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Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor
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Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor E. Vergara-Castan˜edaa, D.R. Grattanb, H. Pasantes-Moralesc, M. Pe´rez-Domı´nguezc, E.A. Cabrera-Reyesa, T. Moralesd, M. Cerbo´na,n a
Unidad de Investigación en Reproducción Humana, Instituto Nacional de Perinatología-Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico b Center for Neuroendocrinology and Department of Anatomy, University of Otago, New Zealand c Instituto de Fisiología Celular, Mexico d Instituto de Neurobiología, UNAM, Mexico
art i cle i nfo
ab st rac t
Article history:
Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against
Accepted 4 February 2016
excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to
Keywords:
assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model
Prolactin
using primary cell cultures of hippocampal neurons, and to determine whether this effect
Excitotoxicity
is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal
Hippocampus
neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and
Neuroprotection
protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu) – induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures. & 2016 Published by Elsevier B.V.
n
Corresponding author. E-mail address: [email protected] (M. Cerbón).
http://dx.doi.org/10.1016/j.brainres.2016.02.011 0006-8993/& 2016 Published by Elsevier B.V.
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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1.
Introduction
Prolactin (PRL) is a polypeptide hormone produced mainly in the anterior portion of pituitary gland, by the lactotrophs. While there is some evidence that it could also be synthetized in extra-pituitary sites, including some areas of the brain (Clapp et al., 1994; DeVito et al., 1991; Mejía et al., 1997; Torner et al., 2004), this still controversial (Grattan and Kokay, 2008; Kanyicska et al., 2000). PRL secretion is mainly inhibited by hypothalamus and it can be stimulated by suckling. PRL displays several functions in the Central Nervous System (CNS), including the regulation of its own secretion through stimulation of TIDA neurons, stimulation of maternal behavior (Bridges and Mann, 1994; Bridges, 2014), suppression of fertility (Bouilly et al., 2012), and regulation of oxytocin neurons (Vega et al., 2010). Also, it has been described that PRL has several neuroprotective effects, either my stimulating neurogenesis and/or promoting survival, among which are: decreases neuroendocrine and behavioral responses to stress stimulus (Donner et al., 2007; Saltzman and Maestripieri, 2011), anxiolytic effects in male rats (Torner et al., 2001), protects hippocampal neurogenesis in the dentate gyrus of chronically stressed mice (Torner et al., 2009), induces neurogenesis in the sub ventricular zone in both in vivo and in vitro models (Gregg et al., 2007; Mohammad et al., 2002; Shingo et al., 2003), and enhances white matter repair and remyelination (Gregg et al., 2007). The prolactin receptor (PRLR) is a class 1 receptor from the cytokine super family; it is composed of three domains: extracellular, transmembranal and intracellular (Bole-Feysot et al., 1998). Several PRLR isoforms have been described, generated by alternative splicing, proteolysis, intron retention, alternative sites for the end of transcription and partial exon suppression (Brooks, 2012; Ding and Wu, 2010). It is well documented that adult rodent brains express short and long PRLR isoforms (Tejadilla et al., 2010; Torner et al., 2009). In all isoforms, the extracellular domain is identical, but the downstream signal transduction activated by each isoform is different. The long form of the prolactin receptor is required for full activation of the prolactin receptor, including the well documents JAK/STAT5 pathway, while the short isoform can activate the MAP kinase pathway and induce mitogenic responses in some cell types. Recent studies showed that the dorsal hippocampus of the rat is protected during lactation against excitotoxic damage Q4 induced by Kainic Acid (KA) (Cabrera et al., 2009, Vanoye-Carlo et al., 2008). The resistance to cell damage in hippocampal areas occurred either following peripheral or intracerebral administration of KA (Morales, 2011), suggesting that the brain adaptation during lactation modifies the sensitivity of this neural tissue to protect against excitotoxic insults. PRL is markedly elevated during lactation, and previous work has demonstrated that PRL exerts neuroprotective effects in the hippocampus in different experimental models (Franssen et al., 2012; Torner et al., 2009). Treatment with PRL, either through systemic or intracerebral administration, protects against the toxic effects of KA or reduces the epileptic like behavior induced by this glutamate agonist, and this effect is independent of ovarian hormones (Tejadilla et al., 2010; Vanoye-Carlo et al., 2008). Hence, PRL may mediate the neuroprotective effect seen during lactation.
In the present study, by using an in vitro model of primary culture of rat hippocampal cells we evaluated the neuroprotective role of PRL against glutamate, to test the hypothesis that the lactation-induced neuroprotective effect might be mediated by the hyperprolactinemia present at that time. We also examined the expression of PRLR in the cultures, to determine whether PRL actions might be mediated by its cognate receptor.
2.
Results
2.1. PRL did not modify cell neuronal viability in primary cell cultures Prolactin effect on neuronal cell viability was evaluated by MTT assay. The results indicated that PRL did not modify cell viability at any dose used (1–25 ng/mL). The prolactin effect was measured 72 h after hormonal treatment; the results are depicted in Fig. 1A.
2.2. PRL protects hippocampal cells against excitotoxicity induced by glutamate PRL treatment had a preventive effect on glutamate-induced cell death compared with the glutamate-treated group. However, not all the PRL doses significantly prevented cell death induced by Glu, with the optimal dose being 10 ng/ml (po0.01) (Fig. 1B).
2.3. PRLR mRNA expression in primary cell cultures hippocampal cells PRLR expression pattern was assessed by RT-qPCR in all treatments groups. As depicted in Fig. 2, the basal expression of PRLR was determined in the primary neuronal cell cultures. Interestingly, PRL treatment increased PRLR expression. Furthermore, when cells were treated with PRL/GLU, the PRLinduced increase in PRLR mRNA was maintained. Interestingly, PRL mRNA was also detected in these cells, but at very low levels as compared to PRLR mRNA level (data not shown).
2.4. Primary culture hippocampal cells express of prolactin receptor (PRLR) Primary cell cultures were labeled with NeuN to identify neural populations; more than 95% of cells were immunopositive for NeuN and these positive NeuN cell cultures were used in all subsequent experiments. Fig. 3A shows immunostaining with DAPI (A), NeuN (B), PRLR (C) and a merged image for these signals (D). PRLR was detected in almost 90% of the cells, predominantly in a cytoplasmic localization.
2.5.
PRLR isoforms expression on hippocampal cells
Western blot analysis of hippocampal cells revealed the expression of two isoforms of PRLR with molecular weights of 100 and 40 kDa, potentially corresponding to long and short isoforms of PRLR, respectively (see Fig. 4). Mammary gland was used as positive control for PRLR, which express variants of PRLR. A hypothalamic derived cell line from mouse (mHypoEN42 cell line, from Cell Solutions, Canada) presented a shorter
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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2.6.
3
PRLR mediates neuroprotection of hippocampal cells
To inhibit the expression of PRLR in hippocampal cells we performed series of experiments using antisense oligonucleotides against PRLR mRNA. As depicted in Fig. 5, oligonucleotides were able to almost totally block the neuroprotective effect exerted by PRL. In these experiments the expression of PRLR isoforms was assessed by Western blot analysis, and we confirmed that the expression of PRLR isoforms significantly diminished after antisense oligonucleotides treatment. The treatment with these oligonucleotides did not affect cell viability, but when cells were treated with Glutamate, the neuroprotective effect of PRL disappeared (Fig. 5).
3.
Fig. 1 – (A) Effect of PRL treatment on hippocampal cells viability. Cells were incubated with different prolactin doses (1– 25 ng/ml), as described in Section 4. Cell viability was assessed by the MTT method. Results are the mean7SD. (B) Protective effect of PRL against glutamate-induced excitotoxicity. Hippocampal cells were pretreated with PRL at different doses, and Glutamate 100 lM. 10 ng/mL of PRL had a significant protective effect against excitotoxic Glutamate induced cell death º(po0.01). Glutamate had a significant diminution on cell viability compared to control group *(po0.01).
Fig. 2 – PRLR mRNA expression on Hippocampal cells. Treatment with PRL and PRL/GLU induced a significant increase on PRLR mRNA expression compared with control group *(po0.01), mRNA expression data were normalized using HPRT1 as housekeeping gene. isoform with molecular weight of 30 kDa (Fig. 4). However, we could not detect the expression of PRL protein on these hippocampal cells by Western blot experiments.
Discussion
In this study, it has been demonstrated that PRL exerts neuroprotective effects against excitotoxicity in primary cultures of neural hippocampal cells. Cultured hippocampal cells express the mRNA for both PRL and its receptor; however, only the protein for the PRLR has been detected. Besides, the observation that antisense oligonucleotides induced suppression of PRLR expression in the cultured neurons blocks neuroprotection induced by PRL, suggesting that PRL exerts neuroprotection against excitotoxicity through a mechanism mediated by its cognate receptor. These data are consistent, support and extend the previous observation that lactationinduced neuroprotective effects may involve PRLR (Cabrera et al., 2013; Tejadilla et al., 2010; Vanoye-Carlo et al., 2008), furthermore, it has been reported that PRL treatment induces neuroprotection in hippocampus from excitotoxic damage (Morales, 2011; Morales et al., 2014; Tejadilla et al., 2010). In the in vivo condition, the most likely source of PRL to influence hippocampal neurons would be the anterior pituitary gland, with PRL being able to be transported from blood into the cerebrospinal fluid to act on PRLR expressed in the brain. Apart from classical PRL expression in the anterior pituitary gland, PRL expression in the brain has been detected in hypothalamic cells (Mann and Bridges, 2002; Torner et al., 2002), although the physiological role of brain PRL origin is controversial. Recently, our laboratory has detected the expression and regulation of PRL in the hippocampus of lactating rats by microarray (submitted). However, microarray was performed using hippocampal tissues from an in vivo model, having the disadvantage that brain tissue contains many cellular types, including blood cells. To explore PRL and its receptor expression in isolated neurons, in the present investigation we performed a series of experiments in neurons from primary cell cultures, establishing a highly pure population of neuronal cells without contamination by other non-neuronal cells such as glia, that are found in the whole hippocampal tissue (Fath et al., 2009; Zemlyak et al., 2007). By using specific and quantitative PCR, we detected PRL mRNA in the neuronal cultures. However, we were unable to detect the PRL protein either by Western blot analysis or immunehistochemical techniques. It is possible that the inability to detect the protein may be due to a posttranscriptional control of protein synthesis in neurons or to the low concentration in these cells.
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Fig. 3 – Representative fluorescent photomicrograph of hippocampal neurons cells. (A) DAPI, (B) NeuN, (C) PRLR, and (D) Merge NeuN/PRLR. Negative control is shown in inset top right corner (A).
Fig. 4 – Prolactin receptor isoforms in hippocampal neurons. Representative western blot of PRLR is depicted in this figure. Mammary Gland (GM) was used as positive control. Expression of short and long isoforms of PRLR on hippocampal neurons (HN) and hippocampal neurons treated with 10 ng/mL of PRL (HNþPRL). A shorter isoform was detected on a mouse hypothalamic cell line (HyN). Actin was included as loading control.
While we were unable to confirm PRL secretion form these neuronal cultures, we clearly detected the PRLR expression, both at the mRNA level by quantitative RT-PCR, and the protein, by immunoblot and immunohistochemistry. Functionally, we also documented a neuroprotective response to PRL in hippocampal cells. To our knowledge, this is the first report demonstrating the presence and functionality of PRLR in primary neuronal cultures
from the hippocampus. In vivo, in some previous studies PRLR mRNA was not found by in situ hybridization in the hippocampus in adult rats (Chiu and Wise, 1994; Bakowska and Morrell, 1997), Q5 but in contrast our data indicates that these neurons retain the ability to express this receptor under certain conditions. This observation is largely supported by several studies that have documented neuroprotective effects of PRL in vivo, particularly in males during stress conditions and during lactation (Franssen et al., 2012; Morales, 2011; Torner et al., 2009). It might be possible that the receptor is activated in response to cellular damage (Möderscheim et al., 2007). The diversity of PRLR isoforms expressed on different tissues has been related with the specific PRL function on each tissue (Brooks, 2012; Ding and Wu, 2010). Different PRLRs have been described in reproductive (Sangeeta Devi and Halperin, 2014; Stocco, 2012), endocrine system (Ferraris et al., 2012; Radl et al., 2011), cancer tissues (Clevenger et al., 2009), or brain tissues (Kokay et al., 2006; Pathipati et al., 2011). The isoforms detected by immunoblot in the primary culture of neuronal hippocampal cells are different as compared to the hypothalamic cell line and mammary gland; this finding could support the hypothesis that PRL exerts their actions throughout different PRLR variants and regulates the PRLR gene expression in the brain (Tabata et al., 2012). Apart from our work in the hippocampus, various studies about PRL effects in this brain area described the neuroprotective effects of PRL hormone using in vivo models (Franssen et al., 2012; Morales, 2011; Torner et al., 2009), this is the first work in vitro reporting neuroprotective effect of PRL in isolated neurons. The overall results indicate that PRL should play an important role in neuronal hippocampal cell functions, however, further studies are required to determine the mechanisms involved on
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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PRL 10 ng/mL
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Fig. 5 – Cells treated with sense and antisense oligonucleotides for PRLR. (A) Effect of oligonucleotide treatment on PRLR expression. Representative western blot is depicted in this figure. Total protein of hippocampal neurons were immunoblotted with anti-PRLR antibody and anti-Actin antibody to normalize. The percentage was calculated considering both PRLR isoforms. *(po0.01). (B) Effect of oligonucleotides treatment on cell viability. The cells treated with sense (S) and antisense (AS) oligonucleotides did not have any significant change on their viability, with or without PRL. (C) Effect of antisense oligonucleotide on excitotoxicity induced by Glu. The cells treated with S oligonucleotide maintained the neuroprotective effect induced by PRL, whether the cells treated with AS oligonucleotide the effect is nullified. *(po0.01), 1(po0.01).
PRL induced neuroprotection and the signaling pathways implicated on this effect.
The cell cultures were treated with these oligonucleotides daily from 4 DIV to 10 DIV and then received treatment with PRL and glutamate, as described above.
4.
Experimental procedures
4.4.
4.1.
Cell cultures
The viability of hippocampal neurons (100 mM) was determined using a MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium), as described previously (González-Sánchez et al., 2011). Briefly, 5 104 cells/well were seeded in 96well microplates. 24 h after excitotoxic insult, MTT assay was performed and relative viability was calculated.
Hippocampal neurons were obtained from rat embryos of 18 days of gestation, as reported previously (Hernández-Fonseca and Massieu, 2005). Cells were plated at 2.1 106 cells/cm2 density in NB medium supplemented with 2% B27, 0.5 mM LGlutamine and 20 mg/mL gentamicin. AraC (10 mM) was added 4 days after plating to prevent non-neuronal cell proliferation. Cultures were used for experiments at 8 days in vitro (DIV) and were maintained at 37 1C in humidified 5% CO2/95% air atmosphere. The percentage of neuronal cells in the cultures was determined by count and was up 95% of total cells. The animals used in this study were cared in accordance with Norma Oficial Mexicana NOM-062-ZOO-1999.
4.2.
Treatments
At 8 DIV, cells were treated with PRL from sheep pituitary (SigmaAldrich L6520), at doses of 1, 2.5, 5, 7.5, 10, 25, and 50 ng/mL in sterile saline solution, or vehicle (sterile saline solution). Glutamate (100 mM) was used as insult at 10 DIV, and another group of cells were treated with 10 ng/mL PRL and Glutamate.
4.3.
Phosphortioate-modified oligonucleotides
Phosphortioate-modified oligonucleotides to reduce the expression of prolactin receptor were designed as previously described (Amaral et al., 2004). Sequence for the sense oligo (50 AAC ATG CCA TCT GCA C 30 ) and antisense (50 GTG CAG ATG GCA TGT T 30 ) oligonucleotides were produced by SigmaAldrich. Both oligonucleotides were diluted to a final concentration of 0.01 nmol/mL and 0.025 nmol/mL respectively in dilution buffer containing 10 mM Tris–HCL and 1 mM EDTA.
4.5.
Cell viability determination/cytotoxic assay
Immunocytochemistry
Hippocampal cells were plated 5 105 in pre-treated poly-Llysine cover slips in 12 well microplates with culture medium, at 8 DIV cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at 4 1C, washed 3 times with PBS for 5 min. Cells were rinsed 3 times with TBS 0.01% Tween-20 for 5 min and permeabilized with TBS 0.1% Tween-20þ0.1% Triton X-100 for 10 min. Then, cells were incubated for 1 h at 4 1C with TBS 0.1% Tween-20þ0.5% goat serum. Primary antibodies were incubated overnight at 4 1C: PRLR (sc-20992, Santa Cruz Biotechnology, 1:200) and NeuN (MAB 377, Chemicon, 1:200). After washing, primary antibodies were detected incubating fluorescent secondary antibody donkey anti-rabbit for PRLR (AP 182F, 1:200), and goat anti-mouse for NeuN (Millipore AP 181R, 1:200) for 1 h under dark conditions at room temperature. After washing with TBS, coverslips were mounted with DAPI and analyzed using a fluorescence microscopy. Sections without primary antibodies were processed in parallel as negative controls.
4.6.
Western blot
Cells were washed with PBS pH 7.4 three times, and then homogenized mechanically with RIPA (PBS pH 7.4, 1% IGEPAL NP 40, SDS 0.1% and sodium deoxycholate 0.05%) lysis buffer plus 5 mM of protease inhibitors cocktail (Roche, Mannheimm,
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Germany). Protein samples (40 mg) were separated by 10% polyacrylamide gel under denaturalizing conditions. Following transfer onto PVDF membranes, subsequently membranes were blocked with 10% dry non-fat milk in TBS 0.1% Tween 20 for one hour at room temperature. Primary antibody PRLR (sc-20992, Santa Cruz Biotechnology, 1:2000) was incubated overnight at 41 under constant agitation. After washes, secondary antibody goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, 1:10,000) were incubated by one hour at room temperature. The signal was detected by chemi-luminescence using Immobilon Western detection system (Millipore, Billerica, USA). Densitometry analysis was performed using LaunchVisionWorksLS Software (California, USA) and normalized against its respective load control.
4.7.
RT-qPCR
Total RNA was isolated using TRIzol (Invitrogen, USA); the extracted RNA was measured by nanodrop spectrophotometer. 1 mg of cDNA were reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to manufacturer's instructions. PCR primers and probes for PRL and PRLR were designed and manufactured by IDT-DNA (USA). Sequences for PRL are (CCA CCT AGT CCA GTT ATT AGT TGA) and (CCC TAG CTA CTC CTG AAG ACA), for PRLR (GCA GGT GAA TGT TTC CTT GTC) and (CTT GCT TTC GTC CTA CTT GTT C). Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as housekeeping gene to normalize the data, the sequences are (GAC CGG TTC TGT CAT GTC G) and (ACC TGG TTC ATC ATC ACTAAT CAC). The RT qPCR reaction was performed using the Applied BiosystemStepOn (Applied Biosystem, USA). Sequence detection software 1.3 (Applied Biosystem) was used for data analysis. To calculate the relative changes in target gene expression the comparative CT method (2T ΔΔC) was used. The average and standard deviation of 2T ΔΔC were calculated for three independent experiments.
4.8.
Statistical analysis
All numerical data were expressed as the mean7SD of three independent experiments. Statistical analysis of each data series was performed by using one-way analysis of variance (ANOVA) followed by a post-hoc analysis with a Dunnet test, with Graph Prism 6s for windows (GraphPad Software, Inc., La Jolla, CA, USA). A p r0.05 value was considered significant.
Acknowledgments This study was supported by funds from PAPIIT-UNAM IN220315.
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Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Mejı´a, S., Morales, M.A., Zetina, M.E., Martı´nez de la Escalera, G., Clapp, C., 1997. Immunoreactive prolactin forms colocalize with vasopressin in neurons of the hypothalamic paraventricular and supraoptic nuclei. Neuroendocrinology 66, 151–159. Mohammad, Y.N., Perone, M., Wang, L., Ingleton, P.M., Castro, M. G., Lovejoy, D.A., 2002. Expression of prolactin receptors and regulation of cell proliferation by prolactin, corticotropinreleasing factor, and corticosterone in a neuroblastoma cell line. Biochem. Cell Biol. 80, 475–482. Morales, T., 2011. Recent findings on neuroprotection against excitotoxicity in the hippocampus of female rats. J. Neuroendocrinol. 23, 994–1001. Morales, T., Lorenson, M., Walker, A.M., Ramos, E., 2014. Both prolactin (PRL) and a molecular mimic of phosphorylated PRL, S179D-PRL, protect the hippocampus of female rats against excitotoxicity. Neuroscience 258, 211–217. Pathipati, P., Gorba, T., Scheepens, A., Goffin, V., Sun, Y., Fraser, M., 2011. Growth hormone and prolactin regulate human neural stem cell regenerative activity. Neuroscience 190, 409–427. Radl, D.B., Ferraris, J., Boti, V., Seilicovich, A., Sarkar, D.K., Pisera, D., 2011. Dopamine–Induced apoptosis of lactotropes is mediated by the short isoform of D2 receptor. PLoS One 6, 7. Saltzman, W., Maestripieri, D., 2011. The neuroendocrinology of primate maternal behavior. Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 1192–1204. Sangeeta Devi, Y., Halperin, J., 2014. Reproductive actions of prolactin mediated through short and long receptor isoforms. Mol. Cell. Endocrinol. 382, 400–410. Shingo, T., Gregg, C., Enwere, E., Fujikawa, H., Hassam, R., Geary, C., Cross, J.C., Weiss, S., 2003. Pregnancy-stimulated neurogenesis in the adult female forebrain mediated by prolactin. Science 299, 117–120. Stocco, C., 2012. The long and short of the prolactin receptor: the corpus luteum needs them both!. Biol. Reprod. 85, 1–2. Tabata, H., Kobayashi, M., Ikeda, J.H., Nakao, N., Saito, T.R., Tanaka, M., 2012. Characterization of multiple first exons in murine prolactin receptor gene and the effect of prolactin on
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their expression in the choroid plexus. J. Mol. Endocrinol. 48, 169–176. Tejadilla, D., Cerbo´n, M., Morales, T., 2010. Prolactin reduces the damaging effects of excitotoxicity in the dorsal hippocampus of the female rat independently of ovarian hormones. Neuroscience 169, 1178–1185. Torner, L., Toschi, N., Pohlinger, A., Landgraf, R., Neumann, I.D., 2001. Anxiolytic and anti-stress effects of brain prolactin: improved efficacy of antisense targeting of the prolactin receptor by molecular modeling. J. Neurosci. 21, 3207–3214. Torner, L., Toschi, N., Nava, G., Clapp, C., Neumann, I.D., 2002. Increased hypothalamic expression of prolactin in lactation: involvement in behavioural and neuroendocrine stress responses. Eur. J. Neurosci. 15, 1381–1389. Torner, L., Maloumby, R., Nava, G., Aranda, J., Clapp, C., Neumann, I.D., 2004. In vivo release and gene upregulation of brain prolactin in response to physiological stimuli. Eur. J. Neurosci. 19, 1601–1608. Torner, L., Karg, S., Blume, A., Kandasamy, M., Kuhn, H.-G., Winkler, J., Aigner, L., Neumann, I.D., 2009. Prolactin prevents chronic stress-induced decrease of adult hippocampal neurogenesis and promotes neuronal fate. J. Neurosci. 29, 1826–1833. Vanoye-Carlo, A., Morales, T., Ramos, E., Mendoza-Rodrı´guez, A., Cerbo´n, M., 2008. Neuroprotective effects of lactation against kainic acid treatment in the dorsal hippocampus of the rat. Horm. Behav. 53, 112–123. Vega, C., Moreno-Carranza, B., Zamorano, M., QuintanarSte´phano, A., Me´ndez, I., Thebault, S., Martı´nez de la Escalera, G., Clapp, C., 2010. Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, R1701–R1708. Zemlyak, I., Manley, N., Sapolsky, R., Gozes, I., 2007. NAP protects hippocampal neurons against multiple toxins. Peptides 28, 2004–2008.
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Research Report
Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor E. Vergara-Castan˜edaa, D.R. Grattanb, H. Pasantes-Moralesc, M. Pe´rez-Domı´nguezc, E.A. Cabrera-Reyesa, T. Moralesd, M. Cerbo´na,n a
Unidad de Investigación en Reproducción Humana, Instituto Nacional de Perinatología-Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico b Center for Neuroendocrinology and Department of Anatomy, University of Otago, New Zealand c Instituto de Fisiología Celular, Mexico d Instituto de Neurobiología, UNAM, Mexico
art i cle i nfo
ab st rac t
Article history:
Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against
Accepted 4 February 2016
excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to
Keywords:
assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model
Prolactin
using primary cell cultures of hippocampal neurons, and to determine whether this effect
Excitotoxicity
is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal
Hippocampus
neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and
Neuroprotection
protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu) – induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures. & 2016 Published by Elsevier B.V.
n
Corresponding author. E-mail address: [email protected] (M. Cerbón).
http://dx.doi.org/10.1016/j.brainres.2016.02.011 0006-8993/& 2016 Published by Elsevier B.V.
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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1.
Introduction
Prolactin (PRL) is a polypeptide hormone produced mainly in the anterior portion of pituitary gland, by the lactotrophs. While there is some evidence that it could also be synthetized in extra-pituitary sites, including some areas of the brain (Clapp et al., 1994; DeVito et al., 1991; Mejía et al., 1997; Torner et al., 2004), this still controversial (Grattan and Kokay, 2008; Kanyicska et al., 2000). PRL secretion is mainly inhibited by hypothalamus and it can be stimulated by suckling. PRL displays several functions in the Central Nervous System (CNS), including the regulation of its own secretion through stimulation of TIDA neurons, stimulation of maternal behavior (Bridges and Mann, 1994; Bridges, 2014), suppression of fertility (Bouilly et al., 2012), and regulation of oxytocin neurons (Vega et al., 2010). Also, it has been described that PRL has several neuroprotective effects, either my stimulating neurogenesis and/or promoting survival, among which are: decreases neuroendocrine and behavioral responses to stress stimulus (Donner et al., 2007; Saltzman and Maestripieri, 2011), anxiolytic effects in male rats (Torner et al., 2001), protects hippocampal neurogenesis in the dentate gyrus of chronically stressed mice (Torner et al., 2009), induces neurogenesis in the sub ventricular zone in both in vivo and in vitro models (Gregg et al., 2007; Mohammad et al., 2002; Shingo et al., 2003), and enhances white matter repair and remyelination (Gregg et al., 2007). The prolactin receptor (PRLR) is a class 1 receptor from the cytokine super family; it is composed of three domains: extracellular, transmembranal and intracellular (Bole-Feysot et al., 1998). Several PRLR isoforms have been described, generated by alternative splicing, proteolysis, intron retention, alternative sites for the end of transcription and partial exon suppression (Brooks, 2012; Ding and Wu, 2010). It is well documented that adult rodent brains express short and long PRLR isoforms (Tejadilla et al., 2010; Torner et al., 2009). In all isoforms, the extracellular domain is identical, but the downstream signal transduction activated by each isoform is different. The long form of the prolactin receptor is required for full activation of the prolactin receptor, including the well documents JAK/STAT5 pathway, while the short isoform can activate the MAP kinase pathway and induce mitogenic responses in some cell types. Recent studies showed that the dorsal hippocampus of the rat is protected during lactation against excitotoxic damage Q4 induced by Kainic Acid (KA) (Cabrera et al., 2009, Vanoye-Carlo et al., 2008). The resistance to cell damage in hippocampal areas occurred either following peripheral or intracerebral administration of KA (Morales, 2011), suggesting that the brain adaptation during lactation modifies the sensitivity of this neural tissue to protect against excitotoxic insults. PRL is markedly elevated during lactation, and previous work has demonstrated that PRL exerts neuroprotective effects in the hippocampus in different experimental models (Franssen et al., 2012; Torner et al., 2009). Treatment with PRL, either through systemic or intracerebral administration, protects against the toxic effects of KA or reduces the epileptic like behavior induced by this glutamate agonist, and this effect is independent of ovarian hormones (Tejadilla et al., 2010; Vanoye-Carlo et al., 2008). Hence, PRL may mediate the neuroprotective effect seen during lactation.
In the present study, by using an in vitro model of primary culture of rat hippocampal cells we evaluated the neuroprotective role of PRL against glutamate, to test the hypothesis that the lactation-induced neuroprotective effect might be mediated by the hyperprolactinemia present at that time. We also examined the expression of PRLR in the cultures, to determine whether PRL actions might be mediated by its cognate receptor.
2.
Results
2.1. PRL did not modify cell neuronal viability in primary cell cultures Prolactin effect on neuronal cell viability was evaluated by MTT assay. The results indicated that PRL did not modify cell viability at any dose used (1–25 ng/mL). The prolactin effect was measured 72 h after hormonal treatment; the results are depicted in Fig. 1A.
2.2. PRL protects hippocampal cells against excitotoxicity induced by glutamate PRL treatment had a preventive effect on glutamate-induced cell death compared with the glutamate-treated group. However, not all the PRL doses significantly prevented cell death induced by Glu, with the optimal dose being 10 ng/ml (po0.01) (Fig. 1B).
2.3. PRLR mRNA expression in primary cell cultures hippocampal cells PRLR expression pattern was assessed by RT-qPCR in all treatments groups. As depicted in Fig. 2, the basal expression of PRLR was determined in the primary neuronal cell cultures. Interestingly, PRL treatment increased PRLR expression. Furthermore, when cells were treated with PRL/GLU, the PRLinduced increase in PRLR mRNA was maintained. Interestingly, PRL mRNA was also detected in these cells, but at very low levels as compared to PRLR mRNA level (data not shown).
2.4. Primary culture hippocampal cells express of prolactin receptor (PRLR) Primary cell cultures were labeled with NeuN to identify neural populations; more than 95% of cells were immunopositive for NeuN and these positive NeuN cell cultures were used in all subsequent experiments. Fig. 3A shows immunostaining with DAPI (A), NeuN (B), PRLR (C) and a merged image for these signals (D). PRLR was detected in almost 90% of the cells, predominantly in a cytoplasmic localization.
2.5.
PRLR isoforms expression on hippocampal cells
Western blot analysis of hippocampal cells revealed the expression of two isoforms of PRLR with molecular weights of 100 and 40 kDa, potentially corresponding to long and short isoforms of PRLR, respectively (see Fig. 4). Mammary gland was used as positive control for PRLR, which express variants of PRLR. A hypothalamic derived cell line from mouse (mHypoEN42 cell line, from Cell Solutions, Canada) presented a shorter
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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2.6.
3
PRLR mediates neuroprotection of hippocampal cells
To inhibit the expression of PRLR in hippocampal cells we performed series of experiments using antisense oligonucleotides against PRLR mRNA. As depicted in Fig. 5, oligonucleotides were able to almost totally block the neuroprotective effect exerted by PRL. In these experiments the expression of PRLR isoforms was assessed by Western blot analysis, and we confirmed that the expression of PRLR isoforms significantly diminished after antisense oligonucleotides treatment. The treatment with these oligonucleotides did not affect cell viability, but when cells were treated with Glutamate, the neuroprotective effect of PRL disappeared (Fig. 5).
3.
Fig. 1 – (A) Effect of PRL treatment on hippocampal cells viability. Cells were incubated with different prolactin doses (1– 25 ng/ml), as described in Section 4. Cell viability was assessed by the MTT method. Results are the mean7SD. (B) Protective effect of PRL against glutamate-induced excitotoxicity. Hippocampal cells were pretreated with PRL at different doses, and Glutamate 100 lM. 10 ng/mL of PRL had a significant protective effect against excitotoxic Glutamate induced cell death º(po0.01). Glutamate had a significant diminution on cell viability compared to control group *(po0.01).
Fig. 2 – PRLR mRNA expression on Hippocampal cells. Treatment with PRL and PRL/GLU induced a significant increase on PRLR mRNA expression compared with control group *(po0.01), mRNA expression data were normalized using HPRT1 as housekeeping gene. isoform with molecular weight of 30 kDa (Fig. 4). However, we could not detect the expression of PRL protein on these hippocampal cells by Western blot experiments.
Discussion
In this study, it has been demonstrated that PRL exerts neuroprotective effects against excitotoxicity in primary cultures of neural hippocampal cells. Cultured hippocampal cells express the mRNA for both PRL and its receptor; however, only the protein for the PRLR has been detected. Besides, the observation that antisense oligonucleotides induced suppression of PRLR expression in the cultured neurons blocks neuroprotection induced by PRL, suggesting that PRL exerts neuroprotection against excitotoxicity through a mechanism mediated by its cognate receptor. These data are consistent, support and extend the previous observation that lactationinduced neuroprotective effects may involve PRLR (Cabrera et al., 2013; Tejadilla et al., 2010; Vanoye-Carlo et al., 2008), furthermore, it has been reported that PRL treatment induces neuroprotection in hippocampus from excitotoxic damage (Morales, 2011; Morales et al., 2014; Tejadilla et al., 2010). In the in vivo condition, the most likely source of PRL to influence hippocampal neurons would be the anterior pituitary gland, with PRL being able to be transported from blood into the cerebrospinal fluid to act on PRLR expressed in the brain. Apart from classical PRL expression in the anterior pituitary gland, PRL expression in the brain has been detected in hypothalamic cells (Mann and Bridges, 2002; Torner et al., 2002), although the physiological role of brain PRL origin is controversial. Recently, our laboratory has detected the expression and regulation of PRL in the hippocampus of lactating rats by microarray (submitted). However, microarray was performed using hippocampal tissues from an in vivo model, having the disadvantage that brain tissue contains many cellular types, including blood cells. To explore PRL and its receptor expression in isolated neurons, in the present investigation we performed a series of experiments in neurons from primary cell cultures, establishing a highly pure population of neuronal cells without contamination by other non-neuronal cells such as glia, that are found in the whole hippocampal tissue (Fath et al., 2009; Zemlyak et al., 2007). By using specific and quantitative PCR, we detected PRL mRNA in the neuronal cultures. However, we were unable to detect the PRL protein either by Western blot analysis or immunehistochemical techniques. It is possible that the inability to detect the protein may be due to a posttranscriptional control of protein synthesis in neurons or to the low concentration in these cells.
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Fig. 3 – Representative fluorescent photomicrograph of hippocampal neurons cells. (A) DAPI, (B) NeuN, (C) PRLR, and (D) Merge NeuN/PRLR. Negative control is shown in inset top right corner (A).
Fig. 4 – Prolactin receptor isoforms in hippocampal neurons. Representative western blot of PRLR is depicted in this figure. Mammary Gland (GM) was used as positive control. Expression of short and long isoforms of PRLR on hippocampal neurons (HN) and hippocampal neurons treated with 10 ng/mL of PRL (HNþPRL). A shorter isoform was detected on a mouse hypothalamic cell line (HyN). Actin was included as loading control.
While we were unable to confirm PRL secretion form these neuronal cultures, we clearly detected the PRLR expression, both at the mRNA level by quantitative RT-PCR, and the protein, by immunoblot and immunohistochemistry. Functionally, we also documented a neuroprotective response to PRL in hippocampal cells. To our knowledge, this is the first report demonstrating the presence and functionality of PRLR in primary neuronal cultures
from the hippocampus. In vivo, in some previous studies PRLR mRNA was not found by in situ hybridization in the hippocampus in adult rats (Chiu and Wise, 1994; Bakowska and Morrell, 1997), Q5 but in contrast our data indicates that these neurons retain the ability to express this receptor under certain conditions. This observation is largely supported by several studies that have documented neuroprotective effects of PRL in vivo, particularly in males during stress conditions and during lactation (Franssen et al., 2012; Morales, 2011; Torner et al., 2009). It might be possible that the receptor is activated in response to cellular damage (Möderscheim et al., 2007). The diversity of PRLR isoforms expressed on different tissues has been related with the specific PRL function on each tissue (Brooks, 2012; Ding and Wu, 2010). Different PRLRs have been described in reproductive (Sangeeta Devi and Halperin, 2014; Stocco, 2012), endocrine system (Ferraris et al., 2012; Radl et al., 2011), cancer tissues (Clevenger et al., 2009), or brain tissues (Kokay et al., 2006; Pathipati et al., 2011). The isoforms detected by immunoblot in the primary culture of neuronal hippocampal cells are different as compared to the hypothalamic cell line and mammary gland; this finding could support the hypothesis that PRL exerts their actions throughout different PRLR variants and regulates the PRLR gene expression in the brain (Tabata et al., 2012). Apart from our work in the hippocampus, various studies about PRL effects in this brain area described the neuroprotective effects of PRL hormone using in vivo models (Franssen et al., 2012; Morales, 2011; Torner et al., 2009), this is the first work in vitro reporting neuroprotective effect of PRL in isolated neurons. The overall results indicate that PRL should play an important role in neuronal hippocampal cell functions, however, further studies are required to determine the mechanisms involved on
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Fig. 5 – Cells treated with sense and antisense oligonucleotides for PRLR. (A) Effect of oligonucleotide treatment on PRLR expression. Representative western blot is depicted in this figure. Total protein of hippocampal neurons were immunoblotted with anti-PRLR antibody and anti-Actin antibody to normalize. The percentage was calculated considering both PRLR isoforms. *(po0.01). (B) Effect of oligonucleotides treatment on cell viability. The cells treated with sense (S) and antisense (AS) oligonucleotides did not have any significant change on their viability, with or without PRL. (C) Effect of antisense oligonucleotide on excitotoxicity induced by Glu. The cells treated with S oligonucleotide maintained the neuroprotective effect induced by PRL, whether the cells treated with AS oligonucleotide the effect is nullified. *(po0.01), 1(po0.01).
PRL induced neuroprotection and the signaling pathways implicated on this effect.
The cell cultures were treated with these oligonucleotides daily from 4 DIV to 10 DIV and then received treatment with PRL and glutamate, as described above.
4.
Experimental procedures
4.4.
4.1.
Cell cultures
The viability of hippocampal neurons (100 mM) was determined using a MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium), as described previously (González-Sánchez et al., 2011). Briefly, 5 104 cells/well were seeded in 96well microplates. 24 h after excitotoxic insult, MTT assay was performed and relative viability was calculated.
Hippocampal neurons were obtained from rat embryos of 18 days of gestation, as reported previously (Hernández-Fonseca and Massieu, 2005). Cells were plated at 2.1 106 cells/cm2 density in NB medium supplemented with 2% B27, 0.5 mM LGlutamine and 20 mg/mL gentamicin. AraC (10 mM) was added 4 days after plating to prevent non-neuronal cell proliferation. Cultures were used for experiments at 8 days in vitro (DIV) and were maintained at 37 1C in humidified 5% CO2/95% air atmosphere. The percentage of neuronal cells in the cultures was determined by count and was up 95% of total cells. The animals used in this study were cared in accordance with Norma Oficial Mexicana NOM-062-ZOO-1999.
4.2.
Treatments
At 8 DIV, cells were treated with PRL from sheep pituitary (SigmaAldrich L6520), at doses of 1, 2.5, 5, 7.5, 10, 25, and 50 ng/mL in sterile saline solution, or vehicle (sterile saline solution). Glutamate (100 mM) was used as insult at 10 DIV, and another group of cells were treated with 10 ng/mL PRL and Glutamate.
4.3.
Phosphortioate-modified oligonucleotides
Phosphortioate-modified oligonucleotides to reduce the expression of prolactin receptor were designed as previously described (Amaral et al., 2004). Sequence for the sense oligo (50 AAC ATG CCA TCT GCA C 30 ) and antisense (50 GTG CAG ATG GCA TGT T 30 ) oligonucleotides were produced by SigmaAldrich. Both oligonucleotides were diluted to a final concentration of 0.01 nmol/mL and 0.025 nmol/mL respectively in dilution buffer containing 10 mM Tris–HCL and 1 mM EDTA.
4.5.
Cell viability determination/cytotoxic assay
Immunocytochemistry
Hippocampal cells were plated 5 105 in pre-treated poly-Llysine cover slips in 12 well microplates with culture medium, at 8 DIV cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at 4 1C, washed 3 times with PBS for 5 min. Cells were rinsed 3 times with TBS 0.01% Tween-20 for 5 min and permeabilized with TBS 0.1% Tween-20þ0.1% Triton X-100 for 10 min. Then, cells were incubated for 1 h at 4 1C with TBS 0.1% Tween-20þ0.5% goat serum. Primary antibodies were incubated overnight at 4 1C: PRLR (sc-20992, Santa Cruz Biotechnology, 1:200) and NeuN (MAB 377, Chemicon, 1:200). After washing, primary antibodies were detected incubating fluorescent secondary antibody donkey anti-rabbit for PRLR (AP 182F, 1:200), and goat anti-mouse for NeuN (Millipore AP 181R, 1:200) for 1 h under dark conditions at room temperature. After washing with TBS, coverslips were mounted with DAPI and analyzed using a fluorescence microscopy. Sections without primary antibodies were processed in parallel as negative controls.
4.6.
Western blot
Cells were washed with PBS pH 7.4 three times, and then homogenized mechanically with RIPA (PBS pH 7.4, 1% IGEPAL NP 40, SDS 0.1% and sodium deoxycholate 0.05%) lysis buffer plus 5 mM of protease inhibitors cocktail (Roche, Mannheimm,
Please cite this article as: Vergara-Castañeda, E., et al., Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.011
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Germany). Protein samples (40 mg) were separated by 10% polyacrylamide gel under denaturalizing conditions. Following transfer onto PVDF membranes, subsequently membranes were blocked with 10% dry non-fat milk in TBS 0.1% Tween 20 for one hour at room temperature. Primary antibody PRLR (sc-20992, Santa Cruz Biotechnology, 1:2000) was incubated overnight at 41 under constant agitation. After washes, secondary antibody goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, 1:10,000) were incubated by one hour at room temperature. The signal was detected by chemi-luminescence using Immobilon Western detection system (Millipore, Billerica, USA). Densitometry analysis was performed using LaunchVisionWorksLS Software (California, USA) and normalized against its respective load control.
4.7.
RT-qPCR
Total RNA was isolated using TRIzol (Invitrogen, USA); the extracted RNA was measured by nanodrop spectrophotometer. 1 mg of cDNA were reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to manufacturer's instructions. PCR primers and probes for PRL and PRLR were designed and manufactured by IDT-DNA (USA). Sequences for PRL are (CCA CCT AGT CCA GTT ATT AGT TGA) and (CCC TAG CTA CTC CTG AAG ACA), for PRLR (GCA GGT GAA TGT TTC CTT GTC) and (CTT GCT TTC GTC CTA CTT GTT C). Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as housekeeping gene to normalize the data, the sequences are (GAC CGG TTC TGT CAT GTC G) and (ACC TGG TTC ATC ATC ACTAAT CAC). The RT qPCR reaction was performed using the Applied BiosystemStepOn (Applied Biosystem, USA). Sequence detection software 1.3 (Applied Biosystem) was used for data analysis. To calculate the relative changes in target gene expression the comparative CT method (2T ΔΔC) was used. The average and standard deviation of 2T ΔΔC were calculated for three independent experiments.
4.8.
Statistical analysis
All numerical data were expressed as the mean7SD of three independent experiments. Statistical analysis of each data series was performed by using one-way analysis of variance (ANOVA) followed by a post-hoc analysis with a Dunnet test, with Graph Prism 6s for windows (GraphPad Software, Inc., La Jolla, CA, USA). A p r0.05 value was considered significant.
Acknowledgments This study was supported by funds from PAPIIT-UNAM IN220315.
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