Both prolactin (PRL) and a molecular mimic of phosphorylated PRL, S179D-PRL, protect the hippocampus of female rats against excitotoxicity

Neuroscience 258 (2014) 211–217

BOTH PROLACTIN (PRL) AND A MOLECULAR MIMIC OF PHOSPHORYLATED PRL, S179D-PRL, PROTECT THE HIPPOCAMPUS OF FEMALE RATS AGAINST EXCITOTOXICITY T. MORALES, a* M. LORENSON, b A. M. WALKER b AND E. RAMOS a

indirectly modulating input signals to the hippocampus and thus regulating excitability. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

a Departamento de Neurobiologı´a Celular y Molecular, Instituto de Neurobiologı´a, Universidad Nacional Autonoma de Mexico, Quere´taro 76230, Mexico

Key words: prolactin, kainate, hippocampus, STAT5, ERK1/2, amygdala.

b Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA

Abstract—Prolactin (PRL) has many functions in the CNS, including neuroprotection. During lactation, the dorsal hippocampus is protected from excitotoxic kainic acid (KA)-induced cellular damage. We have previously reported that systemic pre-treatment with ovine PRL had similar protective effects in female rats. Here, we asked (1) whether intracerebral human PRL (hPRL) would have the same action, (2) because phosphorylated PRL is high in lactation, whether a mimic of phosphorylated hPRL, human prolactin in which the normally phosphorylated serine at position 179 is replaced with an aspartate (S179D-PRL), had similar activity, and (3) what signaling pathways mediated the protective effect. Female ovariectomized (OVX, 1 month) rats were implanted with micro-osmotic pumps connected to unilateral icv cannulae directed at the right lateral ventricle. The pumps delivered 0.10 ng/h of hPRL, S179D-PRL, a combination of hPRL+S179D-PRL, or saline vehicle for 7 days prior to a systemic dose of 7.5 mg/kg of KA. Rats were sacrificed 48 h after KA injection. Immunostaining for neuronal nuclei (Neu-N) revealed a significant KA-induced decrease in cell number in the CA1, CA3, and CA4 hippocampal areas of rats (55% of control). Treatment with either hPRL or S179D-PRL or the combination prevented the damaging effect of KA in these hippocampal regions (95% of corresponding control), but was not completely effective at preventing early seizure-related behaviors such as staring and wet dog shakes. Analysis of signals generated by hPRL and S179D-PRL showed no activation of signal transducer and activation of transcription 5 (Stat5) or other signaling molecules in the hippocampus, but activation of extracellular-regulated kinase (ERK)1/2 in the amygdala. These results support a central protective effect of both PRL forms and suggest that PRL could be exerting its protective action by

INTRODUCTION Prolactin (PRL) is involved in several categorically different functions in the CNS, such as modulation of neurotransmission of hypothalamic neurons (Brown et al., 2012), suppression of fertility (McNeilly, 2001), stimulation of maternal behavior (Mann and Bridges, 2001), suppression of stress responses (Torner and Neumann, 2002), and trophic actions such as myelinization (Gregg et al., 2007), neurogenesis (Shingo et al., 2003; Mak and Weiss, 2010), and neuroprotection (Tejadilla et al., 2010). Apart from the controversial presence of the PRL gene in the brain (DeVito et al., 1992a), PRL can enter the CNS via PRL receptors located in membranes of the choroid plexus (Walsh et al., 1987), and can also enter the CNS in mice lacking the PRL receptor (Brown et al., 2013). Brain PRL receptors exist in both long and short forms (Nagano and Kelly, 1994; Ben-Jonathan et al., 1996; Pi and Grattan, 1999). During reproduction, the hippocampus of the female rat goes through various morphological and functional adaptations (Leuner et al., 2007; Kinsley and Lambert, 2008; Pawluski et al., 2009; Morales, 2011; Cabrera et al., 2013). Taking advantage of the kainate model of epilepsy (Ben-Ari and Cossart, 2000), our group recently reported protective actions of lactation against excitotoxic damage to hippocampus cells (Vanoye-Carlo et al., 2008; Cabrera et al., 2009). Lactation markedly reduces cell loss caused in the CA1, CA3, and CA4 areas of the hippocampus by kainic acid (KA) administered intraperitoneally or intracerebrally, and this correlates with attenuation of seizure behavior in comparison to rats in the diestrus phase of the estrous cycle. After repetitive seizures, serum PRL concentration rises in epileptic patients (Durand et al., 2008; Siniscalchi et al., 2008), and prolonged exposure to PRL, as occurs in pregnancy and lactation, modulates audiogenic-seizure severity in rats (Doretto et al., 2003). We have previously reported that systemic ovine PRL has preventive effects against excitotoxic damage of the hippocampus of female

*Corresponding author. Address: Instituto de Neurobiologı´ a, UNAM, Boulevard Juriquilla 3001, 76230 Quere´taro, Qro, Mexico. Tel: +5255-5623-4071; fax: +52-55-5623-4005. E-mail address: [email protected] (T. Morales). Abbreviations: ERK, extracellular-regulated kinase; hPRL, human prolactin; KA, kainic acid; Neu-N, neuronal nuclei; oPRL, ovine prolactin; OVX, ovariectomized; PRL, prolactin; S179D-PRL, human prolactin in which the normally phosphorylated serine at position 179 is replaced with an aspartate; STAT5, signal transducer and activation of transcription 5.

0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.11.015 211

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rats, and that these effects are independent of ovarian hormones. Treatment with PRL for 4 days was sufficient to attenuate the cell loss and seizure behavior caused by KA in both intact and ovariectomized (OVX) rats, indicating protective actions of PRL in the kainate model of epilepsy (Tejadilla et al., 2010). Possible mechanisms by which PRL exerts protective actions include astrocytes presence (DeVito et al., 1992b, 1995; Moderscheim et al., 2007), neurogenesis (Torner et al., 2009; Mak and Weiss, 2010), antiapoptosis (Leff et al., 1996), and white matter repair and remyelination (Gregg et al., 2007). Also, PRL promotes neurosteroids production (Haraguchi et al., 2010) and can also affects electrophysiological responses (Brown et al., 2012) by inducing hyperpolarization in hypothalamic neurons (Sirzen-Zelenskaya et al., 2011) and by stimulating GABAergic neurons (Kokay et al., 2011). Human prolactin in which the normally phosphorylated serine at position 179 is replaced with an aspartate (S179D-PRL) is a molecular mimic of phosphorylated PRL (Chen et al., 1998). Because of its ability to block signal transducer and activation of transcription 5 (STAT5) signaling from PRL, it has been successfully used as a PRL antagonist both in vitro and in vivo (Schroeder et al., 2003; Naylor et al., 2005). However, as a mimic of a naturally occurring form of PRL, it generates an alternate intracellular signal via activation of extracellular-regulated kinase (ERK)1/2 (Wu et al., 2003). More recently therefore, S179D-PRL has been characterized as a selective PRL receptor modulator as it may function as an antagonist or agonist dependent upon the signaling mechanism required to activate a particular cellular response, i.e. it acts as an antagonist to STAT5-mediated responses and as an agonist for ERK1/2-mediated responses (Walker, 2007). Phosphorylation of PRL is increased during lactation (Huang et al., 2008), suggesting the possibility that phosphorylated PRL may be a superior protective agent for KA-induced damage. In the present study, we used the molecular mimic of phosphorylated human PRL (hPRL), S179D-PRL, to specifically address this question. We also explored candidate-signaling pathways mediating the protective effect.

EXPERIMENTAL PROCEDURES Animals Adult virgin female Wistar rats (200–250 g) were housed individually under controlled temperature and lighting conditions (12:12-h light:dark cycle, lights on at 06:00 h), with food and water available ad libitum. The Institutional Animal Care and Use Committee of the Institute for Neurobiology at the UNAM approved all experimental protocols. Animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Ovariectomy: Ovaries were surgically removed under ketamine/xylazine anesthesia (0.2 ml/100 g b.w., i.p.), and experiments started 1 month after surgery. Animals were randomly assigned to 8 different groups: Vehicle– Vehicle, Vehicle–KA, hPRL–Vehicle, hPRL–KA, S179D-

PRL-Vehicle, S179D-PRL-KA, hPRL+S179D-PRL– Vehicle, and hPRL+S179D-PRL–KA (N = 5 per group). Procedures OVX rats were implanted with micro-osmotic pumps connected to unilateral 28 gauge icv cannulae directed at the right lateral ventricle via a 4-cm polyethylene cannula. The osmotic pumps delivered 0.10 ng/h of hPRL, 0.10 ng/h of S179D-PRL, a combination of 0.1 ng/h of each (hPRL+S179D-PRL), or sterile saline vehicle. Pump contents were infused for 7 days prior to a systemic dose of 7.5 mg/kg of KA. Rats were sacrificed 48 h after KA injection. These doses of PRL or S179DPRL were taken from studies showing that icv S179DPRL disrupts parturition in rats (Nephew et al., 2007). Behavioral seizure stage observation OVX rats (N = 5 per group) treated with KA were observed over a 4-h period for signs of the characteristic progression through the behavioral seizure stages. The progression of these behavioral manifestations of motor seizures was scored according to the Zhang scale: staring, 1; wet dog shakes, 2; hyperactivity, 3; rearing, 4; rearing and falling, 5; jumping, 6 (Zhang et al., 1997). Histological procedures Rats were deeply anesthetized with a lethal dose of urethane (2 ml of 20% urethane; Sigma–Aldrich, St. Louis, MO, USA) and perfused transcardially with 250 ml of 0.9% saline followed by 250 ml of 4% paraformaldehyde in PBS (pH 9.5, 10 °C). Brains were removed, postfixed in paraformaldehyde solution for 2 h, and cryoprotected in 20–30% sucrose-PBS solution for 2–3 days at 4 °C. Coronal sections (30 lm) were cut through the dorsal hippocampus on a freezing microtome, and 5 series were collected and stored in cryoprotectant solution (30% ethylene glycol and 20% glycerol in PBS) at 20 °C. One of the tissue series was employed for each staining, such that consecutive slices of tissue were analyzed by the different methods. Before any procedure, free-floating sections were rinsed 3 times for 10 min in PBS buffer. Morphology of the different hippocampal areas after all treatments was monitored using NISSL staining. The slide-mounted brain sections were soaked in Cresyl Violet solution for 10 min, dehydrated through a graded series of ethanol– water solutions, coverslipped, and analyzed under a bright-field microscope. Immunohistochemistry Immunoreactivity for neuronal nuclei (Neu-N) and other antigens was detected using a conventional avidin–biotin-immunoperoxidase technique (Sawchenko et al., 1990). Floating sections were treated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity, followed by three rinses in PBS, and incubation in 1.0% sodium borohydride for 6–8 min in order to reduce free aldehydes. The tissue was then

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incubated with blocking solution (5% BSA/2% goat or rabbit serum/1% Triton X-100 in PBS) for 1 h to decrease non-specific labeling. Sections were incubated with monoclonal mouse anti- Neu-N antibody (1:1000; Chemicon International Inc., Temecula, CA, USA) at 4 °C for 48 h. After washing, primary antibody was detected with the biotinylated anti-mouse secondary antibody (1:200) and the avidin/biotin system (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Immunolabeling for other antigens employed the same protocol and the following antibodies: monoclonal rabbit anti-Phospho-Stat 5 (1:500, Cell Signaling Technology Inc., Boston, MA, USA); polyclonal rabbit anti-phospho-Map Kinase, ERK1/2 (Thr 202/Tyr 204, 1:500, Cell Signaling Technology Inc.); anti-AKT1 (phospho S473) rabbit monoclonal antibody (1:1000, Abcam Inc., San Francisco, CA, USA); anti-CREB (phospho S133) antibody rabbit monoclonal antibody (1:500, Abcam Inc.); polyclonal rabbit antibody c-Fos, (1:4000, Santa Cruz Biotechnology Inc., Dallas, TX, USA). Sections without primary antibodies were processed in parallel as negative controls. Cell counting Morphometric analysis was performed by NeuN+ cell number quantification, in every fifth section (150 lm separation distance, 6 sections per animal) in the CA1, CA3, and CA4 subfields of the dorsal hippocampus (Larriva-Sahd, 2002). Hippocampal areas were delimited according to the atlas of Swanson (1998) (2.5–4.0 posterior to bregma), and only tissue sections corresponding to these coordinates were included in the quantification, ensuring that the regions of interest were equivalent among animals and experiments. Sections were photographed with a digital camera attached to a Leica microscope, and images were analyzed by using Image-J 1.43 software (NIH, Bethesda, MD, USA). NeuN-positive pyramidal cells were counted in a 500  400 pixel area (410  328 lm) in photomicrographs of the middle portion of the hippocampal CA1, CA3, and CA4 subfields of brain slices from each animal, as described previously (Tejadilla et al., 2010). Neu-Npositive label is expressed as the number of neurons per 0.135 mm2.

CA1, CA3, and CA4 hippocampal areas of rats previously treated with saline (55% of control, p < 0.01–0.001, Fig. 1). In contrast, icv treatment with hPRL prevented the damaging effect of KA in these hippocampal regions, especially in CA4 (p < 0.0001). S179D-PRL, and the combination of both hPRL and S179D-PRL, had the same protective action than either one alone (95% of corresponding control). Candidate cellular signaling pathways for PRL/S179D-PRL action in the hippocampus To investigate the signaling pathway/s responsible for mediating the protective action, we performed immunostaining for signaling molecules known to mediate various actions of these forms of the hormone, such as p-STAT5, p-Map kinase (ERK1/2), pAKT, pCREB, and Fos. Scattered, positively-labeled cells for the different signaling molecules were observed in the hippocampus of rats treated for 7 days with hPRL, S179D-PRL, or both, but the results were not clearly different from the control rats. As previously observed for chronic and acute ovine PRL (oPRL) (Morales, 2011; Sapsford et al., 2012), pSTAT5 signal was induced by hPRL in a few cells of the arcuate nucleus and the periventricular hypothalamic area, but not within the hippocampus (Fig. 2, panel A). However, a clear increase in ERK1/2 labeling was detected in the central subdivision of the amygdala with chronic PRL treatment, especially with S179D-PRL (Fig. 2, panel B). PRL and S179D-PRL attenuated seizure severity The progression of KA-induced seizure expression (behavioral epileptoid markers registered at 4 h after KA injection) included staring, wet dog shakes, hyperactivity and rearing and falling in rats that were previously treated with vehicle. Those manifestations reached only to the early stages in rats treated with either hPRL or S179D-PRL or the combination of both (Fig. 3). hPRLand S179D-PRL-treatment resulted in lower behavioral epileptoid manifestations such as staring and wet dog shakes (p = 0.0019 vs control).

DISCUSSION

Statistical analysis All numerical data are expressed as the mean ± SEM. Statistical analysis of data corresponding to each hippocampal area was performed by using a one-way analysis of variance (1-way ANOVA) followed by a post hoc analysis with a Tukey’s multiple comparison test, with Prism 5.0 software. Values of p < 0.05 were considered statistically significant.

RESULTS PRL and S179D-PRL diminished cell damage induced by KA treatment in the hippocampus Immunostaining for Neu-N revealed a significant KAinduced decrease in the number of NeuN+ cells in

The present study confirms previous observations of the protective effects of PRL in the kainate model of epilepsy and extends them in several important ways. First, we have demonstrated that human PRL, in addition to ovine PRL, had this activity. With respect to behavioral seizure stages observed after KA injection, chronic treatment with hPRL or S179D-PRL decreased the progression of this behavior, indicating protective actions of PRL not only as assessed by morphological indicators, but also by the physiology of neurons of the hippocampus. The combination of both forms of hPRL did not further decrease the progression of the behavioral manifestations, suggesting that incomplete protection was not the result of the use of insufficient hormone.

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80 60 40

**

20 0

80 60 40

Vehicle-Vehicle

Veh-KA

hPRL-Vehicle

hPRL-KA

***

20 0

80 60 40

***

S179D-PRL-KA

hPRL+S179D-PRL-KA

20

Ve hi c

le Ve Veh hi ic cl le ehP K A R LVe hP hic S1 R le 79 LD K PR A hP S1 LR V 79 e L+ D hic S PR l hP 179 L- e R DP K L+ R A S1 LV 79 e D hic PR l L- e K A

0

Fig. 1. Number of cells positive for NeuN immunolabel in CA1, CA3, and CA4 hippocampal areas of OVX rats chronically treated with either vehicle, hPRL, S179D-PRL, or the combination of both, hPRL and S179D-PRL, that received 7.5 mg/kg kainic acid (KA) or vehicle. KA treatment decreased the number of NeuN-positive cells in vehicle-treated rats, while this deleterious effect was not observed in rats previously treated with either form of PRL. To illustrate these effects, CA3 area of rats from the different experimental groups is shown. Bars represent means ± SEM; ⁄⁄p < 0.01, ⁄⁄⁄ p < 0.001, different from corresponding control (N = 5). Scale bar = 100 lm.

Second, a previous study from our group showing that chronic systemic ovine PRL exerted protective effects in the hippocampus against KA excitotoxicity (Tejadilla et al., 2010) could have resulted from an indirect activity of PRL through progesterone from the ovary or the adrenal gland. By using OVX animals and central infusion, the present study provides evidence that intracerebral PRL also has protective actions similar to those seen with systemic PRL. This is evidence in support of a more direct effect of PRL and suggests that

this hormone is a significant player in the neuroprotection that has been demonstrated in reproductive stages such as lactation (Vanoye-Carlo et al., 2008). However, when comparing lactating vs PRL-treated rats wounded with KA, hippocampal cell density is well-preserved with PRL treatment or during lactation, but the progression of seizure behavior is greater in PRL-treated than in lactating rats (Vanoye-Carlo et al., 2008). This suggests possible additional agents necessary for complete normalization of seizure activity.

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215

A

B

Fig. 2. Representative photomicrographs of immunohistochemistry for pSTAT5 and pERK1/2. Chronic infusion of hPRL induced pSTAT5 in the arcuate nucleus of the hypothalamus, but not in the hippocampus (panel A); while both PRL forms induced positive label for ERK1/2 in the amygdala, specifically in the central and lateral subdivisions (marked in the rectangle in the drawing and by the arrows), but not in vehicle-treated rats (panel B).

Seizure Score

6

4

2

hP R L S1 hP 79 R D L PR +S L 17 9D PR L

Ve hi cl e

0

Fig. 3. Graphs showing the observational evaluation through behavioral seizure stages after KA injection. KA-treated rats that received PRL treatment showed a significantly lower seizure score (stage 2–3) than the vehicle-treated rats, which reached stage 3–5. Bars represent means ± SEM; ⁄p < 0.05, different from control (N = 5).

Third, immunostaining for signaling molecules elevated by PRL treatment was used to investigate the molecular pathways leading to the protective effect. In the hippocampus there were no clear effects: chronic

treatment with the PRLs did not induce a signal for intracellular messengers of either the canonical JAK-STAT family, namely pSTAT5, or Map kinases. The presence of PRL receptors in the hippocampus is controversial because they have been observed in this region (Torner et al., 2009; Mak and Weiss, 2010; Tejadilla et al., 2010), but negative results have also been reported (Chiu and Wise, 1994; Bakowska and Morrell, 1997; Pi and Grattan, 1999). Interestingly however, chronic PRL infusion induced ERK1/2 in the amygdala, specifically in the central subdivision. The amygdala and the hippocampus are brain structures damaged in temporal lobe epilepsy (Ben-Ari, 1985). These two areas are interconnected by topographically organized pathways by which they orchestrate several functions, including emotional responses and memory (Pitka¨nen, 2000). The temporal end of the CA1 subfield has substantial reciprocal connections with the amygdala (Pitka¨nen, 2000). It has been proposed that the principal cell projection from the basal nucleus of the amygdala to the CA1/subiculum region of the hippocampus is a candidate pathway for the spread of seizure activity from the amygdala to the hippocampal formation (Kemppainen and Pitka¨nen, 2004; Munasinghe et al., 2010). Furthermore, restraint stress induces c-fos expression within CA3 and the dentate gyrus of the dorsal hippocampus and central

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and medial amygdala (Donner et al., 2007). Such restraint-stress activational responses are blocked by PRL, as well as basal unstressed expression of fos in CA1 of ventral hippocampus and central amygdala, suggesting modulation of inputs to the hippocampus by PRL (Donner et al., 2007). Based on this literature and the fact that no intracellular signal was detected in the hippocampus of rats with chronic PRL, but ERK1/2 activation was seen in the amygdala, we speculate that PRL could be exerting its protective action by indirectly modulating input signals to the hippocampus and thus regulating excitability. Focus on likely signaling and effector pathways is further enhanced by the fact that hPRL and S179D-PRL had similar effects. While S179D-PRL, a mimic of naturally phosphorylated PRL, is often an antagonist, it does not accomplish this by simply blocking the receptor. Instead, it achieves much of its activity by the generation of an alternate signal to unmodified PRL. While an antagonist to PRL-stimulated growth/ antiapoptotic activity, S179D is actually more effective at stimulating a number of different functions (Walker, 2007; Huang et al., 2008). In the present study, both PRL and S179D-PRL had similar effects diminishing the neuronal loss caused by KA. Based on work in other systems, this suggested that PRL was not having its effect on neuronal loss by direct stimulation of proliferation or antiapoptotic activities in the neurons themselves, since S179D-PRL did not antagonized it. This also suggested that the protective effects of both PRL and S179D-PRL were mediated by their activation of ERK 1/2 and by the promotion of some differentiated function in another cell within the brain. S179D-PRL antagonizes STAT5 signaling, but is a better activator of ERK1/2, especially under circumstances of prolonged exposure (Walker, 2007). This seems to be the case for the amygdala in the current set of experiments.

Conclusion In summary, PRL and a mimic of phosphorylated PRL, S179D-PRL, contribute to the preservation of neurons in the hippocampus after an excitotoxic insult. Therefore, protection observed during lactation is due in part to PRL. At the moment, it appears that the action of these molecules is central, exerting effects via the amygdala rather than directly on the hippocampus. The present findings raise additional important questions. These include whether similar protective effects can be seen in males. Also, could PRL treatment after seizure activity ameliorate damage, and could PRL production or sensitivity to this hormone be stimulated as a potential therapeutic strategy for neuroprotection?

Acknowledgments—This study was supported by funds from PAPIIT-UNAM IN202812 and CONACYT 128090 grants. We thank Dr. Dorothy Pless for grammatical review, and Nydia He´rnandez (digital imaging), and Martı´n Garcı´a, Alejandra Castilla, and Nilda Navarro (Animal Care) for technical assistance.

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(Accepted 7 November 2013) (Available online 16 November 2013)