Modulation of miR-19 in Aluminum-Induced Neural Cell Apoptosis

Abstract

Neuronal cell death is an important feature of neurodegeneration. Aluminum is associated with neurodegenerative disorders, particularly Alzheimer’s disease. However, the underlying mechanisms by which aluminum induces neuronal apoptosis remain to be elucidated. miR-19 is a key miRNA implicated in regulating cell survival process, while the role of miR-19 in Alzheimer’s disease has not been investigated. In the present study, we showed that Aluminum maltolate (Al-malt), a lipophilic Al complex which is a common component of human diet with the ability to facilitate the entry of Al into the brain, induced apoptosis in human neuroblastoma SH-SY5Y cells, along with downregulation of miR-19a/miR-19b, upregulation of miR-19-targeted PTEN, and alterations of its downstream apoptosis related proteins including AKT, p53, Bax, and Bcl-2. miR-19 overexpression attenuated Al-malt-induced apoptosis as well as changes in the expression of apoptosis related proteins in SH-SY5Y cells. We further revealed that exposure of rats to Al-malt for 12 weeks at doses relevant to human exposure significantly elevated Al concentrations in serum and brain tissues. Al-malt dose-dependently induced apoptosis in rat brain, as evidenced by increased caspase activation and increased TUNEL staining. Consistent with in vitro results, Al-malt reduced miR-19 expression and altered the expression of apoptotic related proteins in rat brain. Taken together, our data suggest for the first time that miR-19 modulation is critically involved in Al-induced neural cell apoptosis. Findings from this study could provide new insight into the molecular mechanisms of Al-associated neurodegenerative pathogenesis.

Keywords

Aluminum apoptosis miR-19 modulation neurodegenerative diseases

INTRODUCTION

Neurodegenerative diseases are characterized by pathological changes in disease-specific areas of the brain and degeneration of distinct neuron subsets. Based on the specific populations of neuronal cells affected, neurodegenerative diseases are classified into several types, and Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, and amyotrophic lateral sclerosis are among the major neurodegenerative diseases. The specific causes of neurodegenerative diseases are still unclear. Numerous genetic and environmental risk factors are involved in the etiology and pathogenesis of neurodegenerative diseases, including alterations in gene expression, protein misfolding and deposition, inflammation, oxidative stress, energy metabolism, and aberrant re-entry into the cell cycle/apoptosis [1 –3]. Aluminum is the third most abundant element and the most abundant metal existed in the biosphere, which has also been widely used in industrial applications, water treatment, food additives, and pharmaceuticals [4]. Environmental exposure of humans to aluminum is ubiquitous and extensive. Aluminum is not the essential element in human body; on the contrary, it has been reported to be neurotoxic [5]. Aluminum can enter and accumulate in the central nervous system, causes behavioral impairments and neurolipofuscinogenesis [6]. Mounting evidence has suggested aluminum as a possible etiologic factor in neurodegenerative disorders, particularly AD [7]. Increased level of aluminum was observed in demented patients compared with normal people [8]. Using X-ray spectrometric, Al was detected in neurofibrillary tangle of AD patients brain [9]. Al was reported to affect the formation of neurofibrillary tangles in AD [10]. Previous studies have postulated aluminum-induced apoptosis as the major cause of its neurotoxicity [11 –13]. However, the underlying molecular mechanisms by which aluminum induces apoptosis of neuronal cells remain to be elucidated.

Aluminum maltolate (Al-malt) is a lipophilic complex of aluminum with maltolate, which is soluble in aqueous solution at physiological pH [14]. As a common component of the human diet, maltolate is a byproduct formed during sucrose pyrolysis or thermal degradation of starch. Maltolate is also an approved food additive used as a flavor enhancer in beverages like coffee and chocolate milk and as a favoring agent in breads and cakes. Due to the high affinity of maltolate for Al, Al-malt complex can be formed in the gastrointestinal tract. Evidence has also suggested that maltolate may facilitate the entry of Al into the brain [15, 16]. The neurotoxic effects of Al-malt have been illustrated in in vitro and in vivo studies. Al-malt induces apoptosis in neuronal cells and brain tissues [12 , 17]. Treatment of Al-maltolate in aged New Zealand white rabbits results in conditions that mimic the neuropathological, biochemical, and behavioral changes observed in AD [18]. Therefore, Al-malt is advantageous for use in the investigation of Al neurotoxicity since it is relevant to human health.

Numerous studies have demonstrated microRNAs (miRNAs) regulation as an important mechanism in gene expression. miRNAs are a class of highly conserved, single-stranded endogenous non-coding small RNAs that function as post-transcriptional regulators of gene expression via targeting the 3′ untranslated regions (UTRs) of target mRNAs, leading to mRNA degradation or translation inhibition [19, 20]. The critical role of miRNAs in regulating biological process such as apoptosis has been established. Emerging evidence has highlighted the functional importance of miRNAs in the pathogenesis of neurological diseases. It has been demonstrated that dysregulation of miRNAs participates in the development of neurodegenerative diseases such as AD, PD, and Huntington’s disease [21 –27]. miR-19 is a key member of the miR-17/92 cluster [28], which targets several critical apoptosis and/or proliferation related genes such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN) [29]. Dysregulation of miR-19 alteration is involved in nervous system disease including PD, spinocerebellar ataxia type 1 (SCA1), neurofibromatosis, and some other neurodegenerative diseases [21 , 31]. To date, however, whether various miRNAs, including miR-19, play a role in Al-induced neural cell apoptosis, has not yet been investigated.

The present study aimed to investigate the regulatory action of miR-19 in Al-malt induced neural cell apoptosis in human neuroblastoma SH-SY5Y cells and rat brain tissues. We revealed for the first time that miR-19 modulation is crucial for Al-induced neural cell apoptosis, a key event in the process of neurodegenerative diseases. This finding could advance our understanding of the molecular mechanism of Al-associated neurodegenerative pathogenesis.

MATERIALS AND METHODS

Chemicals and reagents

Cell culture reagents were purchased from Gibco (Carlsbad, CA, USA) unless otherwise stated. Fetal bovine serum (FBS) was obtained from PAA Laboratories (Pasching, Austria) Aluminum chloride hexahydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Maltolate was obtained from Aladdin Company (Shanghai, China). miR-19a and miR-19b mimics as well as control mimic were purchased from RIBOBIO (Guangzhou, China). The primary antibodies for anti-caspase 3, anti-caspase 9, anti-bax, anti-bcl-2, anti-PTEN, anti-p-AKT, anti-p53, and anti-GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). β-actin antibody was purchased from Bioworld (Shanghai, China). Sources of other materials are noted accordingly in the text.

Cell culture

Human neuroblastoma cell line SH-SY5Y, a cell line that is commonly used in the research of neurotoxicity and neurodegenerative diseases [32 –34], was obtained from Chinese Academy of Typical Culture Collection Cell Bank. SH-SY5Y cells were cultured and maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. The medium was changed every other day.

Preparation of Al-malt

Al-malt was prepared according to the procedure as described previously [35]. Briefly, aluminum chloride hexahydrate (AlCl₃·6H₂O) and maltolate were dissolved in double distilled water, and the stock solutions of AlCl₃ (30 mM) and maltolate (60 mM) were prepared, respectively. Before each experiment, the stock solutions of AlCl₃ and maltolate were mixed in equal volume, adjusted the pH to 7.4 with NaOH and filtered through a 0.22- μm-pore size filter.

MTT assay

SH-SY5Y cells were seeded in 96-well plates at a density of 2×10³ cells/well in 100 μl of medium. Twenty-four hours later, the medium was replaced and cells were treated with various concentrations of Al-malt (0.005, 0.01, 0.1, 0.25, 0.5, 1, and 2 mM) for 3 days. Cell viability was then assessed using MTT assay. Twenty microliter of methylthiazoletetrazolium (Sigma-Aldrich, St. Louis, MO, USA) assay solution (5 mg/mL) was added to each well and the plates were further incubated for 4 h at 37°C. Medium containing MTT was removed and precipitants were solubilized in DMSO. Absorbance was measured at 490 nm using a microplate reader (Titertek, Huntsville, AL). All measurements were performed in triplicate.

Hochest 33258 staining

Hoechst 33258 staining was used to analyze apoptosis induced by Al-malt. SH-SY5Y cells were treated with various concentrations of Al-malt (0.1, 0.25, and 0.5 mM) in six-well plates. At 3 days post-treatment, cells were washed twice with cold PBS, fixed in 4% formaldehyde at 4°C for 10 min, and stained with 5 μg/ml Hoechst 33258 according to the manufacturer’s instructions (Beyotime, China). After washed twice with PBS, the cells were then observed and imaged using fluorescence microscope.

Annexin V staining and flow cytometry analysis

FITC Annexin V Apoptosis Detection Kit I (BD Bioscience USA) was used in Annexin V Staining and flow cytometry analysis. SH-SY5Y Cells were harvested after Al-malt (0.1, 0.25, and 0.5 mM) treatment for 72 h. Cells were washed twice with PBS and then centrifuged at 800 g for 5 min. Cells were resuspend and diluted in 200 μl cold binding buffer. The cells were stained with 10 μl Annexin V-FITC and 10 μl propidium iodide for 15 min at 4°C, respectively. Flow cytometry analysis was performed within an hour. Data were obtained by counting the number of apoptotic cells per 1×10⁵ cells.

Western blot analysis

SH-SY5Y cells were washed twice with ice-cold PBS and scraped into 0.2 mL of lysis buffer (20 mM HEPES (pH 6.8), 5 mM EDTA, 10 mM EGTA, 5 mM NaF, 0.1 μg/mL okadaic acid, 1 mM dithiothreitol, 0.4 M KCl, 0.4% Triton X-100, 10% glycerol, 5 μg/mL leupeptin, 50 μg/mL of phenylmethanesulphonylfluoride, 1 mM benzamidine, 5 mg/mL aprotinin, and 1 mM sodium orthovanadate). In animal study, brain tissues were homogenized in a lysate buffer (5 mmol/L EDTA, 50 mmol/L Tris, 1% SDS, pH 7.5, 10 μg/mL aprotinin, 1% sodium deoxycholate, 1% NP-40, 1 mM PMSF, 1% Triton-X 100, and 10 μg/mL leupeptin) and then centrifuged at 4°C for 20 min. Protein concentrations were measured with the BCA Protein Assay (Pierce, Rockford, IL). Fifty micrograms of proteins were fractionated by electrophoresis through 10–12.5% SDS-PAGE and were transferred to PVDF membrane (Millipore, Billerica, MA). The membranes were blocked with 5% defatted milk and subsequently probed with primary antibody overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibody. GAPDH and β-actin were served as the loading control. For densitometric analyses, protein bands on the blots were measured by the use of Eagle Eye II software.

RNA extraction and quantitative reverse transcription-PCR detection of miR-19

Total RNA was extracted from cells and rat tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For detection of miR-19a and miR-19b, 1 μg of total RNA was reverse transcribed according to the manufacture’s protocol (TAKALA, Japan). Quantitative reverse transcription PCR (qRT-PCR) was performed using the Power SYBR Green Master Mix (Applied Biosystems, USA) and an ABI 7300 real-time PCR detection system (Applied Biosystems, USA). Forward (F) and reverse (R) primers were as follows: miR-19a-F, 5^′-CCTCTGTTAGTTTTGCATAGTTGC-3^′; miR-19a-R,5^′-CAGGCCACCATCAGTTTTG-3^′;miR-19b-F,5^′-CACCATGGCATGCTTTAGATTATATATTCCGC-3^′;miR-19b-R,5^′-GCGGAATATATAATCTAAAGCATGGGTGCCATGGTG-3^′;U6-F,5^′-CGCTTCGGCAGCACATATACTAAAATTGGAAC-3^′;U6-R,5^′-GCTTCACGAATTTGCGTGTCATCCTTGC-3^′. All of the primers were synthesized by RIBOBIO (Guangzhou, China). The U6 snRNA was used as an internal control. Fold changes in expression of each gene were calculated by a comparative threshold cycle (Ct) method using the formula 2^–(ΔΔCt).

Transfection of miR-19 mimics

miR-19a mimic, miR-19b mimic and negative vector control were obtained from RiboBio (Guangzhou, China). SH-SY5Y cells were plated onto 96-well plates with a density of approximately 1.0×10⁴ cells in RPMI 1640 medium containing 10% FBS without penicillin/streptomycin. Following 24 h of incubation, cells were then transfected with miR-19a/miR-19b mimic (50 nM) or negative vector control by lip 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The medium was replaced with fresh culture medium 4 h later. Cells were then treated with or without Al-malt (0.25 mM) for 3 days. Cell viability, apoptosis, and protein expression were determined using MTT assay, flow cytometry analysis, and western blot analysis.

Rats and Al-malt treatment

Six-week-old male Sprague Dawley (SD) rats weighing 120–150 g were purchased from the Vital River Company (Nanjing, China). All rats were housed in polypropylene cages, maintained on a 12-h light/dark cycle, 25 ± 1°C room temperature, 40–60% relative humidity, and free access to water and food. Animals were handled in accordance with the recommendations in the guidelines of the Animal Care and Welfare Committee of Nanjing Medical University. The study protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University.

After 1 week of acclimation, rats were randomly divided into four groups (n = 8 per group): control group, rats were received drinking water; low-dose group, rats were received drinking water containing 20 mg Al/L; middle-dose group, rats were received drinking water containing 100 mg Al/L; high-dose group, rats were received drinking water containing 200 mg Al/L. The body weight, water intake and food consumption were recorded every five days during the experiment. After 12 weeks treatment, rats were sacrificed and the brain tissues were isolated and used for the analyses of western blotting, quantitative RT-PCR, TUNEL, immunohistochemistry, and Alcontents.

Immunohistochemistry analysis

A SABC kit (Boster Biological Technology, Wuhan, China) was used in immunohistochemistry analysis. Rat hippocampus tissues were fixed with formalin, and then paraffin-embedded specimens were cut into 4- μm thick sequential sections. Tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series, respectively. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at room temperature. The slides were then immersed in 0.01 M vitrate salt buffer and microwaved to boiling for 7 min. Following non-specific blocking, sections were incubated with diluted primary anti-PTEN antibody (1 : 100) at 4°C overnight. Sections were subsequently incubated in peroxidase-labeled secondary antibody followed by SABC reagent, each for 30 min at room temperature. DAB (Beyotime, China) was used as chromogen substrate.

TUNEL assay

A fluorometric TUNEL assay kit (Promega, Catology# G3250, Madison, WI, USA) was used to detect apoptosis according to the manufacturer’s instructions. The kit measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP at 3^′-OH DNA ends using the recombinant Terminal Deoxynucleotidyl Transferase (rTdT). Rat hippocampus tissue sections were deparaffinized in xylene and subsequently rehydrated in ethanol. Sections were incubated with 20 μg/ml Proteinase K for 15 min. Following equilibration, sections were incubated with 50 μl of rTdT reaction mixture at 37°C for 60 min in the dark. The reactions were subsequently terminated, and the tissue sections were analyzed immediately under a fluorescence microscope using a fluorescein filter set to visualize the green fluorescence at 520±20 nm.

Determination of Al concentrations in serum and brain of rats

Fresh rat brain tissues were dried in the dryer at 80°C for 24 h. Then approximately 0.1-0.2 g dried samples were put into a polytetrafluoroethylene tank and dissolved with 2 ml HNO₃ and 1 ml H₂O₂. The mixture was heated at 120°C for 3 h to obtain transparent and yellow brown solution. The resulting digested samples were diluted with 18 Ω high purity Al-free water. Rat blood was collected, centrifuged at 3000 rpm for10 min at 4°C, and serum samples were used for determination of Al contents. The concentrations of Al in the brain and serum were determined by high-resolution continuum source graphite furnace atomic absorption spectrometry (Analytik Jena AG, Jena, Germany) using a wavelength of 396.152 nm with an injection volume of 10 μl. Each sample was repeated twice and the average value was taken.

Statistical analysis

Statistical analyses were performed with SPSS 16.0 (SSPS, Inc., Chicago, IL, USA). All data are expressed as mean ± standard deviation. One-way ANOVA was used for comparison of statistical differences among multiple groups, followed by the LSD significant difference test. A value of p < 0.05 was considered significantly different.

RESULTS

Al-malt induces apoptosis in human SH-SY5Y cells

To investigate the effect of Al-malt on the induction of neural cell apoptosis, human SH-SY5Y cells were exposed to various concentrations of Al-malt for 3 days and cell viability was examined by MTT assay. Fig. 1A shows that Al-malt decreased cell viability in a dose-dependent manner. The survival rate of SH-SY5Y cells reduced approximately 50% at the concentration of 0.5 mM Al-malt.

The apoptotic process involves a series of typical cellular events including plasma eversion, cell shrinkage, chromatin condensation, DNA fragmentation, and formation of apoptotic bodies [36]. Our Hoechst 33258 staining assay revealed that compared to untreated controls, SH-SY5Y cells treated with Al-malt for 3 days showed intense fluorescence in the nuclei and nuclear fragmentation, indicating the apoptotic changes (Fig. 1B).

Moreover, flow cytometry analysis also revealed that Al-malt induced apoptosis in SH-SY5Y cells in a dose-dependent fashion. Treatment of SH-SY5Y cells with Al-malt for 3 days significantly resulted in apoptosis of the cells. In comparison with the control cells, an increase in the number of apoptotic cells was observed in Al-malt treated SH-SY5Y cells. The percentages of apoptotic cells were 7.6% for the control, 12.2% for 0.1 mM Al-malt, 18.9% for 0.25 mM Al-malt, and 37.8% for 0.5 mM Al-malt, respectively (Fig. 1C).

Since caspases play essential role in the regulation of cell apoptosis, we examined the effect of Al-malt on the activation of caspases in SH-SY5Y cells. It was shown that Al-malt treatment decreased the expression levels of pro-caspase-9 and pro-caspase-3, whereas the levels of cleaved caspase-9 and cleaved caspase-3 were significantly increased, indicating the activation of these caspases in the cells (Fig. 1D). Collectively, data from cell viability, Hoechst 33258 staining, flow cytometry, and caspase activation demonstrated that Al-malt induced apoptosis in SH-SY5Y cells in a concentration-dependent manner. As we found from above experiments that treatment of SH-SY5Y cells with Al-malt for 3 days at concentrations greater than 0.1 mM caused significant apoptosis, then we chose Al-malt concentrations ranged from 0.1 –0.5 mM, which were higher than that of human exposure, in the following mechanistic investigation conducted in SH-SY5Y cells with 3 days treatment. Nevertheless, the doses of Al-malt relevant to human exposure (average daily Al-malt intakes ranged from 1.73–15.77 mg/kg body weight) were used in the long-term rat experiment in this study to further examine the apoptotic action and the underlying mechanism of Al-malt.

Al-malt-induced apoptosis is associated with miR-19 down-regulation and alteration of miR-19-targeted PTEN/AKT/p53 pathway

miR-19 is a key miRNA implicated in regulating cell survival process. To determine if miR-19 is involved in Al-malt induced apoptosis of SH-SY5Y cells, we examined the expression of miR-19a and miR-19b in cells exposed to Al-malt. As shown in Fig. 2A, Al-malt treatment significantly decreased the expression levels of both miR-19a and miR-19b in SH-SY5Y cells.

miR-19 exerts its regulatory function by targeting critical cell survival-related genes such as PTEN, a gene that inhibits the activity of the PI3K-Akt signaling pathway and subsequently leads to activation of the transcription factor p53, which in turn regulates the expression of apoptosis-related genes such as Bax and Bcl-2. We then examined the alterations in the expression of miR-19-associated cell apoptosis proteins, including PTEN, AKT, p53, Bax, and Bcl-2, in SH-SY5Y cells treated with Al-malt. Our results indicated that, consistent with the reduction of miR-19, Al-malt upregulated the expression of PTEN, along with decreased levels of phosphorylated (p)-AKT, increased p53 and Bax, and decreased Bcl-2 (Fig. 2B). These data suggested the downregulation of miR-19 and alteration of miR-19-targeted PTEN/AKT/p53 pathway in Al-malt induced apoptosis in SH-SY5Y cells.

miR-19 overexpression ameliorates Al-malt-induced apoptosis of SH-SY5Y cells

To determine the role of miR-19 in Al-malt induced apoptosis, SH-SY5Y cells were transfected with miR-19a and miR-19b mimics or control mimic. After transfection, SH-SY5Y cells were treated with Al-malt (0.25 mM) for 3 days and cell viability was measured. As shown in Fig. 3A, overexpression of miR-19a and miR-19b significantly improved the viability of SH-SY5Y cells treated with Al-malt (p < 0.05). Flow cytometry analysis further revealed the protective effect of miR-19 in Al-malt induced apoptosis of SH-SY5Y cells. The percentages of apoptotic cells were reduced from 18.1% in cells treated with Al-malt alone to 9.8% in cells treated with Al-malt and transfected with miR-19a mimic (p < 0.01), and to 9.5% in cells treated with Al-malt and transfected with miR-19b mimic (p < 0.01), respectively (Fig. 3B). Western blotting also showed that the activation of caspase-9 and caspase-3 triggered by Al-malt was suppressed with miR-19a/miR-19b mimics in the cells (Fig. 3C).

In addition, the effect of miR-19 mimic in modulating miR-19-targeted PTEN/AKT/p53 pathway was examined. We found that ectopic expression of miR-19a ameliorated the spectrum of effects associated with Al-malt treatment in the expression of PTEN, p-AKT, p53, Bax, and Bcl-2 (Fig. 4A). Similar results were also observed with miR-19b mimic (Fig. 4B). Together, these data revealed the important role of miR-19 in Al-malt induced apoptosis in SH-SY5Y cells.

Al-malt treatment increases Al contents in serum and brain tissues of rats

Following the illustration of the regulatory function of miR-19 in Al-malt induced apoptosis in vitro, we further investigated whether Al-malt induces neural cell apoptosis and modulates miR-19 in an animal model. Sprague Dawley rats were exposed to Al-malt (20, 100, and 200 mg/L Al) for 12 weeks and the effects of Al-malt on body weight, food and water consumption, Al intake, as well as Al contents in serum and brain tissues were determined. As shown in Fig. 5, there was no difference in terms of body weight with Al-malt treatment. Water consumption was slightly decreased in rats treated with 200 mg/L Al, and similar change was observed in food consumption. The average daily Al intakes for rats in the groups of 20, 100, and 200 mg/L Al were 1.73, 8.49, and 15.77 mg/kg body weight, respectively. Our results revealed that Al-malt exposure increased Al concentrations both in serum and brain tissues in a dose-dependent manner. These data suggested the bioavailibity of Al from Al-malt complex and the entry of Al into the braintissues.

Al-malt induces apoptosis in brain tissue of rats

In order to determine the potential of Al-malt treatment in the induction of neural cell apoptosis in vivo, rat brain tissues were isolated and used for the analyses of western blotting, immunohistochemistry and TUNEL assays after 12 weeks Al exposure. We found that the expression levels of cleaved caspase 9 and cleaved caspase 3, the indicators of caspase 9 and caspase 3 activation, were upregulated in rat brain by Al-malt (Fig. 6A). Moreover, fluorometric TUNEL assay revealed the intensity of green fluorescence in rat hippocampus tissues were dose-dependently increased in Al-malt treated groups with regard to the control group, indicating Al-malt triggered apoptosis (Fig. 6B). These results demonstrated the induction of neural cell apoptosis in rats by Al-malt.

Al-malt reduces miR-19 expression and alters PTEN/AKT/p53 pathway in rat brain tissues

Since above results revealed dysregulation of miR-19 in Al-malt induced apoptosis in in vitro setting, we further investigated miR-19 modulation in Al-malt induced apoptosis in rats. It was showed that the expression levels of both miR-19a and miR-19b in rat brain tissues were significantly reduced in Al-malt treated groups compared with the control group (Fig. 7A). Meanwhile, alterations in the expression of miR-19-associated cell apoptosis proteins were also examined. Western blot analyses indicated that, in agreement with miR-19 downregulation, Al-malt treatment resulted in upregulated expression of PTEN, decreased phosphorylated AKT, increased p53 and Bax, and decreased Bcl-2 (Fig. 7B). Moreover, immunohistochemistry analysis further illustrated that the expression of PTEN in hippocampus of rats treated with Al-malt was significantly increased when compared with the control group (Fig. 7C). These data suggested that consistent with in vitro study, Al-malt reduced miR-19 expression and altered miR-19-targeted PTEN/AKT/p53 pathway in rat brain tissues.

DISCUSSION

Neuronal cell apoptosis is an important feature of neurodegeneration [37, 38]. Environmental risk factors are involved in the etiology and pathogenesis of neurodegenerative diseases. As a well-known neurotoxin, Al has been associated with neurodegenerative disease [39, 40]. In the present study we investigated the molecular mechanisms of neural cell apoptosis induced by Al-malt, a lipophilic complex relevant to human health, in human neuroblastoma SH-SY5Y cells and rat brain tissues. Our data showed that SH-SY5Y cells treated with relative high concentrations of Al-malt for 3 days resulted in significant apoptotic changes, and that Al-malt treatment of rats for 12 weeks at doses relevant to human exposure induced apoptosis in rat brain tissues in a dose-dependent manner. Moreover, we revealed for the first time that miR-19 modulation plays crucial role in the regulatory action of Al induced neural cell apoptosis. This finding could provide new insight into the molecular mechanism by which Al exerts its neurotoxic effects. Mounting evidence has illustrated the induction of neural cell apoptosis by Al, including Al-malt. Since maltolate is a common component of human diet with high affinity for Al and the ability to facilitate the entry of Al into the brain, Al-malt is advantageous for use in the investigation of Al neurotoxicity [12 –18]. Previous studies have shown the effect of Al-malt on neural cell apoptosis. Griffioen et al. reported that Al-malt (0.25–0.5 mM) induced cell death of NT2 cells with 48 h culture [41]. Chen et al. also shown that treatment of SH-SY5Y cells with Al-malt (0.4 and 0.8 mM) for 24 h decreased the cell viability [42]. In agreement with previous studies, we showed in the present study that Al-malt treatment for 3 days at concentrations greater than 0.1 mM caused apoptosis in human SH-SY5Y cells. Our data from cell viability, Hoechst 33258 staining, flow cytometry analysis and caspase activation demonstrated the concentration-dependent induction of apoptosis in SH-SY5Y cells by Al-malt treatment. Moreover, the neurotoxic effect of Al-malt has also been determined in animal studies. Huh et al. found that exposure of rats to Al-malt (100 μmol/L) via drinking water for one year increased caspase activation and induced apoptosis in rat brain [43]. Kaneko et al. showed that mice given drinking water containing Al-malt (1 g/L Al) for 30, 60, and 90 days exhibited oxidative stress and nervous degeneration in brain tissues [44]. In our animal study, we further revealed that exposure of rats to Al-malt (20, 100, 200 mg/L Al) for 12 weeks significantly elevated Al concentrations in serum and brain tissues. Al-malt treatment induced apoptosis in rat brain, as evidenced by increased expression of cleaved caspase 9 and cleaved caspase 3, as well as increased intensity of TUNEL staining. These results suggested the bioavailibity and the potential of Al-malt to enter the brain and to induce apoptosis in vivo. Notably, Al-malt concentrations used for the investigation of the apoptotic effects as well as the molecular mechanism in the 3-days experiments carried out in SH-SY5Y cells were higher than that of human exposure. Nevertheless, Al-malt doses relevant to human exposure were chosen in the long-term rat experiment in this study, which further verified the apoptotic action and the underlying mechanism of chronic Al exposure.

miR-19 is a key component of the miR-17–92 cluster implicated in regulating cell survival process. It has been reported that miR-19 is differently expressed in PD, spinocerebellar ataxia type 1, and some other neurodegenerative diseases [21 , 31]. miR-19 regulates the expression level of ATXN1 and modulates the pathogenesis of SCA1 [21]. miR-19 affects the pathogenesis of PD through regulating PD-related genes PARK2 and PARK8 [22]. However, the function of miR-19 in AD has not been elucidated yet. To determine if miR-19 is involved in Al-malt induced neural cell apoptosis, we examined the expression of miR-19a and miR-19b in SH-SY5Y cells exposed to Al-malt. It was revealed that Al-malt downregulated the expression levels of both miR-19a and miR-19b. The expression of PTEN, a well-known miR-19-targeted gene critical for apoptosis regulation, was upregulated accordingly, along with the alterations of its downstream apoptosis related proteins including AKT, p53, Bax, and Bcl-2. In order to illustrate the role of miR-19 in Al-malt induced apoptosis, SH-SY5Y cells were transfected with miR-19a and miR-19b mimics. We showed that miR-19 overexpression attenuated Al-malt-induced apoptosis of SH-SY5Y cells; Al-malt-mediated changes in the expression of PTEN, p-AKT, p53, Bax, and Bcl-2 were also ameliorated by miR-19 overexpression. These data revealed the important role of miR-19 in Al-malt induced apoptosis in SH-SY5Y cells. Consistent with in vitro study, Al-malt treatment reduced miR-19 expression and altered PTEN/AKT/p53 pathway in rat brain tissues. These results further confirmed miR-19 modulation in Al-malt induced neural cell apoptosis.

The finding from this study is relevant to the chronic Al neurotoxicity in humans. Although Al-malt concentrations used in the short-time exposure in vitro experiments were high, we found that treatment of rats with Al-malt at doses relevant to human exposure for 12 weeks induced neural cell apoptosis in rat brain tissues, an effect that was dose-dependent. It was shown the average daily Al intake for rats in the low-dose group (20 mg/L Al) was 1.73 mg/kg bw, equivalent to 12.11 mg/kg bw/week; low-dose Al-malt treatment resulted in elevated brain Al concentration, increased caspase activation and apoptotic cells, reduced miR-19 expression and altered expression of miR-19-associated apoptosis related proteins in the brain of rats. As a matter of fact, exposure of humans to Al is ubiquitous and extensive [45]. A provisional tolerable weekly intake (PTWI) of 1 mg/kg bw for Al has been established by the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives (JECFA) [46]. European Food Safety Authority (EFSA) reported that mean Al exposures in European countries were 0.2-1.5 mg/kg bw/week [47]. Health Canada reported that mean Al exposure in Canada was 1.211 mg/kg bw/week [48]. Al exposure in Spanish population was 1.2 mg/kg bw/week [49]. In urban residents from cities in South China, the average dietary exposure to Al was 1.5 mg/kg bw/week; while the high-level consumers’ exposure to Al was 11.1 mg kg bw/week, which was much higher than PTWI; the foods contributing most to Al exposure were processed food with Al-containing food additives [50]. It is noteworthy that the Al intake level in the high-level exposure populations in South China (11.1 mg kg bw/week) is similar to that of rats in the low-dose group with 12 weeks treatment in the present study (12.11 mg/kg bw/week). Considering the much smaller relative body surface area and much longer (even life-long) exposure period of time in humans in comparison to the study animals, those populations with high-level Al intake might be at risk of Al-associated neurotoxicity.

In conclusion, the present study demonstrates for the first time that miR-19 modulation is critically involved in Al-induced neural cell apoptosis. Findings from this study could provide new insight into the molecular mechanisms of Al-associated neurodegenerative pathogenesis.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science Foundation of China (No. 81072330, No. 81373005, No. 81202194), and by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Authors’ disclosures available online ().

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