GSK-3β pathway

Accepted Manuscript Neuroprotection by JM-20 against oxygen-glucose deprivation in rat hippocampal slices: Involvement of the Akt/GSK-3β pathway Jeney Ramírez-Sánchez, Elisa Nicoloso Simões Pires, Yanier Nuñez-Figueredo, Gilberto L. Pardo-Andreu, Luis Arturo Fonseca-Fonseca, Alberto Ruiz-Reyes, Estael Ochoa-Rodríguez, Yamila Verdecia-Reyes, René Delgado-Hernández, Diogo O. Souza, Christianne Salbego PII:

S0197-0186(15)30045-0

DOI:

10.1016/j.neuint.2015.09.003

Reference:

NCI 3766

To appear in:

Neurochemistry International

Received Date: 15 April 2015 Revised Date:

3 September 2015

Accepted Date: 4 September 2015

Please cite this article as: Ramírez-Sánchez, J., Simões Pires, E.N., Nuñez-Figueredo, Y., PardoAndreu, G.L., Fonseca-Fonseca, L.A., Ruiz-Reyes, A., Ochoa-Rodríguez, E., Verdecia-Reyes, Y., Delgado-Hernández, R., Souza, D.O., Salbego, C., Neuroprotection by JM-20 against oxygen-glucose deprivation in rat hippocampal slices: Involvement of the Akt/GSK-3β pathway, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Neuroprotection by JM-20 against oxygen-glucose deprivation in rat hippocampal slices: Involvement of the Akt/GSK-3β pathway. Jeney Ramírez-Sáncheza, Elisa Nicoloso Simões Piresb, Yanier NuñezFigueredoa, Gilberto L Pardo-Andreuc, Luis Arturo Fonseca-Fonsecaa, Alberto

RI PT

Ruiz-Reyesd, Estael Ochoa-Rodríguezd, Yamila Verdecia-Reyesd, René Delgado-Hernándeza, Diogo O Souzab,e, Christianne Salbegob,e. a

Centro de Investigación y Desarrollo de Medicamentos, Ave 26, No. 1605

Boyeros y Puentes Grandes, CP 10600, La Habana, Cuba

SC

b

Programa de Pós-graduação em Bioquímica, Departamento de Bioquímica,

ICBS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, c

M AN U

2600-Anexo I, Porto Alegre, RS 90035-003, Brazil

Centro de Estudio para las Investigaciones y Evaluaciones Biológicas, Instituto

de Farmacia y Alimentos, Universidad de La Habana, ave. 23 # 21425 e/214 y 222, La Coronela, La Lisa, CP 13600, La Habana, Cuba d

Laboratorio de Síntesis Orgánica de La Facultad de Química de La

Universidad de La Habana (Zapata s/n entre G y Carlitos Aguirre, Vedado

TE D

Plaza de la Revolución, CP 10400, La Habana, Cuba e

Departamento de Bioquímica, PPG em Bioquímica, PPG em Educação em

Ciência, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio

AC C

003, Brazil

EP

Grande do Sul, Rua Ramiro Barcelos, 2600 anexo, Porto Alegre, RS 90035-

Corresponding author: Christianne Salbego, Departamento de Bioquímica, PPG em Bioquímica, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600 anexo, Porto Alegre, RS 90035-003, Brazil E-mail address: [email protected], [email protected]

ACCEPTED MANUSCRIPT Abstract Cerebral ischemia is the third most common cause of death and a major cause of disability worldwide. Beyond a shortage of essential metabolites, ischemia triggers many interconnected pathophysiological events, including excitotoxicity, oxidative stress, inflammation and apoptosis. Here, we investigated the

RI PT

neuroprotective mechanisms of JM-20, a novel synthetic molecule, focusing on the phosphoinositide-3-kinase (PI3K)/Akt survival pathway and glial cell response as potential targets of JM-20. For this purpose, we used organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD) to

SC

achieve ischemic/reperfusion damage in vitro. Treatment with JM-20 at 0.1 and 10 µM reduced PI incorporation (indicative of cell death) after OGD.

OGD

decreased the phosphorylation of Akt (pro-survival) and GSK 3β (pro-apoptotic),

M AN U

resulting in respective inhibition and activation of these proteins. Treatment with JM20 prevented the reduced phosphorylation of these proteins after OGD, representing a shift from pro-apoptotic to pro-survival signaling. The OGDinduced activation of caspase-3 was also attenuated by JM-20 treatment at 10 µM. Moreover, in cultures treated with JM-20 and exposed to OGD conditioning, we observed a decrease in activated microglia, as well as a decrease in

TE D

interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α release into the culture medium, while the level of the anti-inflammatory IL-10 increased. GFAP immunostaining and IB4 labeling showed that JM-20 treatment significantly

EP

augmented GFAP immunoreactivity after OGD, when compared with cultures exposed to OGD only, suggesting the activation of astroglial cells. Our results confirm that JM-20 has a strong neuroprotective effect against ischemic injury

AC C

and suggest that the mechanisms involved in this effect may include the modulation of reactive astrogliosis, as well as neuroinflammation and the antiapoptotic cell signaling pathway.

Keywords: JM-20; Neuroprotection; Oxygen–glucose deprivation; Akt; GSK-3β; Neuroinflammation

ACCEPTED MANUSCRIPT

1. Introduction

Stroke is the third most common cause of death and a major cause of disability

RI PT

worldwide. The overall incidence of stroke is predicted to increase in the coming decades due to the aging population, especially in developing countries (Howells and Donnan, 2010). Approximately 80% of all strokes are ischemic, characterized by a reduction or complete blockade of regional cerebral blood

SC

flow that results in deficient glucose and oxygen supply to the affected region (Thrift et al., 2001). Beyond a shortage of essential metabolites, ischemia triggers many other interconnected pathophysiological events, including

M AN U

excitotoxicity and oxidative stress in the acute phase, and inflammation and apoptosis in the sub-acute period (hours to days; Doyle et al., 2008). After ischemic insult, the balance between cell death and survival signals determines neuronal fate. The phosphoinositide-3-kinase (PI3K) pathway plays a crucial role in signal transduction pathways that regulate cell growth, metabolism and apoptosis (Brazil et al., 2004; Kulik et al., 1997). Serine–

TE D

threonine kinase, Akt (also known as protein kinase B), is a downstream kinase of PI3K that is activated by phosphorylation and plays an important role in cell death and survival processes (Simao et al., 2009). Akt partially mediates its

EP

anti-apoptotic effects by phosphorylating (inhibiting), among other proteins, glycogen synthase kinase-3β (GSK-3β), which is particularly abundant in the central nervous system (CNS) and is a key regulator of several physiological

AC C

processes, such as cell cycle and apoptosis. GSK-3β can induce the activation of pro-apoptotic proteins such as caspase-3 and tumor suppressor gene p53 (Welsh et al., 1996).

In addition, the activation of Akt has been associated with the inactivation

of c-Jun N-terminal kinase (JNK) (Kim et al., 2002; Song and Lee, 2005; Wang et al., 2006) and the activation of NF-κB (Song et al., 2008), both potent mediators of inflammation and apoptosis following cerebral ischemia (Benakis et al., 2010). These events strongly activate neighboring glial cells, leading to the release of inflammatory mediators and the expression of adhesion molecules in vascular endothelial cells. These inflammatory responses trigger

ACCEPTED MANUSCRIPT the acceleration of early deleterious events and are a determining factor in the progression of brain damage, contributing to secondary injury, particularly in the surrounding

viable

tissue—known

as

the

penumbra—caused

by

the

enlargement of the cerebral infarct area (Barone and Feuerstein, 1999). Both pro- and anti-inflammatory cytokines are known to be produced during ischemic

RI PT

events and can exert either detrimental or beneficial effects during recovery and repair periods (del Zoppo et al., 2000).

Astrocytes constitute the most abundant glial cells in the CNS, providing structural, trophic and metabolic support to neurons and modulating synaptic

SC

activity (Belanger and Magistretti, 2009). Astrocyte activation - a process known as “reactive astrogliosis” - occurs in response to many CNS injuries, including cerebral ischemia (Verkhratsky et al., 2013). This phenomenon is evidenced by

M AN U

an increase in glial fibrillary acidic protein (GFAP) expression and by hypertrophic morphology (Pekny and Nilsson, 2005).

Meanwhile, microglia constitute approximately 20% of the total glial cell population of the brain (Kreutzberg, 1995). Activation of resting glial cells and their production of pro-inflammatory factors are induced following neuronal damage, with a timing and extent that varies depending upon the initial insult.

TE D

Together, both types of glial cells, in concert with neurons(Jeong et al., 2013), are involved in neuroinflammation. In this way, the appropriate control of this process may be beneficial to neurons exposed to ischemic conditions.

EP

Given the complex pathophysiology of stroke, the use of multi-target therapies that act at several points during the ischemic cascade could improve the odds of successful neuroprotection. The 3-ethoxycarbonyl-2-methyl-4-(2-

AC C

nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine (JM-20) is a 1,5benzodiazepine fused to a dihydropyridine moiety, which is currently under investigation as a potential neuroprotective drug. JM-20 has been shown to possess anxiolytic and sedative effects in rodents (Figueredo et al., 2013) and cytoprotective activity in different in vitro models related to cerebral ischemia (Nunez-Figueredo et al., 2014b). Moreover, JM-20 markedly attenuates ischemic damage in rats subjected to middle cerebral artery occlusion (MCAO), even with delayed oral administration (Nuñez-Figueredo, 2014a). Antiexcitotoxic and mitoprotective effects may play a role in the mechanism of action of JM-20 (Nuñez-Figueredo, 2014a).

ACCEPTED MANUSCRIPT Because any mediator of the ischemic cascade is a potential target for developing new therapeutic strategies for the treatment or prevention of ischemia-induced brain injury, the aim of this study was to evaluate and expand our knowledge of the neuroprotective effects of JM-20, with a focus on cell signaling and, in particular the Akt/GSK-3β signaling pathway, on the activation

RI PT

of microglia and astrocytes, and on inflammatory responses. For this purpose, we used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) as a model of brain ischemia.

2.1.

SC

2. Materials and methods

Synthesis and chemical characterization of JM-20

(Figueredo et al., 2013).

2.2.

M AN U

JM-20 was synthesized, purified and characterized as previously reported

Organotypic hippocampal slice cultures

All animal procedures were approved by the local Animal Care Committee and were in accordance with the NIH Guide for the care and use of laboratory animals. Organotypic hippocampal slice cultures were prepared according to

TE D

the method of Stoppini et al. (1991), with some modifications (Valentim et al., 2003). Briefly, 400 µm-thick hippocampal slices were prepared from 6- to 8-dayold male Wistar rats using a McIlwain tissue chopper, and separated in ice-cold

EP

Hank’s balanced salt solution (HBSS) composed of 36 mM glucose, 1.26 mM CaCl2, 5.36 mM KCl, 136.89 mM NaCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 0.49 mM MgCl2, 0.44 mM MgSO4, 25 mM HEPES, 1% fungizone and 0.01%

AC C

gentamicin, pH 7.2. The slices were placed on Millicell culture membranes and the inserts were transferred to a 6-well culture plate. Each well contained 1 mL of tissue culture medium consisting of 50% minimum essential medium (MEM), 25% HBSS and 25% heat inactivated horse serum supplemented with 36 mM glucose, 25 mM HEPES and 4 mM NaHCO3, 1% fungizone and 0.01% gentamicin, pH 7.3. The cultures were maintained in a humidified incubator with a 5% CO2/95% O2 atmosphere at 37ºC for 14 days. Culture medium was changed two times a week.

2.3.

Oxygen-glucose deprivation (OGD) and JM-20 treatment

ACCEPTED MANUSCRIPT The induction of OGD to mimic ischemic injury was based on the method described by Strasser and Fischer (1995), with some modifications (Valentim et al., 2003). After 14 days in vitro, the inserts were transferred to a sterilized 6well plate and incubated with 1 mL of OGD medium (glucose-free) for 15 min to deplete glucose from intracellular stores and the extracellular space. The OGD

RI PT

medium was composed of 1.26 mM CaCl2, 5.36 mM KCl, 136.9 mM NaCl, 0.34 mM H2PO4, 0.49 mM MgCl2, 0.44 mM MgSO4, and 25 mM HEPES, pH 7.2. Following this period, the medium was exchanged for OGD medium bubbled with 95% N2/5% CO2 for 10 min. The cultures were then transferred to an

SC

anaerobic chamber with an N2-enriched atmosphere at 37 ºC, where they were maintained for 60 min. During this period, control slices were maintained in an incubator with 5% CO2 atmosphere at 37ºC in culture medium. After the period

M AN U

of OGD, the slice cultures were carefully washed twice with HBSS and then incubated for 24 h in culture medium in the absence or presence of JM-20 (0.1 or 10 µM) at 37ºC in a 5% CO2/95% O2 atmosphere, mimicking the recovery period. Respective controls were produced and maintained without exposure to OGD. JM-20 was dissolved in dimethyl sulfoxide (DMSO) and added to the

2.4.

TE D

culture medium at a 1/1000 (v/v) dilution.

Quantification of cell death

Cellular damage was assessed by fluorescent image analysis of propidium

EP

iodide (PI) uptake (Noraberg et al., 1999). PI is a polar compound that is impermeable to the intact cell membrane, but it is able to permeate the damaged membranes of dying cells, where it binds to nuclear DNA to generate

AC C

a bright red fluorescence (Macklis and Madison, 1990). Two hours before the end of the recovery period, PI (5 µM) was added to the culture medium. Cultures were observed with an inverted microscope (Nikon Eclipse TE 300) using a standard rhodamine filter set. Images were captured and then analyzed using Scion Image software (http://www.scioncorp.com). The area where PI fluorescence was detectable above background levels was determined using the ‘‘density slice’’ function of Scion Image software and compared to the total slice area to obtain the percentage of damage.

2.5.

Determination of cytokine levels in the culture medium

ACCEPTED MANUSCRIPT Twenty-four hours after OGD and treatment with JM-20 (following the recovery period), the culture media was collected, rapidly frozen and stored at 20 ºC until determination of tumor necrosis factor a (TNF-α), interleukin-1β (IL1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels using specific enzymelinked immunosorbent assay (ELISA) kits according to the recommendations of

2.6.

RI PT

the supplier (R&D Systems).

Immunohistochemistry

Cultured hippocampal slices were fixed in 4% paraformaldehyde in 0.1 M

SC

phosphate buffer pH 7.4 for 2 h, blocked for 2 h at 22-24°C in 3% BSA and 0.1% Triton X-100 in phosphate-buffered saline (PBS), and incubated overnight at 4 ºC with Isolectin B4-FITC (IB4, 1:500, Sigma) to microglia labeling, or with

M AN U

primary antibody against glial fibrillary acidic protein (GFAP, 1:500, rabbit Sigma) to label astrocytes, followed by incubation with the appropriate fluorochrome-conjugated secondary antibody (alexa fluor 488 anti-rabbit 1:500, Invitrogen) for 2 h at room temperature. Sections were counterstained with DAPI (1:1000, 0.1%, Sigma) and mounted in DPX Mounting Medium (Sigma). The CA1, CA3 and dentate gyrus (DG) subfields of the hippocampus were

TE D

examined using an Olympus Fluoview FV1000 confocal microscope (Olympus, Japan) and fluorescence was analyzed using ImageJ software in 3 regions of interest (ROI) per subfield of the captured images.

EP

Data are presented as percentage of corrected fluorescence intensity [CFI = Integrated Density - (ROI area)*(Mean fluorescence of background readings)] with respect to control group. We did not observed any non-specific staining in

AC C

control sections prepared by omitting primary antibody.

2.7.

Western blot analysis

To investigate the phosphorylation status of Akt and GSK-3β, hippocampal slices were homogenized in lysis buffer (4% sodium dodecylsulfate (SDS), 2.1 mM EDTA, and 50 mM Tris) 24 h after OGD. Aliquots were taken for protein determination (Peterson, 1979), and β-mercaptoethanol was added to a final concentration of 5%. Proteins were resolved (50 µg per lane) on 12% SDSPAGE. After electrophoresis, proteins were electrotransferred to nitrocellulose

ACCEPTED MANUSCRIPT membranes using a semi-dry transfer apparatus (Bio-Rad Trans-Blot SD, Hercules, CA, USA). Membranes were incubated for 60 min at 4°C in blocking solution (Tris-buffered saline containing 5% powdered skim milk and 0.1% Tween-20, pH 7.4) and further incubated overnight at 4°C with the appropriate primary antibody dissolved in the blocking solution. The primary antibodies anti-

RI PT

phosphorylated Akt (Ser473; p-Akt, 1:1000, Cell Signaling Technology), anti-Akt (1:1000; Cell Signaling Technology), anti-phosphorylated GSK-3β (Ser9; pGSK-3β, 1:1000, Cell Signaling Technology), anti-GSK-3β (1:1000, Cell Signaling Technology) and anti-β-actin (1:1000, Cell Signaling Technology)

SC

were used. The membranes were then incubated with horseradish peroxidaseconjugated anti-rabbit antibody (1:1000, Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 2 hr. Chemiluminescence (ECL, Amersham

M AN U

Pharmacia Biotech) was detected using X-ray films (Kodak X-Omat, Rochester, NY, USA) that were scanned and analyzed using the Optiquant Software (Packard Instruments). Data are expressed as percentage of control values.

2.8.

Determination of Caspase-3 proteolytic activity The

fluorescent

probe

N-Acetil-Asp-Glu-Val-Asp-7-amido-4-

TE D

trifluoromethylcoumarin (Ac-DEVD-AFC; Sigma) was used 24 h after OGD exposure to estimate caspase-3 activation. Active caspase-3 hydrolyzes the peptide substrate Ac-DEVD-AFC, resulting in the release of a fluorescent 7-

EP

amido-4-trifluoromethylcoumarin (AFC) moiety. Following 24 hours of recovery, the slices were taken out of culture membranes and lysed in an ice-cold solution of PBS and 0.2% TritonX-100. The extract was centrifuged at 10,000 g for 5 min

AC C

and the supernatant was collected. For each experiment, 30 mg of protein (Peterson, 1979) was incubated with a reaction buffer containing 300 µM sucrose, 2 µM CHAPS, 0.01% BSA and 92.2 µM HEPES–NaOH, pH 7.5 in a 96 well black plate. The caspase-3 substrate was present at a final concentration of 20 µM. The plate was incubated at 37°C for 10 min under agitation. The linear increase of fluorescence intensity at 520 nm was monitored during the incubation time using a POLARstar Omega fluorescence spectrophotometer (Germany; Kuzelova et al., 2011).

2.9.

Statistical analysis

ACCEPTED MANUSCRIPT GraphPad Prism 5.0 software (GraphPad Software Inc., USA) was used to determine significant differences among experimental groups. The data were expressed as the means ± SEM. Comparisons among different groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. Differences were considered statistically significant

RI PT

at p<0.05.

3. Results

JM-20 treatment protects cells from OGD-induced damage

SC

3.1

The exposure of organotypic hippocampal slice cultures to 60 min of OGD caused a marked increase in PI fluorescence after 24 h of recovery,

M AN U

indicating the induction of cellular death, as observed in Figure 1A. Quantification of PI fluorescence showed that OGD caused approximately 50% cell damage in the hippocampus (p < 0.05) compared to control slices (Figure 1B). Treatment with JM-20 at 0.1 and 10 µM decreased cell death by 23% and 37%, respectively. DMSO, the vehicle for JM-20, had no effect on PI

3.2

TE D

fluorescence in control cultures (i.e., hippocampal slices not exposed to OGD).

Treatment with JM-20 decreased the release of pro-inflammatory and

EP

augmented anti-inflammatory IL-10 cytokine release after OGD We investigated the effects of JM-20 on pro-inflammatory (TNF-α, IL-1β, IL-6) and anti-inflammatory (IL-10) cytokine levels. Figure 2 shows a significant

AC C

increase in TNF-α, IL-1β and IL-6 levels and a significant decrease in IL-10 level in the medium of OGD-exposed cultures at the end of the recovery period (Panels A, B, C and D, respectively). JM-20 treatment counteracted the increase in cytokine release caused by OGD in a concentration-dependent fashion (by 22% and 46% for TNF-α, 23% and 44% for IL-1β, and 22% and 40% for IL-6, at 0.1 and 10 µM, respectively); additionally, JM-20 at 10 µM abolished the decrease in IL-10 release observed in the OGD-exposed cultures. The concentration–response pattern for JM-20 effects observed here was similar, suggesting a relationship between them. JM-20 treatment without OGD damage had no effect (Figure 2).

ACCEPTED MANUSCRIPT 3.3

Effects of JM-20 on glial cells Because the increase in cytokine release is often attributed to glia –

specifically, astrocytes and microglia - the activation of these two cellular types upon OGD was examined using immunohistochemical techniques. After 24 h of recovery (re-oxygenation), slices exposed to OGD

RI PT

conditions displayed almost a 2-fold increase in IB4 staining intensity, a microglia marker, across the entire hippocampus compared to controls (Figure 3), which was more pronounced in the CA3 region (Table 1). This increase was significantly lowered by JM-20 treatment in a region-dependent manner (by

SC

59% and 152% at 10 µM in CA1 and CA3, respectively). OGD insult or treatment with the drug caused no effects on microglia in DG. JM-20 treatment

M AN U

had no effect on control cultures (data not shown).

On the other hand, immunostaining for GFAP showed no alterations in OGD-exposed cultures when compared to control cultures treated with DMSO, as illustrated in Figure 4. Interestingly, GFAP immunocontent was found to be markedly increased in slices exposed to OGD and treated with JM-20 10 µM in both CA1 and DG (Table 2). No change in GFAP immunocontent was observed

3.4

TE D

in control cultures treated with JM-20 (0.1 or 10 µM; data not shown).

JM-20 prevents OGD-induced changes in the phosphorylation states of

Akt and GSK-3β. explore

possible

EP

To

mechanisms

by

which

JM-20

exerts

its

neuroprotective effects after OGD, we analyzed the PI3K cell signaling pathway

AC C

by measuring its effects on the phosphorylation of proteins Akt and GSK-3β via Western blotting.

As shown in Figure 5A, the phosphorylation of Akt was

significantly decreased (by 40%) after exposure of the culture to OGD. This effect was counteracted by JM-20 treatment at both tested concentrations (0.1 µM and 10 µM), suggesting that this molecule could act by activating the Akt protein. We also observed that OGD exposure decreased the phosphorylation of GSK-3β (Figure 5B), resulting in the activation of this pro-apoptotic protein. JM-20 treatment at 0.1 µM prevented such dephosphorylation, and JM-20 at concentrations of 10 µM increased the phosphorylation of GSK-3β, which

ACCEPTED MANUSCRIPT indicated an increase in its inactivation. No differences in the total immunocontent of Akt and GSK-3β were observed.

3.5

Effects of JM-20 on Caspase-3 activation following OGD We explored the effects of JM-20 on caspase-3 activity measured as the

RI PT

rate of cleavage of its experimental substrate, Ac-DEVD-AFC. DEVD constitutes a fluorescent-labeled synthetic tetrapeptide that represents the upstream amino acid sequence of the caspase-3 cleavage site in PARP.

The activation of caspase-3 was evidenced by the increase in fluorescence

SC

intensity (1.12-fold increase) in OGD-exposed slices compared to the control group. JM-20 treatment at 10 µM was able to prevent the induction of caspase3 activity (Figure 6). Treatment at a lower concentration (0.1 µM) had no effect.

M AN U

These results suggest that JM-20 may prevent OGD-induced apoptosis through the suppression of caspase-3 activity, as well as through Akt activation, a prosurvival protein kinase (Figure 5A). Both are critical for neuroprotection (Endo et al., 2006;Thornberry and Lazebnik, 1998; Zhao et al., 2006).

TE D

4. Discussion

The use of hippocampal slice cultures as an experimental system for the study of cellular and molecular mechanisms involved in neuronal death, as well

EP

as for pharmacological evaluations, has increased in recent years (Holopainen, 2005). Cultured slices maintain the morphological, cellular and functional organization of the hippocampus, preserving its laminated structure, synaptic

AC C

contacts, and receptor expression (Nishizawa, 2001). Ischemic injury represented by OGD is an in vitro model widely used to

mimic acute stroke. In this model, the exposure of slice cultures to OGD induces a selective death of pyramidal neurons in the CA1 region, within 4 h, similar to that observed in vivo, and the damage extends to neighboring neurons in CA3 following 72 h (Cho et al., 2007). In accordance with this, we observed that 60 min of OGD produced a noteworthy increase in PI incorporation in CA1 and to a lesser degree in DG, indicating the occurrence of neuronal death. In contrast, the CA3 region showed minor PI uptake at this early stage (24 hours) after reperfusion. Cell injury was

ACCEPTED MANUSCRIPT accompanied by a reduction in phosphorylated Akt, a serine/threonine kinase that plays a critical role in cell survival through the negative regulation of proapoptotic proteins such as GSK-3β (Cross et al., 1995). Here, we confirmed that treatment with JM-20 protected cultured hippocampal slices against OGD insult, which is consistent with our previous

RI PT

results (Nuñez-Figueredo et al., 2014a). We then investigated the potential mechanisms implicated in JM-20 neuroprotection. Treatment with the drug at low micromolar concentrations reduced neuronal death, showing a PI incorporation pattern similar to that observed in non-damage slices. JM-20 also

SC

prevented the decrease of Akt phosphorylation, which is the active form of this pro-survival kinase. Likewise, OGD caused a decrease in the phosphorylation of the PI3K/Akt downstream target, GSK-3β, indicating the activation of this pro-

may

contribute

to

neuronal

M AN U

apoptotic mediator. This effect was also prevented by JM-20 treatment, which survival

by

blocking

GSK-3β-dependent

mechanisms of cell death. GSK-3β is a ubiquitously expressed protein serine/threonine kinase, initially identified by its ability to phosphorylate glycogen synthase, the rate-limiting enzyme of glycogen synthesis in mammals (Welsh et al., 1996). Its activation is related to the induction of apoptosis in

TE D

response to numerous neuronal insults (Al Rahim et al., 2013). The pro-apoptotic actions of GSK-3β have been related to the phosphorylation and regulation of Bax, a pro-apoptotic Bcl-2 family member that

EP

stimulates the intrinsic cell death pathway by eliciting cytochrome C release from mitochondria (Linseman et al., 2004). Cytosolic cytochrome C then interacts with Apaf-1 and pro-caspase-9 to form a functional apoptosome that

AC C

ultimately activates downstream executioner caspase-3 (Zou et al., 1999). In this regard, we recently observed (Nuñez-Figueredo, 2014a) that JM-20 administration 1 h after reperfusion prevented the release of MCAo-induced cytochrome C from rat brain mitochondria, likely through the inhibition of calcium uptake and mitochondrial permeability transition pores formation. Thus, both effects could contribute to the inhibition of apoptotic cell death, converging on the suppression of caspase-3 activity observed here. These results suggest that the neuroprotective effects of JM-20 against OGD-induced cell death in organotypic cultures may involve its ability to maintain the phosphorylated (activated) state of the Akt kinase, which in turn may block apoptosis by

ACCEPTED MANUSCRIPT phosphorylating its downstream substrates, such as GSK-3β, shifting the balance between cell death and survival toward the survival path. It is important to note that this anti-apoptotic effect could also be attributed to an early anti-excitotoxic action of JM-20 previously documented in vitro and in vivo (Nuñez-Figueredo et al., 2014a, 2015). Nevertheless, the

RI PT

neuroprotective effects observed against glutamate-induced neuronal cell death, even when GABA receptors were blockaded with pentylenetetrazole (Nuñez-Figueredo et al., 2014b), and the direct mitoprotective effects displayed on the isolated organelles in vitro (Nuñez-Figueredo et al., 2014b, 2015)

SC

support the possibility of direct anti-apoptotic mechanisms as well.

An abundance of evidence suggests that neuroinflammatory processes play determinant roles in the severity and progression of cerebral damage, at

M AN U

least in the acute phase (Polazzi and Contestabile, 2002). Cytokines are thought to be crucial mediators of these complex inflammatory reactions and are usually used as biochemical markers of inflammation and neuronal damage (Mehta et al., 2007). Pro-inflammatory cytokines are involved in the modulation of ischemic damage in rodents, and their levels increase in cerebrospinal fluids (CSF) and serum after cerebral ischemia in humans (McCoy and Tansey, 2008;

TE D

Whiteley et al., 2009). Although neuroprotective roles have been ascribed to TNF-α (Bruce et al., 1996; Lambertsen et al., 2009), one of the most extensively studied cytokines in ischemia, administration of a neutralizing TNF-α antibody or

EP

soluble TNF-α receptor I exerts protective effects, attributing a pathologic significance to TNF-α (Barone et al., 1997; Lavine et al., 1998). Unlike TNF-α, IL-1β possesses a clearly defined pathologic effect (Touzani et al., 1999; Viviani

AC C

et al., 2003). The mechanisms that underlie the effects of these molecules in neuronal injury have not been definitively elucidated, but several putative pathways have been identified, including the disruption of blood-brain barrier permeability (Kim et al., 1992), the acceleration of neutrophils and leukocyte infiltration into the brain (Pober and Cotran, 1990), the induction of expression and release of several neurotoxic mediators, such as reactive oxygen and nitrogen species, eicosanoids and pro-inflammatory cytokines (Tolosa et al., 2011), increased neuronal NMDA receptor (NMDAR) activity and calcium influx through the NMDAR ion channel (Viviani et al., 2003), and the potentiation of neuronal death (Tolosa et al., 2011; Touzani et al., 1999).

ACCEPTED MANUSCRIPT We also investigated changes in the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and IL-10 in the supernatants of hippocampal slice cultures treated with DMSO or JM-20 at the end of the recovery period. A substantial increase in pro-inflammatory cytokines was induced by OGD, in contrast with the reduction of anti-inflammatory IL-10 (Figure 2A-D), suggesting

RI PT

the establishment of a deleterious inflammatory reaction after the ischemic insult. Treatment with 10 µM JM-20 significantly reduced the secretion of TNFα, IL-1β and IL-6 with respect to that observed in non-treated hippocampal cultures

exposed

to

OGD,

suggesting

that

JM-20

could

modulate

SC

neuroinflammation. Moreover, the drug prevented the reductions in the levels of the anti-inflammatory cytokine IL-10, which can suppress the production of a variety of pro-inflammatory molecules, including the aforementioned cytokines

M AN U

(Howard and O'Garra, 1992). However, more experiments are needed to clarify if the effects of JM-20 were exerted by acting directly on the inflammatory process, or if they were a consequence of its effects, decreasing cell death, which, in turn, also leads to decreased inflammation.

It could also be expected that an early anti-excitotoxic/GABAergic effect of JM-20 may be indirectly involved in its anti-inflammatory actions. After

TE D

ischemia, the massive increase of extracellular glutamate is followed by the activation of resident immune cells, and production of inflammation mediators, which could cause the enlargement of the cerebral infarct (Dirnagl et al., 1999).

EP

Thus, by acting on the initial stages of the ischemic cascade, JM-20 could prevent delayed events such as neuroinflammation or apoptosis. Astrocytes and microglia are the main neural cells involved in brain

AC C

inflammation. When activated, both types of glial cells are able to synthesize, release and respond to released cytokines through autocrine or paracrine mechanisms (John et al., 2003). Here, astroglial and microglial activation was characterized using the immunocontent of GFAP and IB4 staining, respectively (Figures 3 and 4, Tables 1 and 2). We showed OGD-induced microglia activation, in the CA3 area, following 24 h of re-oxygenation, and this effect was effectively inhibited by JM-20 at 10 µM concentrations. However, no significant change in GFAP immunoreactivity was observed in non-treated OGD cultures, suggesting that OGD-induced pro-inflammatory cytokine production was more dependent on microglial activation than on reactive astrocytes in our

ACCEPTED MANUSCRIPT experimental conditions. Interestingly, a 10 µM concentration of JM-20 induced an almost 2.0- fold increase in CA1 and a 2.3-fold increase in DG (when compared to damaged cultures) in GFAP immunoreactivity, suggesting that the drug was able to stimulate astroglial cells in both hippocampal regions. The region-selective activation of glial cells observed here could be

RI PT

caused, at least in part, by a direct effect of OGD on astrocytes and microglia, but it also could be attributed to a selective response to post-ischemic changes caused by the OGD, such as alterations in extracellular ions and amino acid concentrations, cytokines, growth factor release or neuronal dysfunction. In this

SC

regard, it is well known that in injured tissues, cellular death and the resulting accumulation of debris begin a healing process that involves the activation of microglial cells, the brain’s resident macrophages. Unfortunately, the cytotoxic

M AN U

inflammatory mediators that are produced as a result of glial over-activation could play a dual role, maintaining and even worsening the initial tissue damage (Morganti-Kossmann et al., 2002). Thus, a proper balance between beneficial and detrimental effects is required for the temporal and spatial restriction of inflammatory reaction.

It has been reported that the interaction of astrocytes and neurons with

TE D

microglial cells may act as a regulatory mechanism to suppress excessive microglial activation (Kim et al., 2010). Astrocytes appear to participate in the suppression of microglial activation through negative feedback loops, as

EP

demonstrated in several in vitro studies in which astrocyte-conditioned medium or the presence of astrocytes attenuate microglial activation in response to various pro-inflammatory stimuli (Hailer et al., 2001; Min et al., 2006; Vincent et

AC C

al., 1997). Accordingly, the astrocyte activation elicited by JM-20 may help to counteract excessive microglia activation, thus limiting neuroinflammation. Furthermore, we recently observed that JM-20 enhanced glutamate uptake in astrocytes and astrocyte-neuron co-cultures, but not in neurons cultured alone (Nuñez-Figueredo et al., 2015). This suggests that unidentified astrocytederived factors may be responsible for the JM-20 effects, either by eliciting direct neuroprotective effects or by modulating the microglial response to neuronal injury. It is interesting to note that such effects on astrocytes are exclusively dependent upon a cytotoxic insult, as no differences between control slices

ACCEPTED MANUSCRIPT treated with JM-20 (not exposed to OGD) were observed with respect to DMSO-treated cultures. This could be relevant for neurological diseases in which the neuroprotective mechanisms (metabolic support, growth factor and nutrient supply, and extracellular homeostasis) provided by astrocytes to neurons are eventually exhausted, especially in CA1 region where pyramidal

RI PT

neurons have shown increased vulnerability (Belanger and Magistretti, 2009), as has been proposed for cerebral ischemia. In fact, pathological stimuli such as oxidative stress, excitotoxicity, metabolic failure and inflammation, are all known to be counteracted by astrocytes (Belanger and Magistretti, 2009).

SC

Moreover, the JM-20 effect on astroglia could account for its anti-excitotoxic activity observed in rats subjected to 90 min of MCAo (Nuñez-Figueredo et al., 2014a), as astrocytes are believed to be responsible for most glutamate uptake

M AN U

in synaptic areas and consequently are key regulators of glutamate homeostasis (Belanger and Magistretti, 2009).

In summary, the results presented here provide important new insights into the protective effects of JM-20 on ischemic injury. The present data suggest that, in parallel to its neuroprotective effects, JM-20 could modulate the inflammatory process and the cell signaling pathway related to cell survival

TE D

although an indirect contribution mediated by the early anti-excitotoxic action could also account for the overall effects. Moreover, its effect on astrocytes could represent an additional protective mechanism for neurons, considering

EP

the extensive functional cooperation that exists between these two cell types. Altogether, our results provide evidence that may justify the application of JM20 as a new drug for the treatment of the acute phase of ischemic stroke, for

AC C

which further studies are needed.

Acknowledgments

This work was partially supported by CAPES-Brazil/MES-Cuba projects 140/11 and 092/10, INCT-EN/CNPq (Brazil), IBN.Net/CNPq (Brazil), FAPERGS/RS, and the Non-Governmental Organization MEDICUBA-SPAIN.

ACCEPTED MANUSCRIPT References Al Rahim, M., Thatipamula, S., Hossain, M.A., 2013. Critical role of neuronal pentraxin 1 in mitochondria-mediated hypoxic-ischemic neuronal injury. Neurobiol Dis 50, 59-68.

RI PT

Barone, F.C., Arvin, B., White, R.F., Miller, A., Webb, C.L., Willette, R.N., Lysko, P.G., Feuerstein, G.Z., 1997. Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury. Stroke 28, 1233-1244. Barone, F.C., Feuerstein, G.Z., 1999. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab 19, 819-834.

SC

Belanger, M., Magistretti, P.J., 2009. The role of astroglia in neuroprotection. Dialogues Clin Neurosci 11, 281-295.

M AN U

Benakis, C., Bonny, C., Hirt, L., 2010. JNK inhibition and inflammation after cerebral ischemia. Brain Behav Immun 24, 800-811. Brazil, D.P., Yang, Z.Z., Hemmings, B.A., 2004. Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 29, 233-242.

TE D

Bruce, A.J., Boling, W., Kindy, M.S., Peschon, J., Kraemer, P.J., Carpenter, M.K., Holtsberg, F.W., Mattson, M.P., 1996. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat Med 2, 788-794. Cross, D.A., Alessi, D.R., Cohen, P., Andjelkovich, M., Hemmings, B.A., 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. . Nature 378, 785-789.

EP

Cho S., W.A., Bowlby M.R., 2007. Brain Slices as Models for Neurodegenerative Disease and Screening Platforms to Identify Novel Therapeutics. Current Neuropharmacology 5, 19-33.

AC C

del Zoppo, G., Ginis, I., Hallenbeck, J.M., Iadecola, C., Wang, X., Feuerstein, G.Z., 2000. Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol 10, 95-112. Dirnagl, U., Iadecola, C., Moskowitz, M.A., 1999. Pathobiology of ischaemic stroke: an integrated view. Trends in Neurosciences 22, 391-397. Doyle, K.P., Simon, R.P., Stenzel-Poore, M.P., 2008. Mechanisms of ischemic brain damage. Neuropharmacology 55, 310-318. Endo, H., Nito, C., Kamada, H., Nishi, T., Chan, P.H., 2006. Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats. J Cereb Blood Flow Metab 26, 1479-1489.

ACCEPTED MANUSCRIPT Figueredo, Y.N., Rodriguez, E.O., Reyes, Y.V., Dominguez, C.C., Parra, A.L., Sanchez, J.R., Hernandez, R.D., Verdecia, M.P., Pardo Andreu, G.L., 2013. Characterization of the anxiolytic and sedative profile of JM-20: a novel benzodiazepine-dihydropyridine hybrid molecule. Neurol Res 35, 804-812.

RI PT

Hailer, N.P., Wirjatijasa, F., Roser, N., Hischebeth, G.T., Korf, H.W., Dehghani, F., 2001. Astrocytic factors protect neuronal integrity and reduce microglial activation in an in vitro model of N-methyl-D-aspartate-induced excitotoxic injury in organotypic hippocampal slice cultures. Eur J Neurosci 14, 315-326. Holopainen, I.E., 2005. Organotypic hippocampal slice cultures: a model system to study basic cellular and molecular mechanisms of neuronal cell death, neuroprotection, and synaptic plasticity. Neurochem Res 30, 1521-1528.

SC

Howard, M., O'Garra, A., 1992. Biological properties of interleukin 10. Immunol Today 13, 198-200.

M AN U

Howells, D.W., Donnan, G.A., 2010. Where will the next generation of stroke treatments come from? PLoS Med 7, e1000224. Jeong, H.K., Ji, K., Min, K., Joe, E.H., 2013. Brain inflammation and microglia: facts and misconceptions. Exp Neurobiol 22, 59-67. John, G.R., Lee, S.C., Brosnan, C.F., 2003. Cytokines: powerful regulators of glial cell activation. Neuroscientist 9, 10-22.

TE D

Kim, A.H., Yano, H., Cho, H., Meyer, D., Monks, B., Margolis, B., Birnbaum, M.J., Chao, M.V., 2002. Akt1 regulates a JNK scaffold during excitotoxic apoptosis. Neuron 35, 697-709.

EP

Kim, J.H., Min, K.J., Seol, W., Jou, I., Joe, E.H., 2010. Astrocytes in injury states rapidly produce anti-inflammatory factors and attenuate microglial inflammatory responses. J Neurochem 115, 1161-1171.

AC C

Kim, K.S., Wass, C.A., Cross, A.S., Opal, S.M., 1992. Modulation of blood-brain barrier permeability by tumor necrosis factor and antibody to tumor necrosis factor in the rat. Lymphokine Cytokine Res 11, 293-298. Kreutzberg, G.W., 1995. Microglia, the first line of defence in brain pathologies. Arzneimittelforschung 45, 357-360. Kulik, G., Klippel, A., Weber, M.J., 1997. Antiapoptotic signalling by the insulinlike growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17, 1595-1606. Kuzelova, K., Grebenova, D., Brodska, B., 2011. Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes. J Cell Biochem 112, 3334-3342. Lambertsen, K.L., Clausen, B.H., Babcock, A.A., Gregersen, R., Fenger, C., Nielsen, H.H., Haugaard, L.S., Wirenfeldt, M., Nielsen, M., Dagnaes-Hansen, F., Bluethmann, H., Faergeman, N.J., Meldgaard, M., Deierborg, T., Finsen, B.,

ACCEPTED MANUSCRIPT 2009. Microglia protect neurons against ischemia by synthesis of tumor necrosis factor. J Neurosci 29, 1319-1330. Lavine, S.D., Hofman, F.M., Zlokovic, B.V., 1998. Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury. J Cereb Blood Flow Metab 18, 52-58.

RI PT

Linseman, D.A., Butts, B.D., Precht, T.A., Phelps, R.A., Le, S.S., Laessig, T.A., Bouchard, R.J., Florez-McClure, M.L., Heidenreich, K.A., 2004. Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci 24, 9993-10002.

SC

Macklis, J.D., Madison, R.D., 1990. Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. J Neurosci Methods 31, 43e46.

M AN U

McCoy, M.K., Tansey, M.G., 2008. TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation 5, 45. Mehta, S.L., Manhas, N., Raghubir, R., 2007. Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Res Rev 54, 34-66.

TE D

Min, K.J., Yang, M.S., Kim, S.U., Jou, I., Joe, E.H., 2006. Astrocytes induce hemeoxygenase-1 expression in microglia: a feasible mechanism for preventing excessive brain inflammation. J Neurosci 26, 1880-1887. Morganti-Kossmann, M.C., Rancan, M., Stahel, P.F., Kossmann, T., 2002. Inflammatory response in acute traumatic brain injury: a double-edged sword. Curr Opin Crit Care 8, 101-105.

EP

Nishizawa, Y., 2001. Glutamate release and neuronal damage in ischemia. Life Sci 69, 369-381.

AC C

Noraberg, J., Kristensen, B.W., Zimmer, J., 1999. Markers for neuronal degeneration in organotypic slice cultures. Brain Res Brain Res Protoc 3, 278290. Nuñez-Figueredo, Y., Pardo Andreu, G.L., Oliveira Loureiro, S., Ganzella, M., Ramírez-Sánchez, J., Ochoa-Rodríguez, E., Verdecia-Reyes, Y., DelgadoHernández, R., Souza, D.O., 2015. The effects of JM-20 on the glutamatergic system in synaptic vesicles, synaptosomes and neural cells cultured from rat brain. Neurochem Int 81, 41-47. Nuñez-Figueredo, Y., Ramírez-Sánchez J., Hansel G., Nicoloso E., Merino N., Valdes O., Delgado-Hernández R., Lagarto-Parra A., Ochoa-Rodríguez E., Verdecia-Reyes Y., Salbego C., Costa S. L., Souza D.O. and Pardo-Andreu G. L., 2014a. A novel multi-target ligand (JM-20) protects mitochondrial integrity, inhibits brain excitatory amino acid release and reduces cerebral ischemia injury in vitro and in vivo. Neuropharmacology 85, 517–527.

ACCEPTED MANUSCRIPT Nuñez-Figueredo, Y., Ramirez-Sanchez, J., Delgado-Hernandez, R., PortoVerdecia, M., Ochoa-Rodriguez, E., Verdecia-Reyes, Y., Marin-Prida, J., Gonzalez-Durruthy, M., Uyemura, S.A., Rodrigues, F.P., Curti, C., Souza, D.O., Pardo-Andreu, G.L., 2014b. JM-20, a novel benzodiazepine-dihydropyridine hybrid molecule, protects mitochondria and prevents ischemic insult-mediated neural cell death in vitro. . Eur J Pharmacol 726C, 57-65.

RI PT

Pekny, M., Nilsson, M., 2005. Astrocyte activation and reactive gliosis. Glia 50, 427-434. Peterson, G.L., 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 100, 201-220.

SC

Pober, J.S., Cotran, R.S., 1990. Cytokines and endothelial cell biology. Physiol Rev 70, 427-451. Polazzi, E., Contestabile, A., 2002. Reciprocal interactions between microglia and neurons: from survival to neuropathology. Rev Neurosci 13, 221-242.

M AN U

Simao, F., Zamin, L.L., Frozza, R., Nassif, M., Horn, A.P., Salbego, C.G., 2009. Protective profile of oxcarbazepine against oxygen-glucose deprivation in organotypic hippocampal slice culture could involve PI3K cell signaling pathway. Neurol Res 31, 1044-1048.

TE D

Song, J.J., Lee, Y.J., 2005. Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop. J Cell Biol 170, 61-72. Song, Y.S., Narasimhan, P., Kim, G.S., Jung, J.E., Park, E.H., Chan, P.H., 2008. The role of Akt signaling in oxidative stress mediates NF-kappaB activation in mild transient focal cerebral ischemia. J Cereb Blood Flow Metab 28, 1917-1926.

EP

Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37, 173-182.

AC C

Strasser, U., Fischer, G., 1995. Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation. J Neurosci Methods 57, 177-186. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 281, 1312-1316. Thrift, A.G., Dewey, H.M., Macdonell, R.A., McNeil, J.J., Donnan, G.A., 2001. Incidence of the major stroke subtypes: initial findings from the North East Melbourne stroke incidence study (NEMESIS). Stroke 32, 1732-1738. Tolosa, L., Caraballo-Miralles, V., Olmos, G., Llado, J., 2011. TNF-alpha potentiates glutamate-induced spinal cord motoneuron death via NF-kappaB. Mol Cell Neurosci 46, 176-186.

ACCEPTED MANUSCRIPT Touzani, O., Boutin, H., Chuquet, J., Rothwell, N., 1999. Potential mechanisms of interleukin-1 involvement in cerebral ischaemia. J Neuroimmunol 100, 203215.

RI PT

Valentim, L.M., Rodnight, R., Geyer, A.B., Horn, A.P., Tavares, A., Cimarosti, H., Netto, C.A., Salbego, C.G., 2003. Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures. Neuroscience 118, 379-386. Verkhratsky, A., Rodriguez, J.J., Parpura, V., 2013. Astroglia in neurological diseases. Future Neurol 8, 149-158.

SC

Vincent, V.A., Tilders, F.J., Van Dam, A.M., 1997. Inhibition of endotoxininduced nitric oxide synthase production in microglial cells by the presence of astroglial cells: a role for transforming growth factor beta. Glia 19, 190-198.

M AN U

Viviani, B., Bartesaghi, S., Gardoni, F., Vezzani, A., Behrens, M.M., Bartfai, T., Binaglia, M., Corsini, E., Di Luca, M., Galli, C.L., Marinovich, M., 2003. Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci 23, 86928700.

TE D

Wang, R., Zhang, Q.G., Han, D., Xu, J., Lu, Q., Zhang, G.Y., 2006. Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats. J Neurochem 99, 1543-1554. Welsh, G.I., Wilson, C., Proud, C.G., 1996. GSK3: a SHAGGY frog story. Trends Cell Biol 6, 274-279.

EP

Whiteley, W., Jackson, C., Lewis, S., Lowe, G., Rumley, A., Sandercock, P., Wardlaw, J., Dennis, M., Sudlow, C., 2009. Inflammatory markers and poor outcome after stroke: a prospective cohort study and systematic review of interleukin-6. PLoS Med 6, e1000145.

AC C

Zhao, H., Sapolsky, R.M., Steinberg, G.K., 2006. Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke. Mol Neurobiol 34, 249-270.

ACCEPTED MANUSCRIPT Figure Legends Figure 1. JM-20-attenuated cell death caused by exposing organotypic hippocampal slice cultures to OGD. (A) Representative photomicrographs of propidium iodide (PI) uptake by slices incubated without (DMSO) or with 0.1 or

RI PT

10 µM of JM-20. (B) Quantification of PI uptake. JM-20 or DMSO (vehicle) was added to the culture medium immediately after OGD and maintained during the recovery period (24 h). Bars represent the means ± SEM (n = 9 cultures per group). Different letters indicate significant differences (p < 0.05) between

SC

groups, by ANOVA and post hoc Tukey Multiple Comparison Test.

Figure 2. Cytokine levels (pg/mL) in a culture medium of organotypic

M AN U

hippocampal slices. Tumor necrosis factor-α (TNF-α, A), interleukin-1β (IL-1β, B), interleukin-6 (IL-6, C) and interleukin-10 (IL-10, D) were measured at the end of the recovery period (24 h). JM-20 or DMSO (vehicle) was added to the culture medium immediately after OGD and maintained during the recovery period (24 h). Bars represent the means ± SEM (n = 6 cultures per group). Different letters indicate significant differences (p < 0.05) between groups, by

TE D

ANOVA and post hoc Tukey Multiple Comparison Test. Figure 3. Treatment with JM-20-suppressed microglia activation in hippocampal slice cultures. Activation of microglia in vehicle-treated (DMSO) or JM-20 (0.1 or

EP

10 µM)-treated cultures submitted to OGD was visualized by Isolectin-B4 (IB4, green) staining. After the recovery period (24 h) following OGD, microglial

AC C

activation was detected close to the pyramidal layer throughout hippocampus. Scale bar: 100 µm.

Figure 4. GFAP immunostaining of hippocampal slice cultures exposed to OGD treated with DMSO (vehicle) or with JM-20 (0.1 or 10 µM). Representative images of hippocampal slices showing GFAP (green) and DAPI (blue) immunoreactivity 24 h after OGD (recovery period). Scale bar: 100 µm [the insets are high magnification images (scale bar: 10 µm)].

ACCEPTED MANUSCRIPT Figure 5. Effects of treatment with JM-20 on the percentage of phosphorylated Akt and GSK-3β in organotypic hippocampal cultures 24 h after OGD, revealed using specific antibodies. (A) Representative Western blots of pAkt and Akt. (B) Representative Western blots of pGSK-3β and GSK-3β. Histograms represent the quantitative Western blotting analysis of the phosphorylation state

RI PT

of Akt (C) and GSK-3β (D). The densitometric values obtained for phospho- and total-Akt (or GSK-3β) from all treatments were first normalized to their respective vehicle-treated controls (DMSO bar; 100%) and then to β-actin. JM20 (0.1 or 10 µM) was immediately added to the culture medium and maintained

SC

during the recovery period (24 h). Data are presented as the means ± SEM (n = 6). Different letters indicate significant differences (p < 0.05) between groups,

M AN U

by ANOVA and post hoc Tukey Multiple Comparison Test.

Figure 6. Effects of JM-20 on caspase-3 activity. Organotypic hippocampal slice cultures were exposed to 60 minutes of OGD. JM-20 (0.1 or 10 µM) or DMSO (vehicle) was added to the culture medium during the recovery period. After 24 h of OGD exposure, cytosolic proteins were extracted and incubated with

fluorogenic

caspase-3

substrate

Acetil-Asp-Glu-Val-Asp-7-amido-4-

TE D

trifluoromethylcoumarin (Ac-DEVD-AFC). The results are presented as a percentage of DEVDase activity compared to control cultures (DMSO). Bars represent means ± SEM (n = 6). Different letters indicate significant differences (p < 0.05) between groups, by ANOVA and post hoc Tukey Multiple

AC C

EP

Comparison Test.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT HIGHLIGHTS

JM-20 has a strong neuroprotective effect against ischemic injury.

-

JM-20 attenuated the OGD-induced activation of caspase-3.

-

JM-20 suppressed the activation of microglia

-

JM-20 increased the GFAP immunireactivity in both, CA1 and DG areas of hippocampus

-

JM-20 decreased the release of interleukin (IL)-1β, IL-6 and (TNF)-α, and increased IL-10 release.

AC C

EP

TE D

M AN U

SC

RI PT

-