypothesized that VDR might also act as a mitochondrial TF. We conducted the experiments in primary cortical neurons, PC12, HEK293T, SH-SY5Y cell lines, human peripheral blood mononuclear cells (PBMC) and human brain. We showed that vitamin D/VDR affects the expression of mitochondrial DNA (mtDNA) encoded oxidative phosphorylation (OXPHOS) subunits. We observed the co-localization of VDR with mitochondria and the mtDNA with confocal microscopy. mtDNA-chromatin-immunoprecipitation and electrophoretic mobility shift assays indicated that VDR was able to bind to the mtDNA D-loop site in several locations, with a consensus sequence "MMHKCA." We also reported the possible interaction between VDR and mitochondrial transcription factor A (TFAM) and their binding sites located in close proximity in mtDNA. Consequently, our results showed for the first time that VDR was able to bind and regulate mtDNA transcription and interact with TFAM even in the human brain. These results not only revealed a novel function of VDR, but also showed that VDR is indispensable for energy demanded cells.

Okadaic acid-induced tau hyperphosphorylation and the downregulation of Pin1 expression in primary cortical neurons.

Hyperphosphorylation of tau leading to neurofibrillary tangles (NFT) is one of the key pathological hallmarks in neurodegenerative disorders such as Alzheimer disease (AD). Peptidyl-prolyl cis-trans isomerase (Pin1) regulates the phosphorylation of Ser/Thr sites of tau protein, and promotes microtubule assembly. In this study, we aimed to determine the effect of tau hyperphosphorylation on Pin1 expression in primary cortical neurons in order to investigate the results of the pathological process on Pin1, an important enzyme involved in various cellular mechanisms. Primary cortical neurons were prepared from embryonic day 16 -Sprague Dawley rat embryos. The cultures were treated with 25 nM okadaic acid (OKA) on day 7 in order to promote tau hyperphosphorylation. The cytotoxicity was determined with LDH release and measured by ELISA. Tau phosphorylation was confirmed by western blot using anti-tau antibodies Thr231 and Tau-1. Pin1 mRNA expression level was determined by qRT-PCR at 8 and 24 h. Pin1 protein expression was analyzed with immunofluorescent labeling at 8 and 24 h. Tau phosphorylation on Thr231 was increased and non-phosphorylated Tau-1 was decreased in OKA treated group compared with the untreated control at 8 h of treatment. While Pin1 mRNA expression levels at 8 h post-OKA treatment were lower than that of control groups, there were no differences between OKA-treated group and control groups in Pin1 protein expression. Whereas no significant differences for Pin1 mRNA expression, protein expression levels were decreased OKA-treated group compared to control groups at 24 h of treatment. The LDH release of OKA-treated group was significantly increased at 24 h. Our study indicates that although OKA treatment suppressed Pin1 mRNA expression and induced tau phosphorylation at 8 h of treatment, its influence on Pin1 protein expression has 16 h phase delay. Given the important role of Pin1 in many cellular mechanisms these results might indicate that tau hyperphosphorylation involved in many neurodegenerative disorders may cause some alterations in brain microenvironment via Pin1.This is the first demonstration of the alteration of the Pin1 mRNA and protein expression in OKA induced model in primary cortical neurons.

Both secreted and the cellular levels of BDNF attenuated due to tau hyperphosphorylation in primary cultures of cortical neurons.

Intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of taupathies such as Alzheimer's disease. Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is selectively downregulated in AD. In this study, we investigated the effects of OKA induced tau hyperphosphorylation on secreted and cellular levels of BDNF in primary cortical neurons that were treated with 25nM OKA. Tau phosphorylation at threonine 231 (Thr231) sites was assessed by Western blot using antibodies against phospho-Thr231. Non-phosphorylated tau protein was detected with the Tau-1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 8th and 24th hours of treatment. Cellular localization and protein expression of BDNF and tau were assessed by immunofluorescent labeling and fluorescent intensity measurements at 24h of treatment. Tau hyperphosphorylation was confirmed with increase in Thr231 and the decrease in Tau-1 signals after 8h of OKA treatment, compared with the control groups, secreted BDNF levels in the OKA-treated group were significantly lower after 24h of treatment but were not significantly different at 8h of treatment. BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control group. BDNF immunoreactivity significantly decreased in the OKA treated group and this attenuation was significant especially at neurites. Our results suggest that the decrease in BDNF secretion and the BDNF expression might depend on the disruption of microtubule structure caused by tau hyperphosphorylation.