Cell culture and OA treatment

SHSY5Y cells (human neuroblastoma cell line) were obtained from the European Collection of Cell Cultures and cultured in nutrient mixture EMEM/F12 (1:1) medium with 1% non-essential aminoacids, 1% antibiotic and antimycotic solution and supplemented with 10% heat-inactivated foetal bovine serum, all from Invitrogen (Barcelona, Spain). The cells were incubated in a humidified atmosphere with 5% CO₂ at 37°C. OA (CAS No. 78111-17-8) was purchased from Sigma-Aldrich Co. (Madrid, Spain) and dissolved in DMSO prior use.

Flasks with approximately 90% of confluence and 4 × 10⁶ cells were chosen for the experiments. For the treatments, cells were incubated at 37°C for 3, 24 or 48 h in the presence of OA (100 nM) or the control dimethylsulfoxide (DMSO) at 1% of final volume.

Total RNA isolation and cDNA synthesis for SSH

After OA treatments, total RNA was isolated from SHSY5Y cells with TRIZOL^® reagent (Invitrogen, Madrid, Spain) following the manufacturer's instructions, and then dissolved in nuclease-free water. RNA was quantified and quality checked using the NANODROP™ 1000 spectrophotometer (Thermo Scientific, Madrid, Spain). One microgram of total RNA from each sample was used as template to synthesize the first strand cDNA with the SMART™ PCR cDNA Synthesis Kit (Clontech, Madrid, Spain), a method for producing high quality cDNA from a low amount of starting material [29]. The double stranded cDNA was amplified with the same Kit according to the manufacturer's protocol.

Construction of subtracted cDNA libraries

SSH was carried out with the PCR-Select™ cDNA subtraction kit (Clontech, Madrid, Spain), as described by the manufacturer. Briefly, the double stranded cDNAs obtained from the step described above were digested with the restriction enzyme Rsa I to obtain blunt-ends that are necessary for adaptor ligation. cDNA subtraction was carried out in two directions for the different exposure times. The forward subtracted libraries were made with the control cells cultured for 3, 24 or 48 h as the driver and OA-exposed cells (also cultured for 3, 24 or 48 h) as the tester. These forward reaction libraries were designed to produce clones that are overexpressed or up-regulated in OA-treated cells. The reverse libraries were made in the same way, but in this case the adapter ligated cDNA from OA-exposed cells were the driver and control cells were the tester. The reverse reaction library was designed to produce clones underexpressed or down-regulated in OA-treated cells. In either case the driver cDNA was added in excess during each hybridization to remove common cDNAs by hybrid selection and leaving overexpressed and novel tester cDNA to be recovered and cloned. The subtracted cDNA fragments were then inserted into yT&A^® cloning vector, transformed into Escherichia coli ECOS707 (strain JM109) competent cells, and plated on LB agar plates containing 100 μg/ml ampicillin, 100 μl IPTG (100 mM) and 20 μl X-gal (50 mg/ml). The yT&A^® cloning vector and the E. coli ECOS707 competent cells were purchased from Yeastern Biotech. Co., Ltd., (Taipei, Taiwan). From the six libraries, a total of 384 white recombinant colonies (corresponding to four 96-well plates) were picked.

Sequencing of the subtracted cDNA clones and bioinformatics analysis

Sequencing of all the cDNA clones from the six SSH libraries was carried out using the BigDye^® Terminator v3.1 (Applied Biosystems) and an AB3730 sequencer (Applied Biosystems) at Secugen (Madrid, Spain). After excluding redundant and false-positive sequences, nucleic acid homology searches were performed against nucleotide databases at the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLASTX and BLASTN) http://www.ncbi.nlm.nih.gov/BLAST to provide gene annotation. Homologies that showed identities over 60% and E-values of less than 1 × E^-10 with more than 100 nucleotides were considered to be significant. The differentially expressed genes identified through expression analysis were classified according to the definition of Gene Ontology (GO) http://www.geneontology.org/ related to the aspects of biological and molecular function.

Differential screening of the subtracted libraries

With the aim of checking the level of background corresponding to common mRNAs in reverse and forward libraries we carried out a differential screening of subtracted libraries using the PCR select differentially screening kit (Clontech, Madrid, Spain), following the manufacturer's instructions. Briefly, PCR products from positive colonies were immobilized in nylon membranes and hybridized with forward- and reverse- probes. Those clones representing mRNAs truly differentially expressed should hybridize only with its corresponding forward-probe. Prior to hybridization forward- and reverse-probes were digested for removing adaptors. More than 90% of the clones tested resulted positive for the virtual Northern analysis (data not shown).

Simple gene set enrichment analysis

A simple gene set enrichment analysis was performed using FatiGO tool (Babelomics 4.2 suite, http://babelomics.bioinfo.cipf.es/). FatiGO takes two lists of genes and convert them into two lists of GO annotations. Then a Fisher's exact test for 2 × 2 contingency tables is used to check for significant over-representation of GO annotations in one of the sets with respect to the other one. Multiple test correction is applied as a measure of control for false positives. In our case, we conducted two single gene set enrichment analysis for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways comparing our set genes from forward and reverse libraries with the rest of annotations in human genome (additional files 1 and 2).

Quantitative PCR

Statistical analysis

Experimental data were expressed as mean ± standard error. Statistical analyses between groups were carried out using Student's t-test and a P-value of < 0.05 was considered significant. Statistical analysis was performed using the SPSS for Windows statistical package (version 16.0).

Identification of genes with different transcript levels in OA-exposed SHSY5Y cells

These characterized genes were associated with various functions including metabolism, signal transduction, and cytoskeleton and cell adhesion. The genes altered after the 3 h OA treatment were related to electron transport chain and redox homeostasis, signal transduction, metabolism, transcription, translation, cell cycle and apoptosis, and cytoskeleton and cell adhesion (Figure 1). Most of these genes are apparently involved in metabolism including electron transport chain and redox homeostasis. A few studies have previously reported the OA effects on the cell metabolism. Cable et al.[38] observed that OA affected the heme metabolism of human hepatic cell lines. Also, Shisheva and Shechter [39] showed that OA mimicked some of insulin bioeffects stimulating the glucose and lipid metabolism in rat adipocytes, and Tanti et al.[40] found that glycolysis was stimulated and glucose transport was increased after OA treatment in mouse skeletal muscle. More recently, another study showed that OA depressed the metabolic rate of rat hepatocytes and changed glucose uptake in these cells [41]. Related to electron transport chain, OA was previously found to induce alterations in mitochondrial membrane potential [42] and increased oxidative stress in the rat brain after intracerebroventricular injection [43], and in different cell types in vitro[10, 44]. The altered expression levels in genes related to cell metabolism and electron transport chain found in this study could help to explain the effects described in all these works. Besides, 8% of the genes altered after the 3 h OA treatment were related to cellular transport processes. OA was previously found to interfere in the secretion of newly synthesized proteins and exocytosis in rats [45]; both effects could be related to the expression alterations found in the present study.

When cells were treated with OA for 24 h, the obtained genes were also categorized into different groups including translation, signal transduction, electron transport chain and redox homeostasis, metabolism, cell cycle and apoptosis, transcription and nuclear specific proteins, transport, and cytoskeleton and cell adhesion (Figure 2). Similar to the 3 h OA treatment, an important number of these genes are involved in metabolism including electron transport chain, but also a great percentage of genes related to translation were observed. The expression alterations found in the genes involved in processes of translation and transcription might be related to the previously reported OA-induced inhibition of protein synthesis [45, 46].

Among the genes altered after the 48 h OA treatment, most were related to signal transduction, translation, cell cycle and apoptosis, electron transport chain and redox homeostasis, metabolism, cytoskeleton and cell adhesion, transcription and nuclear specific proteins, and transport (Figure 3). Fewer genes related to metabolism and transcription were found altered at 48 h, but similarly to 24 h an important percentage of altered genes are involved in cell cycle and apoptosis. In another previous study, the gene expression alterations in mouse BALB/c3T3 cells after different OA treatment times (from 4 h to 24 days, 7.8 ng/ml) were evaluated by microarray analysis, and a total of 177 differentially expressed genes were identified [47]. The authors focused this study on the 31 genes found to be functionally involved in cell growth and/or maintenance, and observed that numerous genes associated with cell proliferation and cell cycle progression were down-regulated after OA treatment. Several genes related to apoptotic processes, some of them involved in the mitochondrial pathway of apoptosis, were also found to be altered. On the basis of their results, they concluded that multiple molecular pathways could be involved in OA-induced proliferation inhibition and apoptosis in these cells [47].

Two simple gene set enrichment analysis were performed using FatiGO tool to find which cellular KEGG pathways could be affected by OA exposure in SHSY5Y cells. The results obtained for the forward libraries revealed a total of 3 KEGG pathways altered: oocyte meiosis (hsa04114, adjusted p-value = 0.003), Parkinson's disease (hsa05012, adjusted p-value = 0.035), and cell cycle (hsa04110, adjusted p-value = 0.003). The genes corresponding to reverse libraries were significantly associated with KEGG pathways related to: glycolysis (hsa00010, adjusted p-value = 0.044), oxidative phosphorylation (hsa00190, adjusted p-value = 0.012), Vibrio cholerae infection (hsa05110, adjusted p-value = 0.044), pathogenic Escherichia coli infection (hsa05130, adjusted p-value = 0.044), Alzheimer's disease (hsa05010, adjusted p-value = 0.022), and ribosome (hsa03010, adjusted p-value < 0.001). Since most effects of OA come from binding to PP1 and PP2A, a possible explanation for the altered pathways could be the protein phosphatases inhibition induced by this toxin. In fact, inhibition of PP2A by OA has been previously demonstrated to increase tau phosphorylation, a pathological hallmark of Alzheimer's disease, in SHSY5Y cells [48].

NEFM, TUBB2A and SEPT7 expression: OA effects on neuronal cytoskeleton

The key role of cytoskeletal organization in several important neural processes such as neurite outgrowth [49], synaptogenesis [50], structural polarity and neuronal shape [51], axonal transport [52], and neurotransmitter release [53] has been characterized. Cell shape and structural polarity are lost in neurodegenerative diseases and neural aging [54, 55].

OA was previously reported to induce several cytoskeleton alterations in different cell systems [56, 57]. It has been hypothesized that these alterations could be due to different mechanisms that involve disruption of F-actin and ⁄or hyperphosphorylation and activation of kinases that stimulate tight junction disassembly, but the exact molecular mechanism has not been elucidated yet [3].

The cytoskeleton is made up of three kinds of protein filaments: actin filaments (also called microfilaments), intermediate filaments (IF) and microtubules, and other associated proteins. NEFM is the human gene which encodes for the neurofilament medium polypeptide. Neurofilaments (NEFs) are a neuron-specific type of intermediate filament proteins (10 nm in diameter) found at especially high concentrations along the axons, where they regulate axonal diameter. NEFs consist of three proteins according to their molecular weight: light neurofilament (NEFL), medium neurofilament (NEFM), and heavy neurofilament (NEFH) [58]. NEFM gene is often employed as a marker of neuronal differentiation [59]. NEF protein levels are correlative to neurite outgrowth, and its gene expression is dramatically altered in neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease [60]. NEF protein levels have also been suggested as a potential biomarker in organophosphorous neurotoxicity [36]. Furthermore, neurite outgrowth can be promoted by nerve growth factor (NGF) via the regulation of NEF gene expression and NEF protein phosphorylation [61]. In the present study a statistically significant underexpression of this gene was found after 3 h OA treatment, but an overexpression was observed after 24 h, and no effects after 48 h, suggesting that OA deregulates NEFM expression at the short term (within at least the first 24 h), but then it stabilizes and return to control levels. It was previously described that tight coordination of the expression of neurofilament subunits is integral to the normal development and function of the nervous system. Imbalances in their expression are implicated in the induction of neurodegeneration in which formation of neurofilamentous aggregates is central to the pathology [62]. To our knowledge, no studies were reported before on the expression of this gene in human neuronal cells exposed to OA; nevertheless incubation of human fibroblasts [63] or rat brain tumour cells [64] with OA promoted the hyperphosphorylation of major intermediate filaments (IF) proteins, leading to the disassembly of IF networks, solubilisation of IF proteins, and disruption of desmosomes.

Microtubules are involved in many cellular functions, including mitosis, intracellular transport, determination of cell morphology, and differentiation [65]. In neurons, microtubules participate actively in the initial steps of neuronal polarization, the organization of intracellular compartments, the modelling of dendritic spines and the trafficking of cargo molecules to pre-, post- or extrasynaptic domains [66]. Tubulin, the subunit protein of microtubules, is a heterodimer, with both α- and β-tubulin isotypes, differing from each other in amino acid sequence [67]. In our study, the expression levels of TUBB2A, the gene encoding for the tubulin β chain 2A, were analyzed in OA-treated neuronal cells. Data obtained from the real-time PCR analysis showed that this gene is down-regulated in OA-treated SHSY5Y cells at 3 h and 24 h, significantly only in the second case, but at 48 h its expression levels were not different from the control. Microtubules were found to be altered after OA exposure in some previous studies mainly due to the hyperphosphorilation of tau, a microtubule-associated protein which promotes microtubule enlargement. Inhibition of PP2A activity by OA was suggested to produce the abnormal tau hyperphosphorylation in vivo after hippocampal injection in rats [68] and in vitro in metabolically competent brain rats [69], in mouse hippocampal HT22 cell line [70] and in human neuronal NT2N and SHSY5Y cells [48, 71]. Besides, Yano et al.[56] found that OA induces reorganization of microtubules in human platelets via the phosphorylation of a microtubule-associated 90 kDa protein, and Benitez-King et al.[72] showed that OA produces cytoskeletal disorganization and microtubule disruption in N1E-115 neuroblastoma cells, as described in other neuronal cell culture models and in rat brain [20, 73, 74]. TUBB2A was characterized primarily as a neuronal β-tubulin isotype [75] and possess a high expression level in brain, peripheral nerves and muscles [76]. Tubulin isotype composition may be a determinant factor on microtubule functions. Therefore, changes in expression levels of tubulin subtypes would alter the microtubule dynamics. In this sense, Falconer et al.[77] demonstrated that TUBB2 is preferentially incorporated into stable microtubules during neuronal differentiation, and Hoffman and Cleveland [78] reported that the isotype TUBB2 is polymerized more efficiently than other isotypes. The higher expression observed in several types of tumours [79] and in cancer cells resistant to microtubule-binding drugs [80] could be related with the more stability of TUBB2A isotype. The underexpression of TUBB2A observed in this work might contribute to cytoskeletal disruption effects of OA in a similar way, since the major isotype in neuronal cells and the more would be incorporated in a lesser extent to microtubules of SHSY5Y OA-exposed cells.

Septins are an evolutionarily conserved family of cytoskeleton GTP-binding proteins [81]. They play putative roles in cytokinesis, cellular morphogenesis, polarity determination, vesicle trafficking and apoptosis [82–85]. Septins have been identified in all eukaryotic cells. Although yeast septins are better understood, the function of mammalian septins remains largely undefined [86]. SEPT7 is a member of the septin family which is abundantly expressed in the central nervous system [81], but its functional role has not been reported yet [87]. However, a previous study showed that SEPT7 is critical for spine morphogenesis and dendrite development during neuronal maturation [87] and other study confirmed that SEPT7 directly interacts with CENP-E via the C-terminal coiled-coil region. This SEPT7-CENP-E interaction is critical for a stable CENP-E localization to the kinetochore and for achieving chromosome alignment at the equator [88]. SEPT7 has been also related to oncogenesis. After investigating SEPT7 expression in a large number of human glioma tissue samples, Jia et al.[86] found that the expression of this gene was generally down-regulated in gliomas or even absent in some high-grade tumours. Furthermore, they showed that SEPT7 induces cell apoptosis through down-regulation of Bcl-2 and up-regulation of caspase-3, and increased cell apoptosis also contributes to the inhibitory effect of SEPT7 on glioma cell growth. Other studies showed that SEPT7 was much less expressed in brain tumours than in normal brain tissues [81, 89] and that neuroblastoma patients with higher SEPT7 mRNA expression might have better prognosis [90]. In our study a significant decrease in the SEPT7 expression levels were found at 3 h, however no differences with regard to the control cells were observed after 24 or 48 h OA treatment.

Therefore, the results obtained from the genes related to cytoskeleton evaluated in this study suggest that the cytoskeleton disruption induced by OA described in previous works are due not only to the hyperphosphorylation of specific proteins caused by phosphatases inhibition, but also to short term alterations (mainly down-regulation) in the expression levels of relevant genes involved in the maintenance of the cell structure as the TUBB2A, NEFM or SEPT7 genes. The fact that no effects of OA were observed after 48 h treatment in any of these genes could be related to cells ability to recover and return to their normal expression levels. However, cell viability was enormously reduced after 48 h OA treatment (microscopic observations). Thus, the absence of gene expression alterations found at that time might also be due to the fact that those cells intensely altered by OA in their gene expression at 3 and 24 h treatments underwent apoptosis or necrosis, being absent at 48 h.

SYT4 and NPY expression: OA effects on synaptic neurotransmission

Synaptic neurotransmission is one of the most highly regulated of all vesicle trafficking events. Although many of the molecular components of synaptic vesicles, the presynaptic cytosol, and presynaptic plasma membrane have been identified, the mechanisms by which these components regulate stimulus evoked vesicle fusion and recycling are not completely understood yet. In this study, expression levels of two genes related to the neuronal signal transduction (SYT4 and NPY) after OA exposure were investigated.

The synaptotagmins (SYTs) are a family of proteins characterized by a short luminal NH₂ terminus, one transmembrane region, and tandem C2A and C2B domains [91]. These synaptic proteins are also important in depolarization-induced, Ca²⁺-dependent fusion of the synaptic vesicles and presynaptic membrane [92]. Currently, it is thought that SYTs participate in the regulation of various steps during membrane fusion, primarily at the plasma membrane [93]. There are at least 17 SYTs isoforms that have the potential to act as modulators of membrane fusion events. SYT4 is particularly interesting since it has been found to be potentially involved in a wide variety of activities in the brain [94]; it is an immediate early gene that is up-regulated following neuronal depolarization [95] and maps to a region of human chromosome 18 associated with schizophrenia and bipolar disease [96]. Data obtained from real-time PCR showed that the SYT4 expression is inhibited by OA at 3 and 24 h exposure, but it recovers normal levels at 48 h treatment. In a previous study, loss of SYT4 results in a reduction of synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons [94]. Golgi disruptions were also found in rat pancreatic cells after OA exposure [45]. Besides, SYT4 affects a number of vesicle recycling properties in peptidergic nerve terminals in the posterior pituitary [97]. Interestingly, SYT4 also appears to play a role in the maturation of secretory granules in neuroendocrine cells [93], suggesting that it may also function in the movement of vesicles [91].

Neuropeptide Y (NPY) is a 36-amino acid peptide produced by neurons throughout the brain and by other secretor cells of the body. NPY has been associated with a number of physiological processes in the brain, including the regulation of energy balance, memory and learning, and epilepsy [98]. Similarly to SYT4, NPY expression levels after OA exposure were found to be down-regulated at 3 and 24 h, but expression levels similar to control were observed at 48 h. A deregulation of the hypothalamic NPY system has been proposed to be related to several pathological and pathophysiological states including cancer cachexia [99], hyperinsulinemia and hypercorticism [100], obesity and metabolic syndrome [101], and anorexia [102].

So far, no studies on SYT4 or NYP expression after OA exposure were reported, but several previous studies described neurotransmission alterations after OA exposure, and the down-regulation of genes involved in the synaptic processes found in this study could help to explain them. OA was found to inhibit mobilization of synaptic vesicles and depress Ca²⁺ release from sarcoplasmic reticulum in mouse neuromuscular junctions [103], to disrupt synaptic vesicle trafficking in goldfish bipolar cells [104], and to interfere with the formation of synaptic vesicle clusters in nerve terminals of frog neuromuscular junctions [105]. In vivo, OA significantly reduces electrically induced inhibitory non-adrenergic, non-cholinergic (NANC) neurotransmission responses in the rat gastric fundus, while leaving direct muscular effects of the inhibitory NANC neurotransmitters vasoactive intestinal peptide and nitric oxide unaffected, suggesting a neural site of action [106]. It was also reported that presynaptic clusters of synaptic vesicles at the frog neuromuscular junction can be disrupted by exposure to OA [105]. Furthermore, our data involving genes related to neurotransmission could also underlie the OA effects previously reported on the rodent nervous system in vivo such as hyperexcitation [107], spatial memory deficit and neurodegeneration [22] and cognitive deficits [48]. Similarly to the results obtained for the cytoskeleton genes expression, the expression levels of both SYT4 and NPY were highly depressed at 3 and 24 h OA exposure, but they went back to basal levels after 48 h, suggesting that surviving cells were able to recover from OA-induced gene expression alterations.