miR-302 Attenuates Amyloid-β-Induced Neurotoxicity through Activation of Akt Signaling

Abstract

Deficiency of insulin signaling has been linked to diabetes and ageing-related neurodegenerative diseases such as Alzheimer’s disease (AD). In this regard, brains exhibit defective insulin receptor substrate-1 (IRS-1) and hence result in alteration of insulin signaling in progression of AD, the most common cause of dementia. Consequently, dysregulation of insulin signaling plays an important role in amyloid-β (Aβ)-induced neurotoxicity. As the derivation of induced pluripotent stem cells (iPSC) involves cell reprogramming, it may provide a means for regaining the control of ageing-associated dysfunction and neurodegeneration via affecting insulin-related signaling. To this, we found that an embryonic stem cell (ESC)-specific microRNA, miR-302, silences phosphatase and tensin homolog (PTEN) to activate Akt signaling, which subsequently stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) elevation and hence inhibits Aβ-induced neurotoxicity. miR-302 is predominantly expressed in iPSCs and is known to regulate several important biological processes of anti-oxidative stress, anti-apoptosis, and anti-aging through activating Akt signaling. In addition, we also found that miR-302-mediated Akt signaling further stimulates Nanog expression to suppress Aβ-induced p-Ser307 IRS-1 expression and thus enhances tyrosine phosphorylation and p-Ser 473-Akt/p-Ser 9-GSK3β formation. Furthermore, our in vivo studies revealed that the mRNA expression levels of both Nanog and miR-302-encoding LARP7 genes were significantly reduced in AD patients’ blood cells, providing a novel diagnosis marker for AD. Taken together, our findings demonstrated that miR-302 is able to inhibit Aβ-induced cytotoxicity via activating Akt signaling to upregulate Nrf2 and Nanog expressions, leading to a marked restoration of insulin signaling in AD neurons.

Keywords

Alzheimer’s disease amyloid-β insulin signaling miR-302 Nanog phosphatase and tensin homolog

INTRODUCTION

Insulin, a hormone made and secreted by the pancreas, has great potent effects on our brains [1]. Insulin resistance represents a loss or reduction in its normal functionality on target tissues and hence affects our cognitive and memory functions, ultimately leading to the onset of Alzheimer’s disease (AD) [2]. Insulin resistance has been linked to several previously identified risk factors that accelerate the cognitive dysfunction and ageing process, including diabetes, obesity, hypertension, hyperlipidemia, and metabolic syndrome [3]. Particularly, brains exhibit defective insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) show alteration or aberrant activation of insulin signaling in progression of AD, the most common cause of dementia [4]. These findings suggest that neuronal insulin signaling becomes dysfunction in the AD brains similar to the dementia symptoms of Type 2 diabetes. The pathogenesis of AD is initially triggered by the presence of extracellular amyloid-β (Aβ) peptides, which impair mitochondrial membrane potential (MMP) and contribute to an increase in the accumulation of intracellular reactive oxygen species (ROS), ultimately resulting in neuronal cell death [5, 6]. It has been well established that Aβ deposition may play a pathogenic role in age-associated AD pathogenesis [7]. In addition, our previous studies have indicated that Aβ induces p-Ser307 IRS-1 expression and inhibits IRS-1 tyrosine phosphorylation and its downstream target protein kinase B (PKB, also known as Akt) [8]. Subsequently, Aβ further suppresses Ser9 phosphorylation of glycogen synthase kinase 3β (GSK3β), which is one of the enzymes responsible for causing tau hyperphosphorylation and neurotoxicity [9]. These findings all indicate that insulin signaling plays an important regulatory role in Aβ-induced neurotoxicity.

MicroRNAs (miRNAs) are short single-stranded noncoding RNAs (approx. 20- to 25-nucleotide long) representing a class of small regulatory RNAs. miRNAs inhibit targeted gene expression by suppressing mRNA translation and thus they play an regulatory role in a wide range of cellular processes [10]. Emerging evidence has shown that aberrant miRNA expression is involved in the development or progression of neurodegenerative disorders including AD [10 –12]. Indeed, alterations in miRNA expression have been reported to potentially modulate the levels of toxic Aβ species and subsequently induce neuronal death [10]. However, the function of these miRNAs involved in Aβ-induced neurotoxicity as well as neurodegenerative diseases remains unclear. As recent studies also showed that embryonic stem cell (ESC)-specific miRNAs play a pivotal role in somatic cell reprogramming [13, 14], of which the reprogramming process may help to reset diseased gene profiles into a relatively normal state. To this, regenerative medicine using such a reprogramming mechanism holds a great promise in developing therapies for treating degenerative diseases [13, 14].

Previously, we have shown that an ESC-specific miRNA, the miR-302 family, was responsible for regulating the pluripotency of human ESC and induced pluripotent stem cells (iPSCs) [15 –17]. The genomic sequence encoding for miR-302 is located in the intron of the La ribonucleoprotein domain family member 7 (LARP7) gene on human chromosome 4, a conserved region frequently associated with longevity [18]. Functional studies also identified that miR-302 governs self-renewal by dually regulating oxidative stress [19] and apoptosis [15, 20]. It suggested that miR-302 may prevent apoptosis via downregulating apoptotic-associated genes and upregulating anti-apoptotic genes [19]. In addition, miR-302 also inhibits oxidant-induced cell death, likely by targeting oxidative-associated genes. Hence, miR-302 is capable of regulating self-renewal and pluripotency simultaneously through modulating oxidative stress, and apoptosis. Yet, it is still unclear whether miR-302 plays a role in preventing Aβ-induced neurotoxicity. To this, we herein investigated whether miR-302 exhibits a neuroprotective effect against Aβ-induced neurotoxicity in human neuronal cells.

Maintaining cell survival is dependent on external factors such as growth factors, the lack of which may initiate cellular apoptosis. The Akt signaling pathway has been established as a major downstream effector of growth factor-mediated cell survival, and has been shown to inhibit apoptosis [21]. Akt promotes cell survival by inactivating certain pro-apoptotic mediators such as Bid, a pro-apoptotic member of the Bcl-2 family involved in the induction of death receptor-mediated apoptosis [22]. In addition, Akt signaling is able to reduce oxidative stress, which in turn may play a role in activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) axis [23]. Activation of Nrf2/HO-1 antioxidant pathway leads to the prevention of Aβ-induced neurotoxicity [24]. Both oxidative stress and apoptosis are the underlying mechanisms involved in Akt signaling pathway in neurodegeneration and mitochondrial dysfunction. Taken together, these observations strongly suggest that the Akt signaling pathway plays an important role in cell survival, anti-oxidative stress, neuroprotection, and pluripotency. Interestingly, miR-302 has been reported to mediate Akt activation through downregulating phosphatase and tensin homolog (PTEN) in maintaining the pluripotent status of ESCs [25]. PTEN is a key negative regulator of phosphoinositide 3-kinase (PI3K)/Akt signaling. PI3K/Akt pathway is also a common mediator of pluripotency and self-renewal in ESCs. Moreover, Akt signaling regulates the pluripotency-associated gene Nanog to maintain stem cell self-renewal, pluripotency [26] and anti-ageing [27]. In the present study, we postulate that neuronal insulin resistance represents one of the mechanisms underlying Aβ-mediated neurotoxicity, and miR-302 can protect neuronal cells by restoring impaired insulin signaling via the prevention of Aβ-inducedneurotoxicity.

MATERIALS AND METHODS

Materials

3-(4, 5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT), 4^′,6-diamidino-2-phenylindole (DAPI), dihydroethidium (DHE), and JC-1 were purchased from Sigma (Munchen, Germany). Amyloid-β (Aβ) 1-42 was acquired from AnaSpec Inc. (San Jose, CA, USA), and solutions were prepared according to our previous report [6]. Antibodies used were directed against Akt, p-Akt, GSK3β, p-GSK3β, IRS-1, Nrf2, HO-1, tBid, Bcl-2, Caspase 3, poly (ADP-ribose) polymerase (PARP) (from Santa Cruz, CA, USA), p-tyrosine, p-Tau, Tau (from Merck Millipore, Darmstadt, Germany), β-actin (from Novus Biologicals, Littleton, CO, USA), p-IRS-1, Nanog, and PTEN (from Cell Signaling Technology, Danvers, MA, USA), respectively.

Cell culture

Human neuroblastoma SK-N-MC cells were obtained from the American Type Culture Collection (ATCC, Bethesda, MD, USA). Cells were maintained in Minimal Eagle’s medium (MEM, Gibco), supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100μg/mL streptomycin, and 2 mM L-glutamine at 37°C, 5% CO₂. For inducing miR-302 expression, a pLVX-miR-302 vector was modified from Clontech’s pLVX-AcGFP plasmid as previously reported [28]. Then, the SK-N-MC cells were transfected with the pLVX-mir302 vector to form miR-302-overexpressed cells, using a lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions. The miR-302-overexpressed cells were identified by the presence of a co-expressed AcGFP green fluorescent protein. For silencing Nanog expression, another shRNA gene silencer vector directed against human Nanog mRNAs, called shRNA-Nanog, was obtained from Academia Sinica in Taiwan. In some experiments, we further transfected the shRNA-Nanog vector into the miR-302-overexpressed cells with the lipofectamine 2000 reagent.

Cell viability assay

Cells were seeded in 24-well plates overnight and then treated as indicated. After 24 h, the tetrazolium salt MTT was added to the medium following the manufacturer’s instructions. Only viable cells could metabolize MTT into a purple formazan product, of which the color density (OD) was further quantified by a Bio-Rad spectrophotometer at 550 nm. Cell viability was determined by the percentage of OD from treated cells or transfected cells divided by OD from control cells.

Nucleus morphology

Cells were cultivated on coated slides at 60% confluency and then treated with drugs for 24 h. Thereafter, changes in cell nucleus morphology, in particular characteristics of apoptosis, were examined, using a fluorescence microscope. The cells were fixed in 4% paraformaldehyde after 24 h of treatment with the indicated compounds, permeabilized in ice-cold methanol, incubated with 1 ng/mL of DAPI stain for 15 min at room temperature, and then observed under a fluorescence microscope (DP80/BX53, Olympus). Apoptotic cells were quantified by counting four random fields per condition of treatment.

Western blot analysis

Cells were harvested and homogenized with a protein extraction lysis buffer containing 50 mM Tris-HCl, pH 8.0; 5 mM ethylenediaminetetraacetic acid (EDTA); 150 mM sodium chloride (NaCl); 0.5% Nonidet P-40; 0.5 mM dithiothreitol (DDT); 1 mM phenylmethylsulfonyl fluoride (PMSF); 0.15 units/ml aprotinin; 5μg/ml Leupeptin; 1μg/ml pepstatin; and 1 mM sodium fluoride (NaF), and then centrifuged at 12,000 g for 30 min at 4°C. The supernatant cell lysate was used for immunoblotting analysis. Equal amounts (50μg) of total proteins from the cell lysate were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore), and then probed with a primary antibody followed by another secondary antibody conjugated with horseradish peroxidase. Primary antibodies were used at a dilution of 1 : 1000 in 0.1% Tween-20 and secondary antibodies were used at 1 : 5000 dilutions. The immunocomplexes were visualized using enhanced chemiluminescence kits (Millipore). The relative expression levels of proteins were quantified densitometrically using the QuantityOne software (BioRad), further normalized according to the reference bands of β-actin, and then compared to the normalized protein levels from control cells.

Analysis of mitochondrial membrane potential (MMP)

MMP was investigated using a vital mitochondrial cationic dye JC-1, which accumulates in mitochondria in a potential-dependent matter. Cells were treated with 1μM of JC-1 in fresh medium and incubated at 37°C for 30 min. Cell morphology was then observed and photographed using an inverted fluorescence microscope (DP72/CKX41, Olympus). In normal cells, JC-1 remained as red fluorescent aggregations, whereas during the induction of apoptosis the mitochondrial potential collapsed and hence JC-1 formed monomers producing green fluorescence. MMP was quantified by fluorescent intensity using Image J software (NIH, Bethesda, MD). Then, the normalized fluorescence intensity levels from control cells were set as 100% for comparing the relative expression levels of the fluorescent intensities in tested groups.

Detection of ROS by dihydroethidium (DHE) staining

DHE is a fluorogenic reagent used for detecting intracellular superoxide radical anion [29]. Cells were treated in fresh medium containing 10μM DHE and incubated for 30 min in the dark at room temperature. After 30-min incubation, the staining medium was discarded and the cells were washed twice with PBS and then observed and photographed under an inverted fluorescence microscope (DP72/CKX41). ROS levels were determined by oxidized DHE fluorescence intensity using Image J software (NIH, Bethesda, MD). Then, the normalized fluorescence intensity levels from control cells were set as 100% for comparing the relative expression levels of the fluorescent intensities in tested groups.

Study population and blood samples

Blood sampling from AD patients (n = 7) and age-matched healthy individuals (n = 6) was performed according to standardized procedures approved by the Institutional Review Board (IRB) of Chung Shan Medical University Hospital (CSMUH No: CS 13233) (Table 1). Clinical AD diagnosis was determined by the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) criteria and completed with a Mini-Mental State Examination (MMSE) and cognitive abilities screening instrument (CASI) test. MMSE scores were used as a rough measurement of cognitive function. CASI scores ranged from 1 to 100 were used for quantitative assessment on attention, concentration, orientation, short-term memory, long-term memory, language abilities, visual construction, list-generating fluency, abstraction, and judgment. A detailed overview of AD patients (n = 7, mean age 80.0±4.9 years, range 74–86 years) and age-matched healthy individuals (n = 6, mean age 80.0±5.9 years, range 72–86 years) was summarized in Table 1. A number of AD patients (n = 7, Female/Male = 4/3) had moderate dementia under MMSE (mean scale = 19.3±2.6, range 16–23) and CASI (mean scale = 65.1±10.3, range 49–79) measurement scales, showing most differences between AD patients and age-matched healthy controls (n = 6, Female/Male = 3/3) (Table 1). Age-matched healthy individuals were recruited by local advertisement at the Aging Research Unit, Chung Shan Medical University, Taichung, Taiwan. Neither cognitive impairment nor any dementia disorder was detected in all tested healthy individuals. Both AD patients and age-matched healthy individuals were volunteers with written informed consents had been obtained from all participants and/or their closest relatives according to the Declaration of Helsinki and the IRB-approved protocols. Approximately 20 mL of venous peripheral blood mononucleated cells (PBMCs) were obtained from each tested subject and then total RNAs were isolated from each blood sample with an Oiagen RNeasy Kit (Qiagen, Germantown, MD, USA) and further used for spectrophotometric quantification following the manufacturer’s instructions.

Reverse transcription (RT) and quantitative PCR (qPCR)

Total RNAs were extracted from patients’ PBMCs and cells, respectively, using a Qiagen RNeasy Kit (Qiagen) and further quantified spectrophotometrically. RT-qPCR was carried out using 1μg of total RNAs and following the protocols of an ABI High-Capacity cDNA Archive Kit (ABI). Then, we diluted the resulting cDNA into ten folds and used only 5μl of the diluted cDNA in each of triplicate qPCRs run on a Applied Biosystems 7300 Real Time PCR System with Maxima SYBR Green qPCR Master Mix (2X), ROX solution provided (Thermo), according to the manufacturer’s instructions. Primers were listed in Supplementary Table 1. Levels of relative mRNA or miRNA expression were acquired withthe SDS software version 1.2.3 (Sequence Detection Systems 1.2.3-7300 Real Time PCR System, Applied Biosystems) and then further normalized with the level of housekeeping GAPDH expression in the same sample. The normalized mRNA levels from control cells or normal healthy individuals were set as 100% for comparing the relative expression levels of the mRNA expression in tested groups.

Indirect detection of endogenous miR-302

The genomic sequence encoding for the miR-302 cluster is located in the intron of the LARP7 gene on human chromosome 4 [18]. The endogenous miR-302 familial cluster is produced by RNA splicing of the intron between exons 8 and 9 of LARP7. After intron splicing, the two exons were joined together to for mature mRNAs, of which the joined sites was targeted by a pair of specially designed primers for detecting the expression and splicing of the miR-302 cluster RNAs derived from the LARP7 gene transcripts (Supplementary Fig. 1). The primers were named as LARP7-forward and LARP7-reverse in the list of Supplementary Table 1.

Statistical analysis

Each experiment was repeated for three times (n = 3). All data were presented as means±standard error of mean (S.E.M). For cell viability tests, the average population number of control cells was set as 100% for comparing the survival rates of other tested cells. For western blotting, the protein level measured in each blot was first normalized with the expression level of a housekeeping β-actin protein, and then compared to the normalized level of the protein expressed in control cells, of which the control protein level was then set as 100% for further comparison. For RT-qPCR, the measured values of mRNA expression were first normalized with the expression level of housekeeping GAPDH, and then compared to the normalized mRNA levels from control cells or normal healthy individuals, of which the control mRNA levels were set as 100% for comparing the relative expression levels of the mRNA in tested groups. For measuring fluorescence intensity, the normalized fluorescence intensity levels from control cells were set as 100% for comparing the relative expression levels of the fluorescent intensities in tested groups. Statistical significance of differences between compared groups was determined by one-way analysis of variance (ANOVA) following Dunnett’s post-hoc test for multiple comparisons with a SPSS statistical software (SPSS, Inc., Chicago, IL, USA) as well as the two-tailed Student’s t-test. A probability value of <0.05 or <0.01 was taken to indicate statistical significance and hence the significant levels were set at ^*p < 0.05 or ^**p < 0.01, respectively, depending on individual experiments.

RESULTS

miR-302 protects SK-N-MC cells against Aβ-induced apoptosis

Recent studies have demonstrated the crucial functions of miR-302 in regulating oxidative stress-induced apoptosis [20]. To address whether miR-302 exerts any protective effect on neuronal cells against Aβ-induced apoptosis, we transfected human neuronal SK-N-MC cells with a cytomegalovirus (CMV)-promoter-driven miR-302 expression vector as previously reported [17], and then exposed to Aβ (2.5μM) for 24 h. After that, the transfected miR-302-overexpressed cells were identified by the presence of a co-expressed AcGFP green fluorescent protein under an inverted fluorescent microscope (Fig. 1A) and the expression of miR-302 was further confirmed by RT-qPCR (n = 3, p < 0.01, Fig. 1B) and miRNA microarray analysis (Supplementary Figure 2), showing successful transcription of the whole miR-302 familial cluster (i.e., miR-302a, b, c, and d). Notably, Fig. 1C further demonstrated that Aβ treatment triggered massive cell death in control cell groups, whereas miR-302-overexpressed cells showed marked attenuation of such Aβ-induced cell death (n = 3, p < 0.01). To determine which kind of cell death induced by Aβ, we further examined the nuclei fragmentation by DAPI staining. As shown in Fig. 1D, Aβ treatment disrupted nucleus margin and significantly increased the apoptotic cell population in the control groups compared to those of miR-302-overexpressed cells (n = 3, p < 0.01). In addition, Fig. 1E revealed that Aβ treatment markedly increased the cleavage formation of both caspase 3 and PARP in control cells but not in miR-302-overexpressed cells (n = 3, p < 0.01), further confirming this point. Taken together, our data strongly suggest that miR-302 plays a protective role in preventing Aβ-induced cell apoptosis.

Activation of Akt signaling is involved in miR-302-mediated neuroprotection

We have previously reported that restoration of insulin sensitivity in neurons leads to Akt activationand so as to inhibit Aβ-induced apoptosis [8]. To determine whether miR-302 expression can restore neuronal insulin sensitivity and prevent Aβ-induced neurotoxicity, we used western blot analyses to measure the expression levels of major insulin signaling-related proteins, such as pSer307-IRS-1, tyrosine phosphorylation of IRS-1, and their downstream target pSer473-Akt. As shown in Fig. 2A, Aβ treatment in control cells significantly increased p-307 IRS-1 serine phosphorylation (n = 3, p < 0.05) while decreasing IRS-1 tyrosine phosphorylation (n = 3, p < 0.01), both of which are considered as hallmarks of insulin resistance; yet, in miR-302-overexpressed cells this Aβ-induced insulin resistance was markedly attenuated (n = 3, p < 0.05). Moreover, Aβ treatment also led to a significant decrease of p-Ser 473-Akt in control groups but not in miR-302-overexpressed cells (n = 3, p < 0.01) (Fig. 2A). To further elucidate the protective role of PI3K/Akt signaling in miR-302-overexpressed cells, we applied a PI3K inhibitor - LY294002. Figure 2B revealed that co-treatment of Aβ (2.5μM) and LY294002 (20μM) could disrupt miR-302-mediated Akt signaling (n = 3, p < 0.01) and thus resulted in a marked reduction of the viable cell population, as determined by MTT assay (n = 3, p < 0.01, Fig. 2C). All these findings suggest that miR-302 prevents Aβ-induced neurotoxicity and neuronal death via activating PI3K/Akt signaling. Alternatively, Aβ-impaired insulin signaling may also lead to an increase of GSK3β activity as well as tau hyperphosphorylation, a relevant step in AD pathogenesis [30]. To this, we found that miR-302 expression could stimulate Akt signaling to slightly increase p-Ser 9-GSK3β levels and hence may provide a mild inhibitory effect on tau hyperphosphorylation (n = 3, p < 0.05) (Fig. 2D). As a result, Fig. 2D also showed that co-treatment of Aβ and LY294002 totally abolished the inhibitory effect of miR-302 on p-Ser 9-GSK3β expression and tau phosphorylation in control cells compared to those of miR-302-overexpressed cells (n = 3, p < 0.05). Taken together, our data demonstrate that miR-302 may exert its protective effects mainly through activating and/or restoring the Akt/GSK3β signaling pathway.

miR-302 attenuates Aβ-induced oxidative stress through Akt-upregulated Nrf2/HO-1

Emerging evidence suggested that Aβ is able to generate free radicals and oxidative damages [6]; yet, activation of Akt signaling may inhibit such Aβ-induced oxidative stress and apoptosis [31]. To determine whether miR-302-mediated Akt activation can prevent Aβ-induced intracellular ROS accumulation, we performed a fluorometric assay to measure the concentration of hydrogen peroxide accumulated in the cells. As shown in Fig. 3A, Aβ treatment stimulated a significant elevation of intracellular superoxide radical anions in control groups but not in miR-302-overexpressed cells (n = 3, p < 0.01). Co-treatment of Aβ (2.5μM) and insulin (1μM) could restore the normal levels of intracellular superoxide radical anions in control groups (p < 0.05), indicating that miR-302-mediated Akt activation did inhibit Aβ-induced ROS. Furthermore, recent studies also indicated that Nrf2, a redox-sensitive transcription factor, can confer protection against ROS damage by upregulating antioxidant-response elements, such as HO-1 [23, 32]. Since PI3K/Akt signaling has been reported to elevate HO-1 expression and Nrf2-dependent transcription [24], we further elucidate this possible anti-oxidant effect of miR-302 by western blot assays. As a result, Fig. 3B revealed that Aβ treatment reduced both Nrf2 and HO-1 expressions in control groups but not in miR-302-overexpressed cells (n = 3, p < 0.05). To confirm the source of this effect, further treatment of LY294002 (20μM) with Aβ (2.5μM) also decreased Nrf2 expression in miR-302-overexpressed cells (n = 3, p < 0.05) (Fig. 3C), indicating that miR-302 regulates Nrf2 expression via the PI3K/Akt signaling pathway. Moreover, activation of Akt signaling significantly restored the Nrf2 expression after co-treatment of Aβ (2.5μM) and insulin (1μM) in control groups (n = 3, p < 0.05) (Fig. 3C), further suggesting that miR-302-mediated Akt activation can prevent Aβ-induced ROS accumulation through the upregulation of Nrf2 and HO-1.

To investigate the miR-302 effect on Aβ-mediated mitochondria dysfunction and apoptosis, we examined MMP with JC-1 staining assays and the expression of apoptotic-associated marker truncated Bid (tBid) and anti-apoptotic-associated marker Bcl-2 with western blotting assays. As shown in Fig. 3D, control cells displayed a significant deficiency of mitochondrial membrane depolarization in response to Aβ treatment (n = 3, p < 0.05), which was however not found in miR-302-overexpressing cells, as indicated by the concurrent loss of cytoplasmic red J-aggregate fluorescence and elevation of diffused green fluorescence. Yet, this miR-302-mediated protective effect on MMP integrity could be totally abolished by co-treatment of Aβ (2.5μM) and LY294002 (20μM) for 24 hours (n = 3, p < 0.05), indicating the involvement of Akt/PI3K signaling. In addition, Aβ treatment resulted in a marked increase of tBid expression (p < 0.01) and decrease of Bcl-2 (p < 0.05) in control groups, but not in miR-302-overexpressed cells (Fig. 3E). All these findings clearly suggest that miR-302-mediated Akt activation can inhibit Aβ-induced oxidative stress, mitochondria dysfunction and apoptosis via upregulating Nrf2 activities.

miR-302 regulates Akt signaling by targeting PTEN and inducing Nanog expression

After having determined the important role of miR-302 in activating Akt signaling to prevent Aβ-induced neurotoxicity, we further investigate the molecular mechanism underlying such miR-302-mediated Akt activation. Recent studies have indicated that miR-302 promotes pluripotency through Akt signaling bytargeting PTEN [25]. To search the miR-302 target site in PTEN, we performed screening analyses using a prediction program, TargetScan (http://www.targetscan.org/), and identified a specific miR-302 binding site located in the 3’UTR of human PTEN gene (Fig. 4A). As our western blotting data have shown a significantly decrease of PTEN expression in miR-302-overexpressed cells (n = 3, p < 0.05) (Fig. 4B), it suggests that miR-302 may target this 3’UTR binding site to suppress PTEN expression. Also, since knockdown of PTEN can increase the pluripotency-associated gene Nanog expression [26], which is further mediated by PI3K/Akt signaling in ESCs [25], we herein examined the miR-302 effects on PTEN, pSer473 Akt, and Nanog expressions with western blot assays. As a result, Fig. 4C showed a marked elevation of Nanog expression only detected in miR-302-overexpressed cells (n = 3, p < 0.05), while Aβ treatment (2.5μM for 24 h) stimulated a significant increase of PTEN as well as decreases of pSer473 Akt and Nanog expressions in control groups but not in miR-302-overexpressed cells (n = 3, p < 0.05) (Fig. 4D). Interestingly, further studies revealed that blocking Akt signaling with LY294002 (20μM for 24 h) could restore Aβ-mediated inhibitory effects on pSer473 Akt and Nanog expressions in miR-302-overexpressed cells (n = 3, p < 0.05) (Fig. 4E), demonstrating that miR-302 activates Akt signaling to induce Nanogexpression.

To determine whether Nanog plays a protective role in Aβ treatment, we further performed shRNA-mediated knockdown of Nanog in miR-302-overexpressed cells. As shown in Fig. 4F, downregulation of Nanog resulted in an increase of p-Ser307 IRS-1 expression as well as a decrease of both tyrosine phosphorylation and p-Ser 473-Akt/ p-Ser 9-GSK3β levels in miR-302-overexpressed cells after Aβ treatment. Taken together, our results strongly suggest that miR-302 may confer protection against Aβ-induced neurotoxicity by downregulating PTEN to activate Akt and the downstream Nanog signaling.

In vitro and in vivo gene expression levels of Naong and LARP7

We have observed that impaired Nanog expression is associated with Aβ-disrupted insulin sensitivity. To investigate this point, we performed RT-qPCR to show that Aβ treatment significantly decreased Nanog mRNA expression in control neurons in vitro (n = 3, p < 0.05, Fig. 5A). Next, we addressed the relevance of this finding to human AD patients in vivo by measuring the mRNA expression levels of Nanog in AD patients’ PBMCs. A detailed overview of the testing subjects’ characteristics is summarized in Table 1. A number of AD patients (n = 7) had moderated dementia by MMSE and CASI measurement scales, which can differentiate between AD patients and age-matched healthy controls (n = 6). As a result, both scales of MMSE and CASI were decreased in these AD patients (Table 1). Figure 5B further showed that the level of Nanog mRNA was significantly decreased in AD patients compared to normal age-match controls (p < 0.05). This observation confirmed our hypothesis that AD patients exhibit reduced Nanog expression, which may contribute to the pathogenesis of AD-associated neurodegeneration.

In addition, recent studies have indicated that miR-302 is encoded in the LARP7 gene on the chromosome 4 of human genome [33]. To determine whether the endogenous level of miR-302 was affected by Aβ-induced neurotoxicity during the progression of AD, we examined the expression of miR-302-encoding LARP7 gene by RT-qPCR with a special primer directed against the joining region of exons 8 and 9, as described in Materials and Methods. As a result, Fig. 5C showed that Aβ treatment markedly decreased LARP7 mRNA expression in control neurons in vitro (n = 3, p < 0.05). Further detection of LARP expression in AD patients’ PBMCs also revealed that the expression of LARP7 mRNA was significantly decreased in AD patients compared to normal age-match controls (Fig. 5D, p < 0.05). These results proved that endogenous LARP7/miR-302 expression likely plays an important role in preventing the progression of AD. Taken together, our findings indicated that an impaired Nanog expression may take place in age-associated AD, of which the detail mechanism remains to be determined.

DISCUSSION

Impairment of insulin signaling not only presents a serious threat to neuron survival but also plays a critical role in aging-related diseases such as AD. Our study, for the first time, demonstrated that miR-302 regulates cell survival and anti-aging processes via activating the Akt signaling pathway, which may confer protection against Aβ-induced neurotoxicity in human neuronal cells. We herein concluded that: (i) miR-302 silences PTEN to activate Akt signaling, which the stimulates Nrf2/HO-1 elevation and hence attenuates Aβ-induced apoptosis, and (ii) miR-302-mediated Akt activation also stimulates Nanog expression to suppress p-Ser307 IRS-1 expression and thus enhance IRS-1 tyrosine phosphorylation and p-Ser 473-Akt/ p-Ser 9-GSK3β formation. Conceivably, both of these newly identified miR-302 effects are useful for developing AD-related therapies.

We also found that miR-302-stimulated Akt signaling is able to attenuate many Aβ-associated AD symptoms, including neuronal insulin resistance, tau hyperphosphorylation, oxidative stress, and neuronal death. Our recent studies have reported that Aβ treatment causes tau hyperphosphorylation [8] through activating GSK3β [9] in SK-N-MC cells [8]. Moreover, the Aβ-induced upregulation of pSer307 IRS-1 has been shown to inhibit the insulin-downstream Akt/GSK3β signaling in cognition-related brain areas, leading to brain insulin resistance [4]. As a result, GSK3β activation regulates tau binding to microtubules and causes tau aggregation, subsequently resulting in tau hyperphosphorylation [9] as well as neuronal death [8]. In contrast, upregulation of Akt has been reported reduce Aβ-induced neurotoxicity, such as insulin resistance, tau hyperphosphorylation and neuronal death [8]. In view of these previously established evidences, our results further indicated that miR-302 expression can stimulate Akt activation and hence may improve all these Aβ-associated ADsymptoms.

Aβ has been reported to trigger mitochondrial dysfunction and induce the generation of ROS [31], both of which represent typical characteristics of aging [5 –7]. As oxidative stress is one of major causes for the onset of many degenerative disorders and aging [8], Aβ-induced ROS accumulation may eventually lead to neuronal death. Interestingly, recent studies observed that miR-302 is able to inhibit oxidant-induced cell death in adipose tissue-derived mesenchymal stem cell [19]. Similarly, we found that miR-302 can attenuate Aβ-induced oxidative stress through activation of Nrf2 and HO-1. Nrf2 is a redox-sensitive transcription factor responsible for regulating the induction of several important anti-oxidant enzymes, including NAD(P)H quinone oxidoreductase 1, glutamate-cysteine ligase, and particularly HO-1 [23]. Since PI3K/Akt signaling has been reported to mediate Nrf2/HO-1 activities to reduce cytotoxicity in oxidative stress-damaged neurons [24], it is conceivable that Akt plays a pivotal role in regulating neuronal survival. To this, our results have clearly demonstrated this point by showing that miR-302 activates Akt signaling to upregulate Nrf2/HO-1 activities, so as to inhibit Aβ-induced oxidative stress and maintain the intracellular redox balance toward cell survival.

Most importantly, our study revealed that miR-302 expression prevents Aβ-induced neurotoxicity through activation of Akt-mediated Nanog expression. We also found that miR-302 activates Akt/GSK3β signaling by knocking down a PI3K/Akt inhibitor, PTEN. It has been known that miR-302 targets PTEN’s mRNA 3^′ UTR translational suppression [25 , 35]. This finding is also supported by a recent study showing that miR-302 promotes human ESC pluripotency by knocking down PTEN to activate Akt signaling [25]. PTEN is a well-known negative regulator of PI3K signaling and positively regulates insulin signaling [36]. In addition, PTEN is a key negative regulator of PI3K/AKT signaling that is arguably the most important pro-survival pathway and ageing-associated disease in neurons [37]. However, loss of PTEN antagonizes the progression of ageing through promoting regeneration and preventing oxidative stress-induced cell death [36, 38]. Recent studies have also demonstrated that knockdown of PTEN results in a significant increase of the pluripotency-associated gene Nanog expression [26]. Particularly, the PTEN-targeted PI3K/Akt signaling has been shown to play an important role in maintaining iPSC status [39]. Nanog is a key determinant that maintains self-renewal of undifferentiated stem cells and hence its expression may be regulated by Akt signaling in differentiated cells [40]. To this, Chen et al. have reported that retinol enhances the expression of Nanog, which directly activates IRS-1 by engaging Akt and preventing differentiation of ESCs [41]. Therefore, deciphering the underlying mechanisms of Nanog-mediated Akt signaling may have a wide impact on many biomedical research fields.

Taken together, in addition to numerous evidence showing that Akt/GSK3β are critical regulators of AD progression, our findings further indicated that miR-302 is able to target and downregulate PTEN to re-activate Akt/GSK3β signaling and Nanog expression, so as to prevent Akt/GSK3β-associated AD pathogenesis and progression. Furthermore, the discovery of concurrent downregulation of Nanog and miR-302-encoding LARP7 gene expressions in AD patients may be useful for developing a diagnostic or therapeutic means for treating AD. In light of our findings, it is conceivable that Akt/GSK3β signaling is associated with regulation of ageing, and hence may serve as a novel target to treat AD. Since Akt signaling is an important regulator for pluripotency and ageing-associated disorders [39], miR-302 expression may confer anti-apoptotic and anti-oxidative effects through activation of Akt signaling [15 , 39]. In summary, we conclude that an elevated expression of miR-302 prevents Aβ-induced neurotoxicity through activation of Akt/GSK3β signaling by downregulating PTEN and upregulating Nanog, as summarized in Fig. 6. Authors’ disclosures available online (http://www.j-alz.com/manuscript-disclosures/15-0741r1).

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Science and Technology (101-2320-B-040-015-MY3 and 104-2314-B-040-007-MY2) of Taiwan. The fluorescence microscope and imaging analyzer were provided by the Instrument Center of Chung Shan Medical University, which is supported by Ministry of Science and Technology, Ministry of Education and Chung Shan Medical University.

Appendix

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