Prenatal stress affects viability, activation, and chemokine signaling in astroglial cultures

Accepted Manuscript Prenatal stress affects viability, activation, and chemokine signaling in astroglial cultures

Joanna E. Sowa, Joanna Ślusarczyk, Ewa Trojan, Katarzyna Chamera, Monika Leśkiewicz, Magdalena Regulska, Katarzyna Kotarska, Agnieszka Basta-Kaim PII: DOI: Reference:

S0165-5728(17)30192-3 doi: 10.1016/j.jneuroim.2017.08.006 JNI 476617

To appear in:

Journal of Neuroimmunology

Received date: Revised date: Accepted date:

11 May 2017 26 July 2017 18 August 2017

Please cite this article as: Joanna E. Sowa, Joanna Ślusarczyk, Ewa Trojan, Katarzyna Chamera, Monika Leśkiewicz, Magdalena Regulska, Katarzyna Kotarska, Agnieszka Basta-Kaim , Prenatal stress affects viability, activation, and chemokine signaling in astroglial cultures, Journal of Neuroimmunology (2017), doi: 10.1016/ j.jneuroim.2017.08.006

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ACCEPTED MANUSCRIPT Prenatal stress affects viability, activation, and chemokine signaling in astroglial cultures Joanna E. Sowa1,2#, Joanna Ślusarczyk2#, Ewa Trojan2, Katarzyna Chamera2, Monika Leśkiewicz2, Magdalena Regulska2, Katarzyna Kotarska2, Agnieszka Basta-Kaim2* 1

Institute of Pharmacology, Polish Academy of Sciences, Department of Physiology, 12

Smętna St, 31-343 Krakow, Poland 2

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Institute of Pharmacology, Polish Academy of Sciences, Department of Experimental Neuroendocrinology, 12 Smętna St, 31-343 Krakow, Poland

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# Joanna E. Sowa and Joanna Ślusarczyk contributed equally to this work.

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Keywords: prenatal stress, primary astrocytes, nitric oxide, fractalkine (CX3CL1), SDF-1α (CXCL12), chemokine receptors

Corresponding author: *Agnieszka Basta-Kaim: Department of Experimental Neuroendocrinology, Polish Academy of Sciences, 12 Smętna St., 31-343 Kraków, Poland, Tel.: +4812 6623273, Fax: +4812 6374500 e-mail: [email protected] Highlights

ACCEPTED MANUSCRIPT  Astrocytes showed increased cell death and GFAP expression after prenatal stress  Prenatal stress increased NO production, which was caused by enhanced iNOS levels  CX3CL1 levels were upregulated in astrocytes derived from prenatally-stressed pups  Prenatal stress altered the CXCL12/CXCR4-7 axis in astroglia

Abstract

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CXCL12/SDF-1α and CX3CL1/fractalkine are constitutively expressed in the brain, which indicates their significant functions. Emerging evidence highlights the role of astrocytes and the immune system in the pathophysiology of stress-related disorders. The aim of this study was to assess whether prenatal stress affects chemokine signaling, cell viability/activation, and the iNOS pathway in astroglial cultures. Our results showed that prenatal stress lowered astrocyte viability and simultaneously increased GFAP expression. Furthermore, CX3CL1 production and the CXCL12/CXCR4-7 axis were also altered by prenatal stress. Taken together, malfunctions caused by prenatal stress may adversely influence brain development, leading to long-term effects on adult brain function and behavior.

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1. Introduction

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A growing body of evidence supports the contention that stress is an important risk factor for many neuropsychiatric disorders, including depression (Baram and Joels, 2009). Among others, stressful events during the prenatal period, which is critical for the development of the central nervous system (CNS), may result in long-lasting neuroanatomic, metabolic and functional changes (Brunton, 2015; Budziszewska et al., 2010; Darnaudéry and Maccari, 2008). Epidemiological studies have confirmed that stressful events experienced during pregnancy are associated with disturbances in neurodevelopment, and they lead to cognitive dysfunction, emotional problems, increased negative temperament, attention deficit/hyperactivity disorder (ADHD), and neuro-immuno-endocrine disturbances in the offspring (Blair et al., 2011; King et al., 2012; Pereira and Ferreira, 2015; Schuurmans and Kurrasch, 2013). Considering these data, the molecular mechanisms underlying these changes have become the subject of many studies in recent years. Therefore, animal models based on stress are widely used.

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The prenatal stress procedure in rats is a well-documented animal model of depression. It has been shown that prenatally-stressed rats exhibit long-lasting behavioral changes, such as increased immobility time in the forced swim test (Basta-Kaim et al., 2014; Maccari et al., 2003; Szczesny et al., 2014), enhanced anxiety-like behavior (Patin, Lordi, Vincent, & Caston, 2005; Szczesny et al., 2014) and cognitive dysfunction (Abdul Aziz et al., 2012). Stress during pregnancy in rats also leads to prolonged neurobiological changes, such as hypothalamic-pituitary-adrenal (HPA) axis disturbance (Budziszewska et al., 2010; Maccari et al., 2003; Szymańska et al., 2009), neuroplasticity (Grigoryan and Segal, 2016; Lemaire et al., 2000; Sowa et al., 2015), and neurotransmitter network dysfunction (Fine et al., 2014). Moreover, unfavorable events that occur early in life lead to immune system malfunction not only in the peripheral but also in the central nervous system (Diz-Chaves et al., 2013; Ślusarczyk et al., 2015). In line with these data, we demonstrated that prenatal stress impairs the release of insulin-like growth factor 1 (IGF-1), which regulates immune cell function by influencing the ratio of pro-inflammatory cytokines in the brain (Basta-Kaim et al., 2014; Szczesny et al., 2014; Trojan et al., 2016). Recently, chemokines, which are chemoattractant cytokines shed new light on the immune malfunction associated with stress-related disorders, and they have become a promising area of research. Chemokines are a diverse family of small (7-11 kDa) proteins. They were originally identified as serving chemotactic functions on immune cells; however, recent evidence has elucidated novel, brain-specific functions of these proteins. In fact,

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chemokines may act as neuromodulators and regulate phenomena such as neurodevelopment, synaptic transmission and neuroendocrine functions, such as thermoregulation, drinking and feeding (Adler, Geller, Chen, & Rogers, 2005; Rostène et al., 2011). Moreover, chemokines are present in virtually all brain inflammation pathologies, including neurodegenerative and neuroinflammatory diseases. Under such conditions, activated glial cells express high levels of chemokines and chemokine receptors, suggesting the involvement of these proteins in brain defense mechanisms (Rostène et al., 2011). Interestingly, fractalkine (CX3CL1) and stromalderived factor 1α (SDF-1α, CXCL12) are two chemokines secreted not only in pathological conditions but they are also constitutively expressed and distributed in many brain areas (Banisadr et al., 2002; Hesselgesser and Horuk, 1999; Schönemeier et al., 2008), which may indicate their significant functions in brain homeostatic processes (Stuart and Baune, 2014). Unlike other chemokines, levels of fractalkine in the brain are higher than those in the periphery (Bajetto et al., 2001), which may suggest its specific role in the CNS. Recent data indicate that neurons are the main source of CX3CL1, although in some cases, astroglia may also express this chemokine, while CX3CR1 is localized mainly on microglia (Lindia et al., 2005).

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On the other hand, SDF-1α/CXCL12 has attracted a lot of attention because of its extensive expression by both neurons and glial cells in the CNS (Banisadr et al., 2003; Heinisch and Kirby, 2010; Stumm et al., 2002). SDF-1α/CXCL12 exerts its biological function through two receptors, CXCR4 and CXCR7. Recent data have demonstrated CXCR4 and CXCR7 expression in astroglia, microglial cells, Schwann cells, distinct neuronal populations, and endothelial cells (Levoye et al., 2009; Li and Ransohoff, 2008). Both the abovementioned chemokines are widely distributed in the brain and constitutively expressed under physiological conditions, while their expression is strongly upregulated during inflammation.

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Our results show that prenatal stress changes the morphological and biochemical profile of microglia as well as modulates the CX3CL1/CX3CR1 axis in distinct brain areas (Ślusarczyk et al., 2016, 2015). Astroglia regulate synaptic transmission and influence the external environment. Astroglia are also trophic and metabolic support for neurons (Perea, Navarrete, & Araque, 2009). It is speculated that prenatal stress leads not only to biological activity malfunction but also to chemokine-chemokine receptor expression in astroglia (Rajkowska and Miguel-Hidalgo, 2007; Sanacora and Banasr, 2013). To the best of our knowledge, there are currently no reports regarding the impact of prenatal stress on astroglia. Therefore, the present study was designed to investigate not only the effects of prenatal stress procedure on viability/death parameters and glial fibrillary acidic protein (GFAP) expression but also nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) levels in astroglial cultures. Moreover, to determine the effects of prenatal stress on the chemokines that are constitutively expressed in the brain, we evaluated the mRNA and protein levels of fractalkine (CX3CL1) and SDF-1α/CXCL12, as well as those of both of its receptors, CXCR4 and CXCR7, in primary astroglial cultures.

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Animals

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All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Ethics Committee in Krakow, Poland. Sprague-Dawley rats (200–250 g upon arrival) were obtained from Charles-River Laboratories (Germany) and housed under standard conditions (a room maintained at 23°C in a 12/12 h light/dark cycle, with the lights on at 6:00) with food and water available ad libitum. One week after the rats arrived, vaginal smears were obtained from the female rats daily to determine the phase of their estrous cycle. During proestrus, the females were placed with males for 12 h. Then, the vaginal smears were evaluated for the presence of sperm. On approximately the 10th day of pregnancy, the females were randomly assigned to either a control group or a stress group. 2.2 Stress procedure

2.3 Cell culture

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Prenatal stress was performed as previously described (Maccari et al., 1995; Ślusarczyk et al., 2016). Briefly, the pregnant female rats were subjected to three stress sessions at 9:00 a.m., 12:00 p.m., and 5:00 p.m. daily from the 14th day of pregnancy until delivery. During this time, the rats were placed in plastic cylinders (d=7 cm; l=19 cm) and exposed to a bright light (150 W; 1800-2000 lx) for 45 min. The control pregnant females remained undisturbed in their home cages.

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The primary astroglia cultures were prepared from the cortices of 1-2-day-old SpragueDawley rats as previously described (Piotrowska et al., 2016; Zawadzka and Kaminska, 2005). Briefly, cells isolated from the cerebral cortex were plated in a poly-L-coated 75 cm2 culture bottle at a density of 3 x 105 cells/cm2 in DMEM/GlutaMAX/high glucose (Gibco, USA) culture medium supplemented with heat-inactivated 10% fetal bovine serum, 0.1 mg/ml streptomycin and 100 U/ml penicillin (Gibco, USA). Cultures were maintained at 37°C and 5% CO2. After 3 days, the culture medium was changed. On the 9th day, the cultures were shaken gently by a horizontal shaker (80 rpm for 1 h and 100 rpm for 15 min) to recover loosely adherent cells, specifically microglial cells. Then, on the 12th day, the non-adherent cells were once again removed, the culture medium was changed, and the culture was left for 2 additional days. Next, the cultures were shaken gently for 3 h (80 rpm). The culture medium was removed, and the astroglial cells were trypsinized (0.05% trypsin EDTA solution, SigmaAldrich, USA). The cells were plated in culture medium at a final density of 1.2 × 106 cells/well in 6-well plates for protein analysis via Western blotting, 2 × 105 cells/well in 24well plates for mRNA and protein analysis via ELISA, or 4 × 104 cells/well in 96-well plates for the NO, MTT and LDH assays and then incubated for 48 h. Two days after plating, the cells were used in experiments. The cultures from both groups of animals (control animals and animals subjected to the prenatal stress procedure) were obtained according to the method described above and simultaneously grown under the same conditions.

ACCEPTED MANUSCRIPT 2.4 Immunofluorescence staining

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To assess the purity of astroglial cell cultures, astroglia obtained from control rats were cultured on sterile cover slips in 6-well plates (1.2 × 106 cells/well). The cells were rinsed with PBS and fixed with 4% paraformaldehyde (Sigma Aldrich, USA) for 20 min at room temperature and washed twice more with PBS solution. The fixed cells were then permeabilized with 0.1% Triton™ X-100 (Sigma Aldrich, USA) in PBS for 30 min at room temperature, washed with PBS and blocked with 5% goat serum in PBS. The astroglia were stained overnight at 4°C with an antibody against GFAP (an astroglia marker; sc-33673, Santa Cruz Biotechnology Inc., USA) and an anti-Iba1 antibody (a microglial marker, sc-32725, Santa Cruz Biotechnology Inc., USA) After being washed with PBS, the cells were incubated for 2 h at room temperature with the appropriate fluorescent-conjugated secondary antibody (Alexa Fluor, Jackson ImmunoResearch, USA). Images were captured using a fluorescence microscope (Zeiss, Germany). We obtained a highly homogeneous astroglia population (greater than 95% GFAP positivity) (Fig. 1). 2.5 Cell viability test

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The cell viability was determined by the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Germany) assay. At 48 h after the astroglial cells were plated, MTT (0.15 mg/mL) was added to each well of a 96-well plate and incubated for 1 h at 37°C. Next, the culture medium was discarded, and 0.1 M HCl in isopropanol was added to dissolve the formazan product. The absorbance value was measured using a multi-well Infinite® M200 PRO Detector spectrophotometer (TECAN, Switzerland) at 570 nm. The data were normalized to the absorbance in the control vehicle-treated cells (100%) and expressed as a percentage of the control ± SEM (Slusarczyk et al., 2015). 2.6 Lactate Dehydrogenase (LDH) test

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To quantify the effects of the prenatal stress procedure on the cell death parameters, the amount of lactate dehydrogenase released from damaged cells into the culture medium was measured 48 h after plating the astroglial cells. The cell culture supernatants were collected from each well of the 96-well plates and incubated with the appropriate reagent mixture at room temperature for 20 min according to the supplier’s instructions (Cytotoxicity Detection Kit, Roche, Germany). LDH is a stable cytoplasmic enzyme that is present in all cells, and it is released into the medium upon plasma membrane damage. In this test, the amount of formazan salt, which is formed after lactate is converted to pyruvate and tetrazolium salt is reduced, is proportional to the LDH activity in the sample. The intensity of the red color formed in the assay measured at a wavelength of 490 nm is proportional to the LDH activity and to the number of damaged cells. The data were normalized to the amount of LDH released from the control vehicle-treated cells (100%) and are expressed as a percentage of the control ± SEM (Slusarczyk et al., 2015).

ACCEPTED MANUSCRIPT 2.7 Nitric Oxide (NO) Release Assay

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NO secreted into the astroglial culture medium was measured by a Griess reaction performed as previously described (Hwang et al., 2008). Supernatants (50 µl) were collected and mixed with an equal volume of Griess reagent (0.1% N-1-naphthylethylenediamine dihydrochloride and sulphanilamine in 5% phosphoric acid) in a 96-well plate and incubated for 10 min at room temperature. The absorbance was measured at 540 nm by a multi-well Infinite® M200 PRO Detector spectrophotometer (TECAN, Switzerland). The data were normalized to the absorbance of the control cells (100%) and expressed as a percentage of the control ± SEM (Slusarczyk et al., 2015). 2.8 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

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Total RNA was extracted from the astroglial cells using TRIzol reagent (Invitrogen, USA) as described previously by Chromczynski and Sacchi (Chomczynski and Sacchi, 1987). Next, the RNA concentration was determined using a NanoDrop spectrophotometer (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). Equal amounts of RNA (500 ng per sample) were reverse transcribed into cDNA using a commercial RT-PCR kit (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, USA) according to the manufacturer’s instructions. Subsequently, the cDNAs were amplified using TaqMan probes, the FastStart Universal Probe Master (Rox) kit (Roche, Germany), and primers for the following genes: CX3CL1 (Rn_00593186_m1), CXCL12 (Rn00573260_m1), CXCR4 (Rn01483207_m1), CXCR7 (Rn00584358_m1), iNOS (Rn00661645_m1), GFAP (Rn01253033_m1) (Life Technologies, USA). The amplification was performed in a total volume of 20 µL containing 1x FastStart Universal Probe Master (Rox) mix, the cDNAs used as the PCR template, TaqMan forward and reverse primers, and the hydrolysis probe (250 nM) labeled with the fluorescent reporter dye FAM at the 5’ end and a quenching dye at the 3’ end. The cycle threshold values (Ct) were calculated automatically by the iCycler IQ 3.0 software. The expression levels were normalized to the Ct of the beta-2 microglobulin (B2m) reference gene (Rn00560865_ml). The control vehicle-treated cultures were used as a reference. The errors were calculated according to the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR published by Applied Biosystems. 2.9 Western Blotting

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Western blot analyses were conducted as previously described (Ślusarczyk et al., 2016). Briefly, the cells were lysed in RIPA buffer (Sigma-Aldrich, USA) containing protease and phosphatase inhibitor cocktails. Next, the lysates (equal amounts of protein) were mixed with 4x Laemmli Sample Buffer and boiled for 5 min at 95°C. The proteins were separated by SDS-PAGE (4-20% gels) under a constant voltage (200 V). Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Roche, Germany) with a semi-dry transblotting apparatus (30 min, 25 V). The membranes were blocked with 5% (w/v) non-fat milk in Tris-buffered saline plus Tween-20 (TBST) for 1 h, washed with TBST and then incubated with the appropriate primary antibody overnight at 4°C. The primary antibodies were: anti-iNOS (sc-651; Santa Cruz Biotechnology, Inc., USA), anti-GFAP (sc-166458; Santa Cruz Biotechnology, Inc., USA), anti-CXCR4 (sc-9046; Santa Cruz Biotechnology, Inc., USA); anti-CXCR7 (20423-1-AP; Proteintech, USA). The blots were washed again with

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TBST and then incubated with a horseradish peroxidase-linked secondary antibodies (antirabbit PI-1000, anti-mouse PI-2000, Vector Laboratories, USA) for 1 h at room temperature. Both primary and secondary antibodies were diluted in SignalBoost Immunoreaction Enhancer solution (Merck Millipore, USA). Afterwards, the membranes were washed four times with large volumes of TBST, and the immunoblots were visualized with the Pierce ECL Substrate (Thermo Fisher Scientific) using chemiluminescence. Data from bands corresponding to the molecular weight of the detected proteins were normalized to that of βactin (60008-1-Ig, Proteintech, USA). The semiquantitative analysis of the band intensity was performed using Fujifilm Image Gauge software (Fuji Film, Japan). The density in the prenatally-stressed group was then expressed as the fold change over the control group, which was set as the reference level at 100%. Enzyme-Linked Immunosorbent Assay (ELISA)

Data analysis

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The supernatants were collected from astroglial cells and analyzed to assess the protein levels of CX3CL1 and CXCL12 in the culture medium. The levels were measured using a commercially available enzyme-linked immunosorbent assay kit (ELISA). Briefly, standards and samples were dispensed into 96-well plates coated with rat CX3CL1 and CXCL12 antibodies, and the plates were incubated. After extensive washing, HPR-conjugated streptavidin was pipetted into each well, and the plates were incubated. The wells were washed, and 3,3’,5,5’-tetramethylbenzidine (TMB) was added. In this assay, the color develops in proportion to the measured protein concentration. Each reaction was stopped after 10 min by the addition of a stop solution. The absorbance was determined using the Infinite 200 PRO Detector (TECAN, Switzerland) system set for the appropriate wavelength (450 nm). For all assays, the intra- and inter-assay coefficients of variation were always ˂10% and ˂12%, respectively. The detection limits were 0.055 ng/ml for CX3CL1 and 0.125 ng/ml for CXCL12. The positive control for each assay was provided by the manufacturers.

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Statistical analysis was performed using GraphPad Prism software (GraphPad Software, USA). All data are presented as the means ± SEM (standard error of the mean) obtained from three independent experiments (independent cell cultures established from the offspring of different mothers). All group means were compared by unpaired t-tests. p-values less than or equal to 0.05 were considered statistically significant.

ACCEPTED MANUSCRIPT 3. Results 3.1 The impact of prenatal stress on the cell viability/death parameters in astroglial cultures

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In the first set of experiments, we examined the effects of prenatal stress on the astroglia viability and death parameters. As shown in Fig. 2, prenatal stress significantly reduced the cell viability, as measured by the MTT reduction assay (p<0.0001, t=7,593 df=176; Fig. 2A), and increased cell death, as measured by the LDH assay (p<0.0001, t=17,05, df=225; Fig. 2B).

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3.2 The impact of prenatal stress on GFAP mRNA expression and protein level in astroglial cultures

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To assess the influence of the prenatal stress procedure on astroglia activation, glial fibrillary acidic protein (GFAP) gene expression and protein level were quantified using qRTPCR and Western Blot, respectively. In cell cultures obtained from prenatally-stressed rats, we observed significantly enhanced expression of the GFAP marker (p<0.05, t=2.698, df=26; Fig. 3a) and protein level (p<0.0001, t=7.462, df=22; Fig. 3b) in comparison to that of cells obtained from control animals. 3.3 The impact of prenatal stress on nitric oxide (NO) release and iNOS production in astroglial cell cultures

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We also evaluated the influence of prenatal stress on the level of the neurotoxic factor NO released in astroglial cell cultures using the Griess reaction. As shown in Fig. 4, astroglia obtained from prenatally-stressed rats showed enhanced NO secretion (p<0.0001, t=16.49; df=261; Fig. 4A).

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In the next set of experiments designed to investigate the molecular mechanism by which prenatal stress increased NO production, we determined the inducible nitric oxide synthase (iNOS) expression. Consistent with the upregulation of NO production, the prenatal stress procedure significantly elevated the level of iNOS protein (p<0.05, t=2.418 df=22; Fig. 4C), while iNOS mRNA gene expression remained unchanged (p>0.05, t=0.7887, df=7; Fig. 4B). 3.4 The impact of prenatal stress on the mRNA and protein levels of fractalkine (CX3CL1) in astroglial cultures The present results revealed no observable changes in CX3CL1 gene expression (p>0.05, t=0.4926, df=26, Fig. 5A) in astroglial cultures obtained from prenatally-stressed rats. Importantly, we found that the prenatal stress procedure significantly elevated the level of fractalkine (CX3CL1) protein (p<0.05, t=2.256, df=28, Fig. 5B) in comparison with that in the control astroglial cultures.

ACCEPTED MANUSCRIPT 3.5 The impact of prenatal stress on the mRNA and protein levels of SDF1α/CXCL12 and their receptors CXCR4 and CXCR7 in astroglial cell cultures In the next set of experiments, we evaluated the mRNA and protein levels of SDF1α/CXCL12 in astroglial cells obtained from both control and prenatally-stressed animals. As demonstrated in Fig. 6, prenatal stress significantly upregulated SDF-1α/CXCL12 mRNA expression (p<0.05, t=2.338, df=15; Fig. 6A). On the other hand, the stress procedure had no impact on the CXCL12 protein level (p>0.05, t=1.022, df=30; Fig. 6B) in astroglial cultures.

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Since SDF-1α/CXCL12 exerts biological activity via its CXCR4 and CXCR7 receptors, we determined the gene expression and protein levels of both receptors in control and prenatally-stressed astroglial cultures using qRT-PCR and Western blot, respectively.

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We found that prenatal stress significantly upregulated the mRNA expression of CXCR4 (p<0.05, t=2.389, df=15, Fig. 7A), while it did not affect the gene expression of CXCR7 (p>0.05, t=1.828, df=15, Fig. 8A).

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Importantly, we observed that the prenatal stress procedure lead to the malfunction of both chemokine receptors that were measured. We demonstrated an enhanced level of CXCR4 expression (p<0.05, t=2.509, df=30; Fig. 7B), while reduced levels of CXCR7 (p<0.01, t=3.293, df=26; Fig. 8B) were observed in astroglial cultures obtained from prenatally-stressed rats.

ACCEPTED MANUSCRIPT 4. Discussion

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Our present study was conducted using the prenatal stress model that is verified in the literature as a model of depression with predictive, constructive, and face validity (Nester and Hyman, 2011). Although earlier investigations have revealed that this procedure leads to many dysfunctions in the nervous, endocrine, and immune systems, including changes in the biological functions of microglia (Ślusarczyk et al., 2016), no studies have assessed the impact of prenatal stress on astroglia thus far. The aim of the present study was to elucidate the potential impact of stress during the critical, late pregnancy period on astroglia viability and death parameters, activation, NO release and iNOS expression. Moreover, we evaluated the level of chemokines and their receptors in primary astroglial cultures obtained from prenatally-stressed animals.

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We found that the prenatal stress procedure lowered astroglia viability and metabolic activity and simultaneously increased cell death. In addition, those changes were accompanied by enhanced GFAP gene expression and protein level, NO release and iNOS levels. We demonstrated that prenatal stress increased fractalkine/(CX3CL1) production and upregulated SDF-1α/CXCL12 mRNA expression. Prenatal stress also led to the malfunction of both SDF1α/CXCL12 receptors: it enhanced CXCR4 expression and downregulated CXCR7 expression in the astroglial cultures. Our data clearly indicate the important role of prenatal stress in the modulation of the biochemical profile and function of primary astroglia. If these changes last for extended periods, they may affect adult brain function and behavior and provide an interesting background for stress-related disorders, including depression.

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The present study showed that the primary astroglial cultures obtained from prenatallystressed animals displayed diminished viability and metabolic activity. Because the MTT assay is dependent on mitochondrial dehydrogenase activity and not on the membrane integrity of cells, the changes observed in astroglia in our study may indicate their decreased metabolic activity. On the other hand, we demonstrated increased astroglia death using the LDH assay. Hence, by evaluating two distinct aspects of the astroglia death and viability processes, we have provided an accurate and complete assessment of the unfavorable impact of prenatal stress on the vital status of astroglial cells. In pathological conditions, astroglia can enter a state called reactive astrogliosis in the brain, which is characterized by hypertrophy (enlarged cell bodies and thick processes) and increased GFAP expression (Goswami et al., 2015; Gupta et al., 2015). Consistent with these findings, our results showed increased GFAP gene expression and protein level in astroglia after the prenatal stress procedure. Observations from this study signify prenatal stress as a potent activator of glial cells and correlate with our previous finding on microglial cultures (Ślusarczyk et al., 2016). Interestingly, the clinical data demonstrated the presence of hypertrophic cortical astroglia in depressed, suicidal patients, indicating their possible activation in the course of this stress-related disorder (Torres-Platas et al., 2011). On the other hand, the exposure to chronic, unpredictable stress in rats leads to behavioral dysfunction and

ACCEPTED MANUSCRIPT decreased GFAP mRNA expression in the prefrontal cortex (Banasr et al., 2010), which may suggest that the profile of astroglia activation depends on the experimental conditions.

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It is well accepted that astroglia play an important role in the regulation of brain homeostasis. Several lines of evidence suggest that neurodegenerative and stress-related disorders are correlated with functional changes in astroglia, including the secretion of immune modulators and toxic factors (Moriyama et al., 2016). Among them, the release of NO seems to be crucial because it serves as a signaling molecule and participates in a plethora of biological functions (Pannu & Singh, 2006). NO may be generated from L-arginine by three different isoforms of NOS: endothelial, neuronal, and inducible NOS (Guix et al., 2005). In relation to our study, the most important isoform is iNOS, as it is primarily expressed in glial cells. In astroglia, an initial drop in NO content triggers NF-kappaB activation, followed by iNOS expression, which acts in a calcium-independent manner. In the present study, prenatal stress did not change mRNA iNOS expression but significantly upregulated the iNOS protein level. Therefore, it can be assumed that the increased NO secretion in astroglia observed in our study is a result of iNOS activation. Data obtained in this report are consistent with our previous observations in that the same changes were observed in NO production and iNOS concentration levels after prenatal stress exposure in primary microglial cultures (Ślusarczyk et al., 2015).

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The main finding of our study is that prenatal stress affects the chemokine-chemokine receptor axis in astroglia. To the best of our knowledge, our data are the first to demonstrate that prenatal stress potentiates fractalkine (CX3CL1) release by astroglia. It is worth emphasizing that there is still some debate regarding the types of cells that express fractalkine and its receptor in the CNS. Mounting evidence shows that this chemokine is principally expressed by neurons, while its receptor, CX3CR1, is primarily located in microglial cells (Cardona et al., 2006). The majority of former research has indicated that astroglia do not constitutively express the fractalkine/CX3CL1 protein; however, upregulation of its expression may occur after stimulation by pro-inflammatory proteins, such as TNF-α and IL1β, or by toxic factors (Maciejewski-Lenoir et al., 1999; O’Sullivan et al., 2016). However, fractalkine expression levels in astroglia are relatively lower than those in neurons, whereas microglia do not appear to express fractalkine transcripts. In fact, fractalkine (CX3CL1) immunoreactivity is upregulated in astroglia, while CX3CR1 expression is elevated in microglia in a prion model of chronic neurodegeneration and inflammation (Hughes et al., 2002). Fractalkine (CX3CL1) expression in astroglia has also been demonstrated in neuropathic pain (Lindia et al., 2005). So far, fractalkine has been demonstrated to be involved in neuromodulation, neurodevelopmental process regulation and neuroendocrine functions (Limatola et al., 2005; Paolicelli et al., 2011; Polyák et al., 2014). Fractalkine has also been shown to have antiinflammatory action (Corona et al., 2010). Importantly, fractalkine anti-inflammatory action was confirmed by its inhibitory effects on nitric oxide (NO) production as well as the iNOS expression level demonstrated in our previous research (Ślusarczyk et al., 2016). Interestingly, we observed that treatment with exogenous fractalkine was able to normalize stress-evoked

ACCEPTED MANUSCRIPT changes in pro-inflammatory cytokine release (Ślusarczyk et al., 2016). Therefore, our working hypothesis is that the elevated fractalkine release observed in our present study may be considered to be a compensatory anti-inflammatory mechanism of astroglia in response to elevated pro-inflammatory cytokines as well as to NO production evoked by prenatal stress.

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In addition to the effects of fractalkine (CX3CL1) release on astroglia, this study showed the impact of prenatal stress on the expression of SDF1-α/CXCL12. We demonstrated that prenatal stress significantly increased mRNA SDF1-α/CXCL12 expression, but did not affect the CXCL12 protein level.

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In general, data concerning the role of SDF1-α/CXCL12 and its receptors in the brain are ambiguous. Astroglial and endothelial cells are thought to be the main source of SDF1α/CXCL12 in the brain. In physiological conditions, SDF1-α/CXCL12 expression seems to be at a relatively low level, whereas upregulation of this chemokine is observed in some pathological states, such as AIDS, brain tumors and inflammation (Li and Ransohoff, 2008; Momcilović et al., 2012; Tiveron and Cremer, 2008). Regarding the effects of prenatal stress, we did not show elevated level of SDF1-α/CXCL12 in astrocytes, but our previous study indicated increased production of this chemokine in microglia (Slusarczyk et al., 2015). It has been shown that elevated level of SDF1-α/CXCL12 may potentiate TNF-α production in astroglia as well as glutamate release, which directly affects astroglia, but also leads to the activation of microglia, enhancing TNF-α production and neurotoxicity in these cells (Bezzi et al., 2001). This action is mediated by CXCR4.

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Our study shows that prenatal stress enhanced CXCR4 level in primary astroglial cultures. CXCR4 is the main SDF1-α/CXCL12 receptor expressed in all cell types in the CNS, however, glia are its main source (Banisadr et al., 2003, 2002). CXCR4 modulates the release of neurotransmitters from neurons and glia as well as hormone secretion from neuroendocrine cells (Banisadr et al., 2003, 2002; Bezzi et al., 2001; Odemis et al., 2010). Regarding the crucial role of the CXCL12/CXCR4 pathway during development, such as regulation of the migration of neural precursors or axonal outgrowth, it is likely that the upregulation of CXCR4 caused by prenatal stress observed in our study may adversely influence brain developmental processes. In the brain, the important role of CXCL12/CXCR4 signaling is not only regulation of neuron-glia but also glia-glia interactions. Therefore, our result showing the enhanced astroglia CXCR4 level together with malfunctions in the mentioned axis in microglial cultures (Ślusarczyk et al., 2015) may indicate a possible explanation for the disturbance of the glia-glia system caused by prenatal stress procedure. On the other hand, we can not exclude that the up-regulation of CXCR4 observed in our study may be related with the dynamic trafficking of this receptor in the presence of other ligands like chemokine C-XC motif ligand 11 (CXCL11) or macrophage migration inhibitory factor (MIF-1) evoked by prenatal stress (Chatterjee et al., 2015; Nishibori et al., 1996). Contrary to CXCR4, our results demonstrated that prenatal stress lowered CXCR7 levels in astroglial cultures. CXCR7 is an alternative SDF-1α/CXCL12 receptor (Schönemeier et al., 2008), and SDF-1α exhibits distinctly higher affinity for CXCR7 than for CXCR4. CXCR7 is a non-classical G-protein-coupled receptor because it fails to signal through Gαi proteins and

ACCEPTED MANUSCRIPT acts as a β-arrestin-biased receptor (Burns, 2006; Kalatskaya et al., 2009). During development, CXCR7 seems to act as an SDF-1α/CXCL12 scavenger, which directs the CXCR4-induced migration of primordial cells by shaping the extracellular SDF-1α/CXCL12 gradient (Odemis et al., 2010). Interestingly, it has been demonstrated that in the developing or injured brain, astroglia rarely express CXCR4, whereas a much larger subset of astroglia express CXCR7 (Schönemeier et al., 2008; Stumm et al., 2002). Furthermore, CXCR7 modulates CXCR4-dependent cell signaling by heterodimerization with CXCR4.

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The identification of CXCR7 as an alternative receptor for SDF-1α/CXC12 and the fact that it forms a heterodimer with CXCR4 provides a supplementary level of complexity to CXCL12/ CXCR4 signaling (Levoye et al., 2009). It seems that the upregulation of CXCR4 and the downregulation of CXCR7 due to the prenatal stress procedure observed in the present study may indicate disparate effects through different signaling pathways. Considering this, it has been suggested that CXCR4 is involved in cell migration, whereas CXCR7 is involved in cell survival (Burns, 2006). Based on our results, we can postulate that prenatal stress affects the CXCL12/CXCR4-7 axis; however, the exact biological importance of these changes requires further investigation.

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In summary, this study showed for the first time that stressful events during critical periods of development influence the astroglia phenotype. It appears that prenatal stress may contribute to processes leading to the onset of affective disorders, including depression, by enhancing neurotoxic processes and causing chemokine signaling malfunction. Considering the limitations of our results, further studies are required to determine the accurate meaning of these multi-factorial changes.

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5. Acknowledgments This work was supported by the Statutory funds of the Department of Experimental Neuroendocrinology and the Department of Physiology at the Institute of Pharmacology at the Polish Academy of Sciences and partially by Grant no. 2013/09/B/NZ7/04096 from the National Science Centre in Poland. Publication charges are supported by KNOW funds (MNiSW-DS-6002-469326/WA/12). Joanna E. Sowa, Ewa Trojan, and Katarzyna Chamera hold scholarships from KNOW, sponsored by the Ministry of Science and Higher Education in Poland. 6. References

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Fig. 1. Fluorescence images of a representative astroglial culture under 40x (A) and 10x (B) optics. Astroglial cells positively stained for GFAP.

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Fig. 2. The effects of prenatal stress on astrocyte viability measured by the MTT assay (A) and death parameters measured by the LDH assay (B). The results are shown as the percentage of the control ± SEM; ****p< 0.0001.

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Fig. 3. The effect of prenatal stress on GFAP expression and protein level in primary astrocyte cultures. (A) The qRT-PCR results are shown as the percentage of the control ± SEM. (B) The Western blot results are normalized with β-actin and presented as the percentage of the control ± SEM. *p<0.05; **** p<0.0001. Fig. 4. The effects of prenatal stress on nitric oxide (NO) production (A) and iNOS mRNA (B) and protein levels (C) in astrocyte cultures. NO production and iNOS mRNA expression are expressed as a percentage of the control ± SEM. The Western blot results are normalized with β-actin and presented as the percentage of the control ± SEM from 3 independent experiments. *p<0.05; **** p<0.0001. Fig. 5. The effects of prenatal stress on CX3CL1 mRNA and protein levels. (A) The qRT-PCR results are expressed as a percentage of the control ± SEM. (B) Protein levels obtained from the ELISA assay are shown as the percentage of the control ± SEM. The presented results are from 3 independent experiments. *p<0.05.

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Fig. 6. The effects of prenatal stress on the mRNA and protein levels of CXCL12. (A) The qRT-PCR results are expressed as a percentage of the control ± SEM. (B) Protein levels obtained from the ELISA assay are shown as the percentage of the control ± SEM. The presented results are from 3 independent experiments. *p<0.05.

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Fig. 7. The effects of prenatal stress on the mRNA and protein levels of the CXCL12 receptor CXCR4. (A) The mRNA expression is presented as a percentage of the control ± SEM. (B) The Western blot results are normalized with β-actin and presented as the percentage of the control ± SEM. The presented results are from 3 independent experiments. *p<0.05, **p<0.01

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Fig. 8. The effects of prenatal stress on the mRNA and protein levels of the CXCL12 receptor CXCR7. (A) The mRNA expression is presented as the percentage of the control ± SEM. (B) The Western blot results are normalized with β-actin and presented as the percentage of the control ± SEM. The presented results are from 3 independent experiments. *p<0.05.

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Graphical abstract