1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

Amyloid beta (Aβ) accumulation in the brain is one of the major pathological features of Alzheimer’s disease. The active form of vitamin D (1,25(OH)2D3), which acts via its nuclear hormone receptor, vitamin D receptor (VDR), has been implicated in the treatment of Aβ pathology, and is thus considered as a neuroprotective agent. However, its underlying molecular mechanisms of action are not yet fully understood. Here, we aim to investigate whether the molecular mechanisms of 1,25(OH)2D3 in ameliorating Aβ toxicity involve an interplay of glial cell line-derived neurotrophic factor (GDNF)-signaling in SH-SY5Y cells. Cells were treated with Aβ(25-35) as the source of toxicity, followed by the addition of 1,25(OH)2D3 with or without the GDNF inhibitor, heparinase III. The results show that 1,25(OH)2D3 modulated Aβ-induced reactive oxygen species, apoptosis, and tau protein hyperphosphorylation in SH-SY5Y cells. Additionally, 1,25(OH)2D3 restored the decreasing GDNF and the inhibited phosphorylation of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) protein expressions. In the presence of heparinase III, these damaging effects evoked by Aβ were not abolished by 1,25(OH)2D3. It appears 1,25(OH)2D3 is beneficial for the alleviation of Aβ neurotoxicity, and it might elicit its neuroprotection against Aβ neurotoxicity through an interplay with GDNF-signaling.


Introduction
Alzheimer's disease (AD) is one of the most commonly occurring neurodegenerative diseases, and it is characterized by two pathologic feature: aberrant deposition of amyloid beta (Aβ) in extracellular plaques and intracellular accumulation of phosphorylated tau proteins in the brain [1][2][3]. This disease clinically presents slow progressive memory loss and cognitive deficits. Whether aberrant Aβ and tau proteins are key mechanisms in response to the AD-associated neuronal loss and death is still poorly understood. Aberrant Aβ has been gaining increasing attention due to the possibility of AD pathogenesis being initiated by this event and as a probable mediator of tau-pathology [4], although with the addition of 1,25(OH) 2 D 3 . In the present study, cell viability, intracellular ROS, apoptosis, and phosphorylated tau protein were determined to reflect the putative neuroprotective effects of 1,25(OH) 2 D 3 on modulating Aβ-related pathology. Moreover, Western blot analysis was employed to examine the Aβ-induced alterations in cellular mediators, including VDR and GDNF, and molecules of the PI3K/AKT/GSK-3β signaling pathway in SH-SY5Y cells.

Discussion
It is recognized that aberrant Aβ exhibits neurotoxicity that contributes to neuronal death, and this event is thought to be the primary factor that initiates the pathogenesis of AD [36,37]. Several neurotoxic effects of Aβ shown in the present study are consistent with previous studies [38,39]. For instance, we observed changes in cell morphology and tau phosphorylation, and increase in the number of apoptotic cells in parallel with the excess generation of ROS after Aβ treatment. These findings support that Aβ-associated oxidative stress was involved in the observed neuronal damage, and thus played an important role in Aβ neurotoxicity [34]. Furthermore, the morphology of neuronal cells is stabilized by the tau protein [40]. Once the tau protein is hyperphosphorylated, as observed after Aβ treatment in this study, it failed to maintain the cell structure [41] and caused cell apoptosis [2,42,43]. It is worth mentioning that neurons are capable of protecting against oxidative damage through secreting neurotrophic factors; as neurotrophic factors decrease, neurons are unable to eliminate the accumulated ROS [15,44]. In this study, we observed that both ROS production and cellular apoptosis increased as GDNF expression decreased after Aβ treatment. Hence, we speculate that Aβ might exert its toxic effects by inhibiting the action of GDNF and augmenting oxidative stress and apoptosis. In this regard, given the strong implication of excessive production of ROS and the reduction in GDNF levels in the mechanisms of Aβ neurotoxicity, it is plausible that antioxidants could be effective in the treatment of Aβ-related pathological processes [45].
In recent years, 1,25(OH) 2 D 3 has received great attention due to its therapeutic potential as a potent antioxidant and neuroprotectant [24,46]. In the brain, 1,25(OH) 2 D 3 regulates the neurotrophic factors via VDR, thereby controlling neuronal survival, development, and function [47]. There is evidence that protein and gene expressions of GDNF can be elevated by the binding of 1,25(OH) 2 D 3 to the VDR [48,49]. As 1,25(OH) 2 D 3 binds to the VDR, the protein and gene expressions of the VDR also increase [50,51]. In contrast, it was found that Aβ suppresses the protein and gene expressions of the VDR [52]. In the present study, we first confirmed that VDR and GDNF expressions were both suppressed by Aβ treatment in our model. This suppression of VDR and GDNF expression was reversed after the addition of 1,25(OH) 2 D 3 , indicating that the upregulation of GDNF may be a consequence of the formation of the 1,25(OH) 2 D 3 /VDR complex. These data suggest that VDR activity may be linked to GDNF production [53]. Taken together, we hypothesized that for 1,25(OH) 2 D 3 to elicit its anti-Aβ cytotoxicity, GDNF-signaling may be required as a cooperating event. Our hypothesis is supported by a previous study reporting that a GDNF mechanism potentially participates in anti-neurotoxicity of 1,25(OH) 2 D 3 , regardless of the type of toxic substances administered [54]. Our observations described below corroborate such statements. Suppression of ROS production and apoptotic cell death after the administration of 1,25(OH) 2 D 3 supports the theory that 1,25(OH) 2 D 3 may act as an antioxidant as well as a neuroprotectant to ameliorate Aβ-induced oxidative damage [22]. To determine whether this protection involves the upregulation of GDNF in response to 1,25(OH) 2 D 3 , we utilized heparinase III to block GDNF signaling and found that the generation of ROS was indeed not affected. A recent study has established that GDNF-signaling in dopaminergic neurons is regulated by 1,25(OH) 2 D 3 [55], which supports our discovery of an interplay between 1,25(OH) 2 D 3 -VDR and GDNF signaling. We therefore postulate that the ability of 1,25(OH) 2 D 3 to decrease ROS produced by Aβ may occur, at least in part, through direct interactions of 1,25(OH) 2 D 3 with GDNF at the cellular levels.
In an attempt to further understand the interaction between 1,25(OH) 2 D 3 -VDR and GDNF-signaling against Aβ neurotoxicity, the PI3K/AKT/GSK-3β pathway was examined due to its involvement in the promotion of cell survival and the pathogenesis of AD [56,57]. The GDNF-stimulated PI3K/Akt pathway regulates phosphorylation of GSK-3β (Ser 9 ) and cell survival [58][59][60][61]. It was indicated that Aβ also decreases GDNF secretion and increases activation of GSK-3β, promoting tau protein hyperphosphorylation and neuronal apoptosis in the brain [62][63][64][65][66][67]. Inactivation of Akt in the brain causes the amyloid protein precursor (APP) to accumulate [64]. In the present study, we observed that Aβ treatment downregulated the activated form of PI3K/Akt and that 1,25(OH) 2 D 3 reversed this dysregulation. Akt is the main regulator of GSK-3β [9]. Activation of GSK-3β causes greater Aβ accumulation and promotion of cell apoptosis through caspase-3 activation [62,68]. Activation of GSK-3β decreases as Akt is phosphorylated (i.e., activated), which results in greater cell survival [62]. Importantly, the major cause of the decrease in activation of Akt is downregulation of neurotrophic factors [62]. In the present study, we found that Aβ treatment enhanced the activated forms of GSK-3β and caspase-3, and tau protein hyperphosphorylation, but 1,25(OH) 2 D 3 administration normalized these hyperactivations caused by Aβ. Altogether, in the present study, 1,25(OH) 2 D 3 potentiated PI3K/Akt activation and subsequently led to the inactivation of downstream GSK-3β upon Aβ challenge. These findings demonstrate a putative neuroprotective role of 1,25(OH) 2 D 3 against Aβ neurotoxicity by acting on the PI3K/AKT/GSK-3β pathway. In addition, blockage of the GDNF upregulation by heparinase III was likely to prevent the aforementioned beneficial effects of 1,25(OH) 2 D 3 . Our data suggest that the protective PI3K/AKT/GSK-3β pathway involving GSK-3β inactivation may be partially mediated through GDNF [65]. Therefore, we propose that GDNF signaling might be an important driving mechanism underlying the 1,25(OH) 2 D 3 -mediated modulating effects on Aβ-induced neurotoxicity.
As mentioned previously, PI3K/AKT pathway activation mediated by GDNF contributes to neuronal survival, making cells resistant to apoptosis [58]. In this study, PI3K/Akt downregulation may have resulted in activation of GSK-3β and caspase-3, thus inducing cell apoptosis. Aβ accumulation to cause toxicity may exacerbate neuronal damage, leading to cell apoptosis [69]. In our study, treatment with 1,25(OH) 2 D 3 appeared to attenuate Aβ-induced apoptosis, supporting that 1,25(OH) 2 D 3 may be anti-apoptotic. In addition, combinational treatment with1,25(OH) 2 D 3 and the GDNF inhibitor showed inhibition of the counteracting of apoptosis in the presence of Aβ. As a possible consequence to these findings, upregulation of GDNF seemed to be a key mechanism through which 1,25(OH) 2 D 3 neutralized Aβ-induced excessive ROS production and apoptotic death in SH-SY5Y cells. Collectively, 1,25(OH) 2 D 3 might elicit its neuroprotection via actions of GDNF-signaling, with the signals subsequently and indirectly leading to the resistance of SH-SY5Y cells to Aβ neurotoxicity.
For the preparation of 1,25(OH) 2 D 3 , the biological concentration of vitamin D in the peripheral circulation in healthy people is around 20 ng/mL (50 nM) [74] and 10 nM was indicated to induce GDNF expression [48,49]. Considering the conversion rate from 25-hydroxyvitamin D 3 to 1,25(OH) 2 D 3 and much lower concentrations in the brain, we treated cells with 0.1 and 10 nM of 1,25(OH) 2 D 3 . For this, 1,25(OH) 2 D 3 (D1530, Sigma, St. Louis, MO, USA) was dissolved in 99.5% ethanol to a concentration of 10 mM as a stock solution before being diluted in 99.5% ethanol to desired concentrations.

Cell Morphology
Cell morphology was observed under a microscope (Nikon, Tokyo, Japan) at 40× and 400× magnifications, and photos were processed with SPOT 4.7 Advanced software (SPOT Imaging Solutions, Sterling Heights, MI, USA).

Cell Viability Analysis
A 3-[4,5-cimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to determine the viability of SH-SY5Y cells that were pre-treated with Aβ(25-35) for 24 h and then further treated with two different concentrations of 1,25(OH) 2 D 3 for 24 h. Briefly, MTT was added to each well of a 24-well plate and incubated at 37 • C for 1 h. Purple-colored precipitates of the living cell metabolite, formazan, were then dissolved in 500 µL of dimethyl sulfoxide (DMSO) and were analyzed in a 96-well plate. The color absorbance was recorded at 590 nm. Cell viability was calculated by the absorbance ratio of the treated group over the control.

Intracellular ROS Analysis
The production of intracellular ROS was determined by the 2',7'-dichlorofluoroescin diacetate (DCFH-DA) probe, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of peroxides. Cells were seeded in 6-well dishes at 5 × 10 5 cells per well before the allotted experimental treatments were performed. After being treated, cells were trypsinized and washed with phosphate-buffered saline (PBS) once by centrifugation at 200× g for 3 min at 25 • C. After removing the supernatant, DCFH-DA dissolved in PBS was added to each sample. Samples were then incubated in the complete absence of light for 60 min. Each sample was moved to a Falcon tube prior to analysis by flow cytometry (Flowcytometer-3, FACSCantoII, BD Biosciences, Franklin Lake, NJ, USA).

Protein Extraction and Quantification
After being treated, cells were harvested, washed three times with PBS, and lysed using cold RIPA buffer supplemented with a protease inhibitor and an EDTA solution at a ratio of 100:1:1, respectively, then centrifuged at 13,000× g at 4 • C for 30 min. The supernatant was collected, and the protein concentration was estimated with a BCA Protein Assay Kit (Milpitas, CA, USA) using bovine serum albumin as the standard.

Western Blot Analysis
A Western blot analysis was performed to examine expression levels of certain proteins. Equal quantities (30 µg) of proteins were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. After being transferred, membranes were blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and 5% non-fat milk for 1 h. Membranes were subsequently incubated with specific primary antibodies: β-actin After washing three times with TBST for 30 min, the membranes were incubated with an anti-mouse (A9024, Sigma) or an anti-rabbit (R5506, Sigma) immunoglobulin G (IgG) secondary antibody for 1 h, and then washed with TBST three times for 30 min. Immunoreactive proteins were detected and quantified using an enhanced chemiluminescence (ECL; Bionovas, Toronto, Canada) Western blot detection system and Image-Pro Plus Software (Cybernetics, Rockville, MD, USA), respectively.

Apoptotic Cell Analysis
Apoptosis cell analyses were performed using flow cytometry by double-staining with propidium iodide (PI) and annexin-V dye. Cells were seeded in 6-well dishes at 5 × 10 5 cells per well before the allotted experimental treatments were performed. After being treated, cells were trypsinized and washed with PBS at least twice by centrifugation at 200× g for 3 min at 4 • C. The supernatant was removed, and the pellet was re-suspended in 1 mL of cold PBS and centrifuged for 3 min at 200× g and 4 • C. After removing the supernatant, 100 µL of binding buffer, 2 µL of PI dye, and 2 µL of annexin-V dye were added to each sample. Samples were then incubated at room temperature in the complete absence of light for 15 min. Each sample was resuspended in 600 µL of cold PBS and moved to a Falcon tube prior to analysis by flow cytometry (Flowcytometer-3, FACSCantoII, BD Biosciences, San Jose, CA, USA).

Conclusions
In conclusion, this study demonstrates the neuroprotective effects of 1,25(OH) 2 D 3 against Aβ neurotoxicity and concomitant changes in phosphorylated tau protein in SH-SY5Y cells. The underlying mechanisms of the action of 1,25(OH) 2 D 3 were attributed to its ability to counteract excessive production of ROS and apoptotic cell death. However, the optimal responses of 1,25(OH) 2 D 3 to Aβ neurotoxicity in SH-SY5Y cells required an interplay with GDNF-signaling by targeting inactivation of the PI3K/Akt/GSK-3β pathway at the cellular level. However, more studies are warranted to explore the additional pathways through which GDNF works to mediate neuroprotection of 1,25(OH) 2 D 3 and to better understand the interactions with the PI3K/Akt/GSK-3β signaling in relation to Aβ neurotoxicity. Nonetheless, we have demonstrated the pivotal role of GDNF in 1,25(OH) 2 D 3 -elicited neuroprotection following an Aβ challenge in SH-SY5Y cells, which could add value as the basis of 1,25(OH) 2 D 3 treatment for limiting Aβ-related pathology.

Conflicts of Interest:
The authors declare no conflict of interest.