MiR-124-3p attenuates hyperphosphorylation of tau protein-induced apoptosis via caveolin-1-PI3K/Akt/GSK3β pathway in N2a/APP695swe cells

Hyperphosphorylation of Tau forming neurofibrillary tangles has been considered as a crucial event in the pathogenesis of Alzheimer's disease (AD). MiR-124-3p belongs to microRNA (miRNA) family and was markedly decreased in AD, however, the functions of miR-124-3p in the pathogenesis of AD remain unknown. We observed that the expression of miR-124-3p was significantly decreased in N2a/APP695swe cells; and transfection of miR-124-3p mimics not only attenuated cell apoptosis and abnormal hyperphosphorylation of Tau protein without any changes of total Tau protein, but also increased expression levels of Caveolin-1, phosphoinositide 3-kinase (PI3K), phospho-Akt (Akt-Ser473)/Akt, phospho-glycogen synthase kinase-3 beta (GSK-3β-Ser9)/GSK-3β in N2a/APP695swe cells. We further found that miR-12-3p directly targeted Caveolin-1; miR-124-3p inhibited abnormal hyperphosphorylation of Tau by regulating Caveolin-1-PI3K/Akt/GSK3β pathway in AD. This study reveals that miR-124-3p may play a neuroprotective role in AD, which may provide new ideas and therapeutic targets for AD.


INTRODUCTION
Alzheimer's disease (AD) is the major neurodegenerative disease related to aging, characterized by progressive impairment of memory and cognition. Excessive accumulation of amyloid-beta (Aβ) protein and formation of neurofibrillary tangles (NFTs) consisting of hyperphosphorylated Tau are the two main pathological features. Hyperphosphorylated Tau is neurotoxic and can promote the neuronal apoptosis and cell death in AD [1,2]. As such, inhibiting Aβ-induced hyperphosphorylation of Tau could be one of therapeutic strategies for AD.
MicroRNAs (miRNA), a class of small noncoding RNAs, approximately 22 nucleotides in length, plays a crucial role in fundamental biological processes, such as apoptosis, proliferation, differentiation, development, and inflammation [3]. The aberrant expression of miRNA were found in AD brains, and the deregulation of miRNA target networks played a key role in AD pathogenesis via regulating genes including APP and BACE1/β-secretase [4]. MiR-124, is highly and specifically expressed in brain of human and rodents [5]. Previously, most studies focused on the role of miR-124 in neuronal differentiation and the ability of learning and memory [6][7][8]. MiR-124-3p is one of subtypes of miR-124, its biology function is similar to the family of miR-124; miR-124-3p is markedly decreased in AD [5]. However, the function of miR-124-3p in the pathogenesis of AD remains unknown.
Caveolae, flask-shaped cavities in cell membrane, is a specialized form of membrane lipid rafts. A few evidences showed lipid rafts might play a key role in neurodegeneration [9,10]. Caveolin-1 (Cav-1) is a protein comprising the portions of caveolae membranes. And it can not only promote the progression of cancers [11], but also has been implicated in the pathogenesis of neurodegenerative disease. In addition, Cav-1 was elevated in Parkin KOMEF cells, a Research Paper cell model of Parkinson's disease [12]. Upregulation of Cav-1 increased alpha-secretase activity which led to increasing α-secretase-mediated cleavage of amyloid precursor protein; down-regulation of Cav-1 increased accumulation of Aβ in AD [13,14]. Furthermore, Cav-1 has been shown as a direct target of miR-124 and miR-124-3p [3,15] . However, the relationship of Cav-1 and miR-124-3p remains unclear in AD.
Glycogen synthase kinase-3β (GSK-3β) is a constitutive serine/threonine kinase, which plays a key role in a number of diseases such as immune disorders, chronic inflammatory diseases and neurodegenerative diseases by regulating cellular processes including cell division, cell differentiation, development and apoptosis via many different signaling pathways [16]. GSK-3β is abundant in CNS and the active GSK-3β is largely confined to degenerated neurons. Overactivation of GSK-3β is associated with diverse aspects of neuronal dysfunction, such as the impairments of neuronal architecture, plasticity and survival; GSK-3β inactivation maintains neuronal polarity, survival and activity [17]. Substantial evidences revealed that functioning as a downstream target of the phosphatidylinositol-3-kinase (PI3K)/threonine/serine protein kinase B (Akt) signaling pathway and a major Tau kinase, GSK-3β regulated both Tau phosphorylation and Aβ production in PI3K/Akt-dependent pathway in AD [18]. Moreover, GSK-3β was closely associated with Cav-1. Expressions of Cav-1 mRNA and protein were increased while phosphorylation of Akt and GSK-3β were significantly decreased in mouse cerebral astrocytes [19], downregulation of Cav-1 inhibited the level of p-GSK3β [20]. In this study, we found that the expression of miR-124-3p was significantly decreased in AD cell model; the transfection of miR-124-3p mimics attenuated cell apoptosis and inhibited abnormal hyperphosphorylation of Tau protein by regulating Caveolin-1-PI3K/Akt/GSK3β pathway. This study reveals that miR-124-3p may play a neuroprotective role in AD, which may provide new ideas and therapeutic targets for AD.
Overexpression of caveolin-1 promoted the apoptosis and increased ratio of Tau-Ser404/ Tau proteins in N2a/APP695swe cells In our study, mRNA and protein expressions of Caveolin-1 were detected in the N2a/WT and N2a/ APP695swe cells by using qRT-PCR and Western blot. The results showed that the expressions of Caveolin-1 was increased both at protein and mRNA levels in N2a/ APP695swe cells (0.95 ± 0.10 and 4.97 ± 0.06), compared to N2a/WT cells (0.60 ± 0.05 and 0.48 ± 0.04) (p < 0.01) ( Figure 3A, 3B and 3C).
MiR-124-3p directly regulates expression of Caveolin-1 in N2a/APP695swe cells To find the relationship between miR-124-3p and Caveolin-1, it was necessary to firstly investigate the function of miR-124-3p on caveolae. We transfected miR-124-3p mimics and NC-miR-124-3p into N2a/APP695swe cells transiently, transmission electron microscopy was used to observe the number of morphologically defined caveolae on the cell membrane ( Figure 6A). Cells (10) were randomly selected from the blank control group, the empty vector (NC-miR-124-3p) group and the overexpression of miR-124-3p group. The cells were amplifying so that it was easy to calculate the number of The ratios of apoptotic cells were calculated to be 12.23 ± 0.58% in N2a/APP695swe cells, which were higher than that in N2a/WT group (6.27 ± 0.44%) (**p < 0.01). (C) Western blot was used to test the expressions of Tau-Ser404, Tau and β-actin proteins. (D) The relative optical density (OD) of at Tau-Ser404 protein level was compared to Tau, respectively. And the ratios of Tau-Ser404/Tau were increased in N2a/APP695swe group, compared to N2a/WT group (*p < 0.05). (E) The expression of miR-124-3p at mRNA were assessed by qRT-PCR. N2a/APP695swe cells exhibited low level of miR-124-3p significantly, compared with N2a/WT cells (**p < 0.01). caveolae on the cell membrane. The data displayed that miR-124-3p-transfected cells (43 ± 6) had a remarkable reduction of caveolae, compared with the blank control group (91 ± 6) and the empty vector group (92 ± 9) (p < 0.01) ( Figure 6B).
Since Caveolin-1 played a key role in the formation of caveolae, qRT-PCR and Western blot ( Figure 6C) were performed to detect the targeted function of miR-124-3p. As shown in Figure 6D and 6E, the expression of Caveolin-1 was observably decreased both at protein and mRNA levels in the miR-124-3p-transfected group (0.21 ± 0.02 and 0.45 ± 0.08), no matter compared with that blank control group (1.58 ± 0.11 and 4.15 ± 0.29) or empty vector group (1.56 ± 0.14 and 4.00 ± 0.43) (p < 0.01).

Figure 3: Expressions of Caveolin-1 between N2a/WT and N2a/APP695swe cells. (A) Western blot of Caveolin-1 and β-actin
in the cells of N2a/WT and N2a/APP695swe groups, respectively. (B and C) The relative optical density (OD) of Caveolin-1 both at protein and mRNA levels were compared to β-actin, respectively. And the ratios of Caveolin-1/β-actin were higher both at protein and mRNA levels in N2a/APP695swe group than that in N2a/WT (**p < 0.01). Furthermore, we performed computational analyses using the miRNA target prediction database targetscan (http://www.targetscan.org/) and PicTar (pictar.mdcberlin.de) to identify the miRNAs which could target mice caveolin-1. The result showed miR-124-3p had a potential target sites in the 3 , UTR of the Caveolin-1 gene, and the binding sites (position 604-610) was highly conserved in mammals( Figure 7A).

DISCUSSION
It is generally acknowledged that the formation of NFTs is a key pathogenesis of AD, which is composed of hyperphosphorylation of Tau protein. The previous study has been found that the abnormal phosphorylated Tau occurs early stage of AD and the impairment of cognition may precede histologically identified NFTs [21]. The hyperphosphorylation of Tau was concerning the neuronal apoptosis and may play a crucial role in the development of AD. There are some phosphorylation sites of Tau related to AD, such as Ser404, Ser396, and Thr181 and so on [22]. The development of NFTs proceeds in three stages, including preneuro fibrillary tangle, intraneuronal neurofibrillary tangle and extraneuronal neurofibrillary tangle. The hyperphosphorylation of Tau occurs at serine 396/serine 404 and threonine 231 in the intraneuronal neurofibrillary tangle stage, compared to those tau which hyperphosphorylated at serines 199, 202 and 409 in the preneuro fibrillary tangle stage [23]. Tau-Ser404 is the major site in paired helical filament (PHF), which is the main fibrous component of NFTs and contains predominantly the abnormally phosphorylated tau [24]. Some researchers have found that tau phosphorylated at Ser404 emerges very early in the disease process, and Tau, Tau-Ser404 and β-actin. After transfection of pcDNA-Caveolin-1, the rates of Caveolin-1/β-actin and Tau-Ser404/Tau were increased, no matter compared to the pcDNA3.1-transfected group or control group ( ## p < 0.01, **p < 0.01) (A and B). But there was an opposite result after transfection of Caveolin-1-siRNA that the ratios of Caveolin-1/β-actin and Tau-Ser404/Tau were decreased, no matter compared to the NC-siRNA-transfected group or control group ( ## p < 0.01, **p < 0.01) (C and D). www.impactjournals.com/oncotarget  the Tau-Ser404 can be considered as a predictor of mild cognitive impairment (MCI) to AD [22,25]. Study has demonstrated neuron apoptosis can be suppressed by reducing other phosphorylation sites of tau at Ser199 and Thr205 [26], but there is still unclear whether the hyperphosphorylation of Tau at Ser404 perform the same effect. In this study, Tau-Ser404 was selected to evaluate the phosphorylation levels of Tau, and was found to increase in N2a/APP695swe cells. The apoptosis of cells was increased along with the higher ratio of Tau-Ser404/Tau in the AD cell model, which implied the hyperphosphorylation of Tau at Ser404 might also inhibit cell apoptosis in AD. However, the precise regulation of Tau protein in AD still remains elusive.
Specific micro-RNAs have an aberrant expression in Alzheimer hippocampus, which may contribute to the development of AD [27]. MicroRNAs regulate the expression of gene through accessing to their target mRNAs via the complementary base pairing. Although 100-200 miRNAs are expressed in lower metazoa, 1000 or more are predicted to function in humans, possibly regulating 30% of human genes [28]. Furthermore, studies have demonstrated that miR-214 can inhibit the cytotoxicity of Aβ [29]. And miR-298, miR-328 and miR-29c can regulate the expression of BACE-1 [30,31]. MiR-15a and miR-128a perform an effect on the phosphorylation of Tau [32,33], leading to affect AD. MiR-124 that is highly expressed in differentiating and  A and B). Conversely, they were all elevated in the Caveolin-1-siRNA-transfected group, compared with the NC-siRNA-transfected group (**p < 0.01) (C and D). At the same time, the ratios of PI3K/βactin, Akt-Ser473/Akt and GSK-3β-Ser9/GSK-3β were markedly elevated in the miR-124-3p mimics-transfected group, compared with the NC group (**p < 0.01) (E and F). mature neurons was firstly found to be abundant in mouse brain ranging from 5% to 48% of all miRNAs [34]. In addition, miR-124 was down-regulated in AD brain [35], which was related to the generation and accumulation of Aβ [35,36]. In recent studies, researchers have identified miR-124-3p, which belongs to miR-124 family, is not only as a tumor suppressor in some cancers, but also involved in both brain development and neuronal function [37]. Even so, the function of miR-124-3p in AD is still largely unknown. In our study, the expression of miR-124-3p was decreased in N2a/APP695swe cells in comparison with N2a/WT cells, which was consistent with the study in human about miR-124 [35]. Then miR-124-3p mimics was transfected into N2a/APP695swe cells, interestingly, both apoptosis of cells and hyperphosphorylation of Tau proteins were significantly decreased. It indicated that miR-124-3p could have neuroprotective effect in AD through inhibiting the hyperphosphorylation of Tauinduced cell apoptosis. As we know, the abundant studies have shown that it is useful to improve the abilities of memory and learning by targeting hyperphosphorylation of Tau. Therefore, the therapeutic target of miR-124-3p, which regulates phosphorylated Tau, may play an essential role in the treatment of AD.
Caveolin-1 is a primary structural component of caveolae in most mammalian cells, and it is also a negative controlled and scaffolding protein in many signaling pathways. A precious study showed that Caveolin-1 was up-regulated both at mRNA and protein levels in AD by approximately two-fold [38], which indicated that Caveolin-1 may play a role in the pathogenesis of AD. Our studies were in accordance with the previous study [38], showing increase of Caveolin-1 in AD cell model. It is intriguing that knockdown of Caveolin-1 attenuated cell apoptosis and decreased Tau-Ser404/Tau proteins in N2a/APP695swe cells. On the contrary, overexpression of Caveolin-1 exacerbated the apoptosis of cells and increased Tau-Ser404/Tau proteins. These results revealed that increased expression of Caveolin-1 may promote the progression of AD by elevating the hyperphosphorylation of Tau protein-induced apoptosis. MiR-124 was shown to reduced caveolar density by targeting Caveolin-1 in porcine kidney epithelial PK15 cells [3]. As one member of miR-124 family, miR-124-3p was found to decrease the density of caveolae and downregulate the expression of caveolin-1 in this study. Furthermore, Caveolin-1 was found to be the direct target of miR-124-3p in our study via miRNA target prediction and validation of dualluciferase reporter assay.
There is no related study about the downstream signaling which miR-124-3p invoved. Caveolin-1 was shown to play a critical role in the inactivation of PI3K/ Akt pathway [39][40][41][42], the upregulation of caveolin-1 could inhibit the PI3K/Akt pathway in the process of IL-6 synthesis in chondrocytes [39]. A previous study reported that Caveolin-1 gene silencing promoted the activation of PI3K/Akt dependent on Eralpha36 and the transformation of MCF10ACE in breast cancers [40]. GSK-3β was phosphorylated when the signaling pathway of PI3K/Akt is activated. And GSK-3β is one of the most important kinases for abnormal phosphorylation of Tau protein, over activation of GSK-3β promotes the expression of hyperphosphorylated Tau which is the key factor in the formation of neurofibrillary tangles in AD brain. Furthermore, hyperphosphorylation of Tau was induced by Aβ through Akt-GSK3β signaling [43]. In our study, overexpression of Caveolin-1 not only increased phosphorylation of Tau without any change of total Tau protein, but also decreased expressions of PI3K, the ratios of Akt-Ser473/Akt and GSK-3β-Ser9/GSK-3β proteins, and vice versa. After transfection of miR-124-3p in the AD cell model, the phosphorylation of Tau was inhibited; meanwhile, the expressions of PI3K, the ratios of Akt-Ser473/Akt and GSK-3β-Ser9/GSK-3β proteins were increased.
In summary, our findings suggest that miR-124-3p plays a protective role through attenuating the hyperphosphorylation of Tau-induced cell apoptosis in AD. And the potential mechanism may be through regulating Caveolin-1-PI3K/Akt/GSK3β pathway, which might provide novel ideas and therapeutic targets for AD.

RNA isolation and quantitative real-time PCR analysis
Primer pairs used for quantitative real-time PCR (qRT-PCR) are presented in Table 1. Total RNA was extracted from cultured cells using Biozol (TaKaRa) according to the manufacturer's instructions. For mRNA expression analysis, the synthesis of cDNA was conducted with 1 ug of total RNA using PrimeSriptTM RT reagent Kit (TaKaRa) and gene expression quantified using SYBR Premix Ex TaqTM II (TaKaRa).

Cell apoptosis assessment by flow cytometry
For cells apoptosis assay, the N2a/WT and N2a/ APP695swe cells were cultured in 6-well plates at a concentration of 1 × 10 6 cells/well. The cells were harvested after 48 h with different treatments. Then ice-cold PBS was used to wash cells three times, and AnnexinV-FITC and propidium iodide (PI, KeyGEN Biotech, Nanjing, China) Abbreviations: F: forward primer; R: reverse primer; RT: loop primer for microRNA reverse transcript. buffer were chosen to incubate them for 30 min at 37°C in the dark. Finally, cells were analyzed by flow cytometry and were considered to be apoptosis no matter early apoptosis (in the fourth quadrant, Annexin V+/PI-) or late apoptosis (in the first quadrant, Annexin V+/PI+).

Validation of MiR-124-3p Target
MiR-124-3p targets were validated by using pGL3 constructs (Promega, Madison, WI, USA). First of all, miR-124-3p target sequences were cloned into pGL3 using XhoI and NotI. Then the gene segments of mouse Caveolin-1 3′UTR were amplified from the genomic DNA using primers as indicated in Table 2. DH-5α bacterial strains were co-transfected with 100 pM miR-124-3p mimics, 400ng pGL3-control or pGL3-Cav1-3′UTR-WT or pGL3-Cav1-3′UTR-MUT, and 200ng pRL-TK vectors in 24-well plates. According to the manufacturer's instructions, the relative luciferase activities of firefly and Renilla were measured using the Dual-Luciferase Reporter Assay System (Promega). 1) 50 ul of Luciferase Assay Reagent II was added to detect the background value generated by the reagent. 2) 20 ul of PLB cracking liquid was added to detect the signal of firefly luciferase which was on behalf of the activity of Caveolin-1. 3) 50 μl Stop & Glo Reagent was used to measure the signal of Renilla luciferase which could be the normalization. 4) The relative activities of Caveolin-1 were the quotient of firefly/ Renilla luciferase activities, and three independent experiments were performed in triplicate.

Transmission electron microscopy
For transmission electron microscopy detection, the N2a/APP695swe cells were cultured in 6-well plates and the concentration is greater than 1×10 6 cells/well at least. The cells were harvested at 48h after transfection with miR-124-3p mimics or miR negative controls. Then 0.1% trypsin-EDTA buffer was used to digest cells, the low speed centrifuge was chose to centrifuge at 800 × g for 5 min. Phosphate buffer saline (PBS, PH7.4) was used to resuspend cells before they were centrifuged at 1200 ×g for 10 min. Furthermore, the cell pellets were fixed in 2.5% electron microscopy-specialized glutaraldehyde for 2 h, washed several times with PBS (0.01 M), stained with 1% osmium tetroxide for 2h, and then dehydrated in a gradient series of alcohol solutions. The samples were placed in propylene oxide, embedded in the epoxy resin Epon812, and cut into ultrathin sections. After uranyl acetate and lead citrate double staining, cells were observed by a transmission electron microscopy of Philips EM208S.
In order to quantify caveolae, the typical flaskshaped structures found on the membranes were scored as caveolae. 10 cells were selected randomly to account for the number of caveolaes each group. And we repeated it three times in three independent experiments.

Bioinformatics and statistical analysis
The miRNA targets predicted by computer-aided algorithms were obtained from TargetScanMouse6.2 (http://www.targetscan.org) and PicTar (pictar.mdcberlin.de). Statistical analysis was performed using SPSS 17.0 software. All the data were presented as means ± S.E.M. And data were statistically analysed by one-way ANOVA, followed by Bonferroni post hoc test, or were analyzed with Student's t test. P < 0.05 was considered to be statistical significance.