Neurotox Res (2014) 26:1–15 DOI 10.1007/s12640-013-9427-y
ORIGINAL ARTICLE
Cross-Talk Between Neurons and Astrocytes in Response to Bilirubin: Adverse Secondary Impacts Ana Sofia Falca˜o • Rui F. M. Silva • Ana Rita Vaz • Ca´tia Gomes • Adelaide Fernandes • Andreia Barateiro Claudio Tiribelli • Dora Brites
•
Received: 11 December 2012 / Revised: 23 September 2013 / Accepted: 24 September 2013 / Published online: 10 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Previous studies using monotypic nerve cell cultures have shown that bilirubin-induced neurological dysfunction (BIND) involves apoptosis and necrosis-like cell death, following neuritic atrophy and astrocyte activation, and that glycoursodeoxycholic acid (GUDCA) has therapeutic efficacy against BIND. Cross-talk between neurons and astrocytes may protect or aggravate neurotoxicity by unconjugated bilirubin (UCB). In a previous work we have shown that bidirectional signaling during astrocyte-neuron recognition attenuates neuronal damage by UCB. Here, we investigated whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB was instead associated with a lower resistance of
Electronic supplementary material The online version of this article (doi:10.1007/s12640-013-9427-y) contains supplementary material, which is available to authorized users. A. S. Falca˜o R. F. M. Silva A. R. Vaz C. Gomes A. Fernandes A. Barateiro D. Brites Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farma´cia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal A. S. Falca˜o R. F. M. Silva A. R. Vaz A. Fernandes D. Brites (&) Department of Biochemistry and Human Biology, Faculdade de Farma´cia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal e-mail:
[email protected] C. Tiribelli Centro Studi Fegato, AREA Science Park, Department of BBCM, University of Trieste, Bld Q, Basovizza Campus, Trieste, Italy C. Tiribelli Clinica Patologie Fegato-Liver Clinic, Ospedale Cattinara, Trieste, Italy
neurons to UCB toxicity, and if the pro-survival properties of GUDCA were replicated in that experimental model. We have introduced a 24 h adaptation period for neuron-glia communication prior to the 48 h treatment with UCB. In such conditions, UCB induced glial activation, which aggravated neuronal damage, comprising increased apoptosis, cell demise and neuritic atrophy, which were completely prevented in the presence of GUDCA. Neuronal multidrug resistance-associated protein 1 expression and tumor necrosis factor-a secretion, although unchanged by UCB, increased in the presence of astrocytes. The rise in S100B and nitric oxide in the co-cultures medium may have contributed to UCB neurotoxicity. Since the levels of these diffusible molecules did not change by GUDCA we may assume that they are not directly involved in its beneficial effects. Data indicate that astrocytes, in an indirect neuron-astrocyte co-culture model and after homeostatic setting regulation of the system, are critically influencing neurodegeneration by UCB, and support GUDCA for the prevention of BIND. Keywords Astrocyte activation Co-culture Glycoursodeoxycholic acid Neuron-astrocyte signaling Neuronal dysfunction Unconjugated bilirubin
Introduction Astrocytes and neurons form an intimate communication network. Early during development, astrocytes act as guiding structures for migratory neurons, and later they are not only the main source for nutrients and growth factors in the brain but are also signaling partners of neighboring neurons (Kirchhoff et al. 2001). Despite intervening in
123
2
preserving the brain from an immediate neurotoxic effect, the neuron-glia communication can aggravate a chronic insult and promote its propagation. Indeed, astrocyte dysfunction was shown to be implicated in brain degeneration (Sidoryk-Wegrzynowicz et al. 2011; Viviani 2006). Therefore, release of pro-survival factors by astrocytes may be neuroprotective by promoting neuron survival, synapse formation and plasticity (Butt 2011; Jones et al. 2012; Meeks and Mennerick 2003), while nitrosative stress resulting from inflammatory activation of astrocytes (Miller et al. 2011) may have detrimental effects on neurons by altering the microenvironment and exacerbating neuroinflammation (Anderl et al. 2009; Allaman et al. 2010; Domenici et al. 2002; Laird et al. 2008; MalchiodiAlbedi et al. 2001). Neonatal unconjugated hyperbilirubinemia is the most common clinical diagnosis during neonatal life (Kaplan and Hammerman 2004; Rubaltelli and Griffith 1992), and mostly occurs in a transient manner (physiologic jaundice) (Ostrow et al. 2002). However, above a still undefined concentration threshold, unconjugated bilirubin (UCB) can cause neurological damage via mechanisms not yet completely clarified (Hansen 2002). High-levels of UCB trigger oxidative stress in neurons through the production of nitric oxide (NO), superoxide anion radical and other reactive oxygen species (ROS), associated with the disruption of glutathione redox status (Brito et al. 2008a, b; Vaz et al. 2011b). These effects are prevented by glycoursodeoxycholic acid (GUDCA), a bile acid with anti-apoptotic, antioxidant and anti-inflammatory properties (Fernandes and Brites 2009), both in astrocytes (Fernandes et al. 2007) and neurons (Brito et al. 2008a). Although the contribution of astrocytes to bilirubin-induced neurological dysfunction (BIND) is still not entirely understood, astrocytes are considered to be a key player (Brites 2012) through the release of pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and IL-6, as well as glutamate, ATP and the production of free radicals (Fernandes et al. 2004, 2006; Falca˜o et al. 2005). All these events may lead to the degeneration of astrocytes in the brain, contributing to neuronal dysfunction (Allaman et al. 2011). The multidrug resistance-associated protein 1 (Mrp1) mediates the ATP-dependent cellular efflux of UCB (Rigato et al. 2004), at least from astrocytes (Gennuso et al. 2004). A reduced expression of Mrp1 can concur to the nerve cell vulnerability to UCB. We observed a decreased expression of Mrp1 in rat immature neurons and astrocytes (Falca˜o et al. 2007a) and its inhibition caused increased secretion of glutamate and cell death. The close interaction between neurons and astrocytes stimulated us to further explore the mechanisms of BIND in the co-culture system that considers the molecular
123
Neurotox Res (2014) 26:1–15
interactions between neurons and astrocytes, instead of the more common monocultures models (Brites 2012; Brito et al. 2008b; Falca˜o et al. 2006). In our previous study we simultaneously exposed astrocytes to neurons and UCB using the co-culture system, and observed that astrocytes prevented the UCB-induced neurotoxicity by reducing the loss of cell viability, dysfunction and death by apoptosis, as well as the impairment of neuritic outgrowth (Falca˜o et al. 2013). Data suggested that if a cell is primarily committed in signaling to a neighboring cell, it may become resistant to the direct effects of a neurotoxin, such as UCB. However, neurons may show reduced resistance to UCB if the bi-directional communication with astrocytes is formerly established (Brown 1999). Thus, in the present study, we aim to investigate whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB could be associated with a reduced resistance of neurons to UCB and if the pro-survival properties of GUDCA can be replicated in this experimental model. Herein, we provide experimental evidence that 48 h incubation with UCB triggered neuronal apoptosis, cell demise, and neuritic dystrophy, which further increased when cells were co-cultured with astrocytes after the homeostatic setting regulation of the system for 24 h. Soluble astrocyte-derived factors increased neuronal Mrp1 expression and TNF-a release. These in vitro results indicate that astrocytes change from a neuroprotective (Falca˜o et al. 2013) to a detrimental effect on neurons when are extensively activated by UCB, as suggested by the increased secretion of S100B and NO to the co-culture media, which was not observed in our prior study. Interestingly, GUDCA was not able to prevent the UCBinduced release of S100B and NO, but sustained neuron survival and neuritic development in mono- and co-culture models exposed to UCB. The present study clarifies that bidirectional communication between astrocytes and neurons may have different functional consequences in neonatal jaundice depending both on the cell–cell recognition momentum and the duration of exposure to UCB.
Materials and Methods Chemicals Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were purchased from Biochrom AG (Berlin, Germany). Neurobasal medium, B-27 Supplement (509), Hanks’ balanced salt solution (HBSS-1), Hanks’ balanced salt solution without Ca2? and Mg2? (HBSS-2), gentamicin (50 mg/mL), and trypsin (0.025 %) were
Neurotox Res (2014) 26:1–15
acquired from Invitrogen (Carlsbad, CA, USA). Antibiotic antimycotic solution (209), human serum albumin (HSA), fraction V, fatty acid free, Hoechst dye 33258, the fluorescent dye propidium iodide [PI; 3,8-diamino-5(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide] and MTT [3-(4,5-dimethylthiazol, 2-yl)-2,5diphenyltetrazolium bromide] were purchased from Sigma Chemical Co (St. Louis, MO, USA). UCB was obtained from Sigma and purified as mentioned (McDonagh 1979). Mouse anti-microtubule associated protein (MAP)-2 antibody (MAB3418) was from Merck Millipore (Darmstadt, Germany), FITC-labeled horse antibody anti-mouse was acquired from Vector (Burlingame, CA, USA). Nitrocellulose membrane and Hyperfilm ECL were from Amersham Biosciences (Piscataway, NJ, USA). Rabbit Mrp1A23 antibody was prepared in Centro Studi Fegato, Trieste, Italy (Fernetti et al. 2001) and mouse anti-b-actin antibody was from Sigma. Horseradish peroxidase-labeled goat antirabbit IgG and anti-mouse IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell lysis buffer and LumiGLOÒ were from Cell Signaling (Beverly, MA, USA). Sigma Fast O-phenylenediamine dihydrochloride tablet set (OPD) and monoclonal anti-S-100 (bSubunit) antibody were acquired from Sigma. Rabbit polyclonal anti-S100B protein was purchased from Dako (Glostrup, Denmark). GUDCA (minimum 96 % pure) was acquired from Calbiochem (Darmstadt, Germany). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). Animals Wistar rats were maintained on a 12 h light/dark cycle under conditions of constant temperature and humidity. Animals were supplied with standard laboratory chow and water ad libitum. Animal care followed the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals). All animal procedures were approved by the Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Methods In the present study we used our established experimental model of hyperbilirubinemia (Silva et al. 2012). Briefly, stock solutions were extemporarily prepared in 0.1 M NaOH, and the pH adjusted to 7.4 by addition of an equal amount of 0.1 M HCl. Preparation of solutions and
3
execution of experiments were performed under light protection (vial wrapped in tin foil and dim light) to avoid UCB photodegradation. In order to more closely mimic clinically relevant conditions, a molar ratio of UCB to HSA of 0.5 was used (50 lM UCB:100 lM HSA) (Brites 2012). Other concentrations were not tested once lower concentrations of UCB did not produce detectable cytotoxicity in our experimental model (Brito et al. 2008b) and doubled levels triggered a high loss of cell viability (Fernandes et al. 2009; Falca˜o et al. 2007a). By consistently using the same experimental model, besides having suitable and stable UCB solutions, we may easily cross link present and prior results in different cell types where the selected UCB concentration has been used (Silva et al. 2011, 2012; Vaz et al. 2010, 2011a, b; Fernandes et al. 2011). Moreover, with this UCB/HSA molar ratio of 0.5 we generate a concentration of free bilirubin of 20 nM (Palmela et al. 2012), which was previously observed in jaundiced newborns with serum bilirubin levels between 116 and 615 lM and C2.5 kg birth weight (Ahlfors et al. 2009), thus assuring that we are reproducing the clinical condition of hyperbilirubinemia that may lead to BIND. Neuron Cultures Neurons were isolated from fetuses of 17–18-day pregnant Wistar rats, as previously described (Silva et al. 2002). In short, pregnant rats were anesthetized and decapitated. The fetuses were collected in HBSS-1 and rapidly decapitated, the brain cortices were mechanically fragmented, and the fragments transferred to a 0.025 % trypsin in HBSS-2 solution and incubated for 15 min at 37 °C. Following trypsinization, cells were washed twice in HBSS-2 containing 10 % FCS, and resuspended in Neurobasal medium supplemented with 0.5 mM L-glutamine, 25 lM L-glutamic acid, 2 % B-27 Supplement, and 0.12 mg/mL gentamicin. Aliquots of 1.0 9 105 cells/cm2 were plated on 12-well poly-D-lysine-coated tissue culture plates and maintained at 37 °C in a humidified atmosphere of 5 % CO2. Neurons were cultured in poly-D-lysine-coated 12-well tissue culture plates or in glass coverslips placed in the bottom of culture plates, for the immunocytochemistry studies. Every 3 days, 0.5 mL of old medium was removed by aspiration and replaced by equal volume of fresh medium without L-glutamic acid. At 8 days-in vitro (DIV), the cultures were highly enriched in neuronal cells as the contaminant GFAP-positive cells were less than 1 % (Falca˜o et al. 2013). Astrocyte Cultures Astrocytes were isolated from 2-day-old rats, as previously described (Blondeau et al. 1993), with minor modifications
123
4
Neurotox Res (2014) 26:1–15
Astrocytes
Neurons Astrocytes
Cerebral cortex of P2 rats
Neurons Cell culture flask
DMEM
Cerebral cortex of E17 fetuses
9 DIV Cell culture insert
DMEM
8 DIV
24h Cell culture insert
DMEM
cell culture plate
Astrocytes
DMEM
A B C D
Neurobasal
Neurons Co-cultures DMEM
24h
A B C D
24h DMEM
DMEM
A B
HSA 100 µM 48 h
24h
DMEM
UCB 50 µM HSA 100 µM 48 h
DMEM
GUDCA 50 µM + UCB 50 µM HSA 100 µM 48 h 1h GUDCA 50 µM HSA 100 µM 48 h
C
D
Fig. 1 Schematic drawing of study design. Astrocytes were isolated from 2-day-old rats and cultured on tissue culture flasks for 9 daysin vitro (DIV) in DMEM. Neurons were isolated from rat fetuses at embryonic day 17 (E17) and plated on 12-well tissue culture plates for 8 DIV in supplemented Neurobasal medium. Astrocytes with 9 DIV were then seeded onto 0.4 lm culture plate inserts where they were left for 24 h in DMEM. Next, the astrocyte-containing inserts were co-cultured with 8 DIV neurons and maintained in that way during 24 h before being incubated in the absence (a) or in the presence of 50 lM UCB (plus 100 lM HSA) (b) for 48 h using DMEM as the incubation medium. In another set of experiments, cells were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA)
prior to the addition of 50 lM UCB during 48 h (c). In parallel experiments, cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h (d). Pure neuronal and astroglial cultures were incubated under similar conditions. After incubation, attached neurons from both pure neuronal cultures and co-cultures were used to determine apoptotic cell death, cellular viability, neurite extension, and ramification, as well as Mrp1 expression. The extracellular medium, free from cellular debris, was used for TNF-a, S100B, and NO determinations. DIV days in vitro, E embryonic day, GUDCA glycoursodeoxycholic acid, HSA human serum albumin, P postnatal day, UCB unconjugated bilirubin
(Silva et al. 1999). Briefly, rats were decapitated and the brains collected in DMEM containing 11 mM sodium bicarbonate, 38.9 mM glucose and 1 % antibiotic antimycotic solution. The cortical fraction was homogenized by mechanical fragmentation; cells were collected after centrifugation (10 min at 700 g) and resuspended in culture medium supplemented with 10 % FCS. Finally, 2.0 9 105 cells/cm2 were plated on tissue culture flasks and maintained at 37 °C in a humidified atmosphere of 5 % CO2, and cultured for 9 DIV. At this time point, the protocol resulted in highly pure astrocyte cultures, with a percentage of contaminant microglia that was less than 5 % (Falca˜o et al. 2013).
Co-cultures and Nerve Cell Treatments
123
Astrocytes with 9 DIV were removed from culture flasks by trypsinization and seeded (4.5 9 105 cells/mL) onto 0.4 lm MillicellÒ culture plate inserts (Merck Millipore, Germany), where they were left in DMEM for 24 h. These astrocyte-containing inserts were then placed on the 12-well plates containing 8 DIV neurons and maintained in co-culture for 24 h before being incubated with 100 lM HSA, in the absence (Control) or in the presence of 50 lM UCB during 48 h, using DMEM as culture medium. Pure neuronal and astroglial cultures were incubated under similar conditions (Fig. 1).
Neurotox Res (2014) 26:1–15
In parallel experiments, cells were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the UCB incubation, or with GUDCA alone, to investigate its protective efficacy on UCB-induced cytotoxicity. The 50 lM GUDCA solution was obtained from a 5 mM stock solution in PBS at pH 7.4. The bile acid GUDCA, together with the taurine-conjugated species, is found in serum of patients with cholestatic liver diseases treated with ursodeoxycholic acid (UDCA) at a dose of 450–600 mg/day (Poupon et al. 1994; Lazaridis et al. 2001; Brites et al. 1998), once it is only residual in humans. The concentration of 50 lM we selected mimics the physiological GUDCA level frequently encountered in such UDCA-treated individuals (Simoni et al. 1995; Podda et al. 1990; Brites et al. 1998; Rudolph et al. 2002). Neonatal jaundice usually increases after the first 24 h of post-natal age and lasts for 2–3 days (Kaplan and Hammerman 2005), where it achieves the highest levels of UCB. Thus, our experimental model of hyperbilirubinemia using 48 h of incubation with UCB at UCB/HSA = 0.5 reproduces the physiological conditions that may lead to BIND (Silva et al. 2012; Brites 2012). After incubation, astrocyte-containing inserts were removed and apoptosis, cell viability, neurite arborization, and Mrp1 expression were determined in the attached neurons. In addition, extracellular medium free from cellular debris was used to evaluate the levels of TNF-a, S100B and NO. Apoptosis
5
non-viable cells, but cannot cross the membrane of viable cells. This dye binds to double-stranded DNA and emits red fluorescence (630 nm). Nonpermeabilized adherent cells, cultured on coverslips, were incubated with a 75 lM PI solution for 15 min in the absence of light. Subsequently, cells were fixed and the nuclei stained with Hoechst dye 33258. Red-fluorescence and UV images of 10 random microscopic fields were acquired per sample and the percentage of PI-positive cells was estimated and expressed as fold change versus the control of pure neuronal cultures. MTT Assay Cellular reduction of MTT was measured in nerve cells to assess cell dysfunction, as previously described by us (Silva et al. 2002). Briefly, after the incubation periods, supernatants were removed and adherent cells (in inserts and culture plates for the evaluation of cellular dysfunction in astrocytes and neurons, respectively) were incubated for 1 h, at 37 °C, with a freshly prepared solution of MTT at 0.5 mg/mL. At the end of the incubation, medium was discarded and MTT formazan crystals were dissolved by addition of isopropanol/HCl 0.04 M and gentle shaking for 15 min at room temperature. After centrifugation, absorbance values at 570 nm were determined in a spectrophotometer Unicam UV2 (Unicam Limited, Cambridge, UK). Results were expressed as percentage of control from pure astroglial or neuronal cultures, which was considered as 100 %.
Cell death by apoptosis was evaluated by the assessment of nuclear morphology. In brief, cells were fixed with freshly prepared 4 % (w/v) paraformaldehyde in PBS and incubated with Hoechst dye 33258 at 5 lg/ml in PBS, for 2 min at room temperature, washed with PBS, and mounted using PBS/glycerol (3:1, v/v). Fluorescent nuclei were visualized using a Leica DFC490 camera adapted to an Axio Scope A1 microscope (Zeiss, Go¨ttingen, Germany), categorized according to condensation and staining characteristics of chromatin, and scored using random blinded counts. Apoptotic nuclei were identified by condensed chromatin contiguous to the nuclear membrane, as well as by nuclear fragmentation of condensed chromatin. At least five random microscopic fields were counted per sample, and the number of apoptotic nuclei was expressed as percentage of total nuclei. The results obtained by Hoechst staining were validated in previous studies by the use of TUNEL labelling that showed similar results (Silva et al. 2001, 2002).
For the immunofluorescence detection of the cytoskeletal protein MAP-2, a widely used neuritic marker known to be mainly located in dendrites, fixation of the cells was performed as described above and a standard indirect immunocytochemical technique was done using a mouse antiMAP-2 antibody (1:100) as the primary antibody and a horse FITC-labelled anti-mouse antibody (1:227) as the secondary antibody (Falca˜o et al. 2007b). Green-fluorescence images of ten random microscopic fields were acquired per sample. Evaluation of neurite extension and number of nodes from individual neurons, excluding apoptotic cells identified by Hoechst dye, were determined as previously described (Falca˜o et al. 2007b). Results were expressed as fold change versus the control of pure neuronal cultures.
Cellular Viability
Mrp1 Neuronal Expression
Cellular viability was assessed by monitoring the cellular uptake of PI, a fluorescent dye. PI readily enters and stains
Mrp1 expression was determined by Western blot, as previously described (Falca˜o et al. 2007a). Membranes were
Neurite Extension and Ramification
123
6
blocked for 1 h at room temperature in 4 % milk-TTBS [0.2 % Tween 20, 20 mM Tris–HCl (pH 7.5), 500 mM NaCl] and incubated overnight at 4 °C with the specific MRP1-A23 rabbit antibody against Mrp1 (1:750), or mouse anti-b-actin (1:10,000), in blocking solution. The membranes were then incubated with horseradish peroxidaselabeled anti-rabbit and anti-mouse secondary antibodies (1:5,000) in blocking buffer for 1 h at room temperature. Protein bands were detected by LumiGLOÒ and visualized by autoradiography with Hyperfilm ECL. The relative intensities of protein bands were analyzed using the Quantity oneÒ 1-D densitometric analysis software (Bio-Rad, Hercules, CA, USA), after scanning using Adobe Photoshop (Adobe Systems Software, Uxbridge, UK) and expressed as fold change versus the control of pure neuronal cultures. TNF-a Determination TNF-a levels were detected in the extracellular medium of pure astroglial and neuronal cultures, as well as in that of the co-culture system using a specific Quantikine ELISA kit (R&D Systems, Minneapolis, USA), according to manufacturer’s instructions and as already described (Fernandes et al. 2004). Absorbance (450 nm) was measured using a microplate reader. Samples were assayed in duplicate and TNF-a concentrations were expressed in pg/mL. S100B Assay S100B concentration was determined by ELISA in the culture medium of pure astroglial and neuronal cultures, as well as in that of the co-culture system, as previously described (Leite et al. 2008). Briefly, samples were incubated for 2 h on a 96-well plate previously coated with a monoclonal anti-S100B antibody (1:1,000). Next, a polyclonal anti-S100B antibody (1:5,000) was added and samples incubated for an additional 30 min. Finally, an anti-rabbit peroxidase-conjugated antibody (1:5,000) was added for a period of 30 min. The color reaction with Sigma Fast OPD tablets was measured at 492 nm using a microplate reader and results were expressed in ng/mL. Nitrite Levels Determination of nitrites, a stable end product of NO, was performed as previously described (Vaz et al. 2011b). Briefly, the extracellular medium from pure astroglial and neuronal cultures, as well as that from the co-culture system free from cellular debris, was mixed with the Griess reagent [1 part 1 % (w/v) sulfanilamide in 5 % H3PO4, 1 part 0.1 % (w/v) N-1-naphthylethylenediamine (v/v)] in 96-well tissue
123
Neurotox Res (2014) 26:1–15
Fig. 2 UCB-induced neuronal apoptosis in neuron monocultures increases when cells are co-cultured with astrocytes and is prevented by GUDCA. Pure neuronal cultures and neuron-astrocyte co-cultures were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments, both cultures were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB during 48 h. In parallel experiments, cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. Cell death by apoptosis was evaluated by nuclear morphological analysis as described in the ‘‘Methods’’ section and expressed as percentage of apoptotic cells. Results are expressed as mean ± SEM. **p \ 0.01 versus respective control; ##p \ 0.01 versus co-cultures at same experimental condition; §§p \ 0.01 versus UCB alone
culture plates for 10 min at room temperature in the dark. The absorbance at 540 nm was determined using a microplate reader and results were expressed in lM. Statistical Analysis Results of at least three different experiments, performed in duplicate, were expressed as mean ± SEM. Statistical analysis was performed using two-tailed Student’s t test, on the basis of equal or unequal variance, or one-way ANOVA as appropriate, and p \ 0.05 was accepted as statistically significant.
Results UCB-Induced Apoptosis and Loss of Cell Viability in Neuronal Monocultures Increase when Cells are Co-cultured with Astrocytes, and are Counteracted by GUDCA in Both Cases We observed that exposure of neurons to 50 lM UCB for 48 h led to a 1.5-fold increase in apoptotic cells (p \ 0.01) (Fig. 2), in accordance with previous data also pointing to UCB-induced neuronal apoptosis as a mechanism of neurotoxicity. Interestingly, a further rise in neuronal apoptosis (p \ 0.01) was obtained when cells were co-
Neurotox Res (2014) 26:1–15
7
Fig. 3 UCB-induced neuronal demise in monocultures increases when cells are co-cultured with astrocytes and is prevented by GUDCA. Pure neuronal cultures and neuron-astrocyte co-cultures were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments, both cultures were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB during 48 h. In parallel experiments, cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. Cellular viability was assessed by propidium
iodide (PI) staining (a), as described in the ‘‘Methods’’ section. The number of PI-positive cells (arrows), as well as the number of total nuclei, was counted in the same merged microscopic field and the percentage of PI-positive neurons was expressed as fold change versus the control from pure neuronal cultures (b). Results are expressed as mean ± SEM. **p \ 0.01 versus respective control; ## p \ 0.01 versus co-cultures at same experimental condition; §§ p \ 0.01 versus UCB alone. Bar represents 20 lm
cultured with astrocytes (1.7-fold, p \ 0.01). Once the conditions used intend to mimic a moderate hyperbilirubinemia, the levels of apoptosis were rather low, as we anticipated, to sustain the main functionality of both cell types and to better observe any alteration caused by the coculture system. Similar to the anti-apoptotic effects already observed in our studies using UCB, neurons, and GUDCA (Vaz et al. 2010), the bile acid prevented the apoptotic effect of UCB on neurons to values near the control ones (5.4 % instead of 9.5 %, p \ 0.01). It also showed efficacy to completely abrogate the apoptotic neuronal death in the co-culture system (5.8 % instead of 11.5 %, p \ 0.01). As neuronal viability is concerned, results with PI reinforced the loss of neuronal viability when cells were treated with UCB. In fact, we found that exposure of neurons to 50 lM UCB for 48 h induced a 2.3-fold increase in unviable neurons (p \ 0.01) (Fig. 3). As observed for apoptosis, the number of PI-positive cells was higher when neurons were co-cultured with astrocytes (3.3fold change, p \ 0.01). Again, GUDCA fully counteracted the loss of cell membrane integrity in the pure neuron culture (near 1-fold change, p \ 0.01) and almost completely avoided such UCB effect when communication with astrocytes was allowed (1.5-fold change, p \ 0.01). Thus, GUDCA is a promising compound in protecting neuronal demise by UCB, not only in monotypic cultures but more importantly also in a co-culture system where the UCB deleterious effects were enhanced. Regarding UCB-induced cellular dysfunction, we observed a further decrease in MTT reduction ability by neurons in the co-culture system (Online Resource 1),
attesting the increased neurotoxicity by UCB in such model thus corroborating the results already shown. In contrast, the ability of astrocytes to reduce MTT was not affected by the presence of neurons which leads us to think that though rather dysfunctional the astrocytes may become more reactive and produce soluble molecules toxic to neurons.
Neuritic Tree Collapse by UCB in (Pure) Neurons is Enhanced if Cells are Co-cultured with Astrocytes, and is Counteracted by GUDCA in Both Models We have shown that UCB-induced neuritic atrophy in immature neurons (Fernandes et al. 2009) was a longlasting phenomenon along cell differentiation (Falca˜o et al. 2007b), and a probable cause of later vulnerability to a new injury. Thus, it was not a surprise to observe a significant reduction on both the length of neurites (0.8-fold change, p \ 0.01) and the number of nodes per cell (0.7-fold change, p \ 0.01) in the pure neuronal cultures exposed to 50 lM UCB for 48 h (Fig. 4). Remarkably, it seems that the presence of soluble factors released by the cells in the co-culture experiments further intensifies the neuritic tree impairment (0.7- and 0.6-fold change for neurite length and ramification, respectively, p \ 0.01). Regarding the effect of GUDCA, we again observed its efficacy in abrogating the UCB-induced reduction of neuritic length and ramification, even in co-cultures where the more drastic effect of UCB seemed to be more difficult to prevent (p \ 0.01 for both vs. UCB alone; p \ 0.01 and p \ 0.05 vs. UCB alone, respectively). Overall, these results indicate that neuronal
123
8
Neurotox Res (2014) 26:1–15
Fig. 4 UCB-induced impairment of neuritic arborization increases in the presence of astrocytes and is prevented by GUDCA. Pure neuronal cultures and neuron-astrocyte co-cultures were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments both cultures were pretreated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB during 48 h. In parallel experiments, cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. Neurites were detected by immunocytochemistry using a mouse anti-
MAP-2 antibody followed by a species-specific fluorescent secondary antibody labeled with FITC. Representative results of one experiment are shown (a). Neurite extension (b) and ramification (c) were evaluated after MAP-2 staining, as described in the ‘‘Methods’’ section and expressed as fold change versus the control from pure neuronal cultures. Results are expressed as mean ± SEM. **p \ 0.01 versus respective control; #p \ 0.05 and ##p \ 0.01 versus cocultures at the same experimental condition; §p \ 0.05 and §§ p \ 0.01 versus UCB alone. Bar represents 40 lm
apoptosis, cell death, and reduced arborisation triggered by UCB are potentiated by UCB-related changes in astrocytic environment and that GUDCA is able to preserve the neuron-astrocyte homeostasis and cell resistance in such conditions.
produced by UCB in both pure neurons and co-cultures, attesting no direct influence of UCB on Mrp1 expression (Fig. 5). However, the expression of Mrp1 was significantly up-regulated when the signaling communication between astrocytes and neurons was allowed (1.5-fold change, p \ 0.01). In addition, our results evidenced that the beneficial effects of GUDCA are not due to an induced expression of Mrp1 mediated by this bile acid.
Mrp1 Neuronal Expression is Increased by Astrocytes Independently from the Presence of UCB Mrp1 may have an important role in protecting cells from UCB-induced cytotoxicity due to its pivotal role in promoting the cellular efflux of UCB, protecting cells from its intracellular accumulation (Falca˜o et al. 2007a; Calligaris et al. 2006; Cekic et al. 2003). Therefore, we wondered whether the increased UCB-induced neuronal dysfunction in the presence of astrocytes was derived from an induced reduced expression of Mrp1 in neurons when facing UCB and glial cells. Our results showed that no changes were
123
Extracellular TNF-a Content Increases in NeuronAstrocyte Co-Cultures as Compared to Neuron Monocultures and Remains Unchanged after the Addition of UCB and GUDCA, either Alone or in Association TNF-a was previously indicated to up-regulate Mrp1 in astrocytes (Ronaldson et al. 2010) and we first demonstrated that astrocytes release increased levels of TNF-a in
Neurotox Res (2014) 26:1–15
Fig. 5 Mrp1 neuronal expression increases in the presence of astrocytes, independently of UCB and GUDCA addition. Pure neuronal cultures and neuron-astrocyte co-cultures were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments both cultures were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB during 48 h. In parallel experiments cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. Total cell lysates were analyzed by Western blotting with antibody specific for Mrp1, as described in the ‘‘Methods’’ section. Representative results of one experiment are shown (a). The intensity of the bands was quantified by scanning densitometry standardized with respect to b-actin protein (b). Results are expressed as mean ± SEM fold change versus the control from pure neuronal cultures. #p \ 0.05 and ##p \ 0.01 versus co-cultures at the same experimental condition
the presence of UCB (Fernandes et al. 2004). Therefore, we questioned if the up-regulation of neuronal Mrp1 in cocultures was a result of UCB-induced secretion of TNF-a by astrocytes. Although astrocytes produced higher TNF-a levels (*600 pg/mL) than neurons (*250 pg/mL), no statistical difference was observed versus controls in any of the tested conditions (Fig. 6). For that it may have accounted the longer time of incubation we here used, once we previously noticed that the UCB-induced TNF-a peak levels are achieved at 12 h, decreasing to control levels at 24 h (Fernandes et al. 2006). As so, the increased values observed in co-cultures, where the TNF-a levels in the culture medium attained values of *800 pg/mL (p \ 0.05), may result from the conjoint secretion of both cells. Since GUDCA did not produce any alteration on the amount of the released TNF-a either in monocultures or in co-cultures, we hypothesize that TNF-a is only transiently, or not directly, involved in the increased UCB-induced
9
Fig. 6 TNF-a concentration in the extracellular medium rises from neuron monocultures to neuron-astrocyte co-cultures, independently of UCB and GUDCA treatment. Pure astrocytes and neurons, as well as neuron-astrocyte co-cultures, were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments, cultures were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB for 48 h. In parallel experiments, cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. After incubation, the extracellular medium free from cellular debris was used to assess the TNF-a concentration by ELISA, as described in the ‘‘Methods’’ section. Results are expressed as mean ± SEM. #p \ 0.05 versus cocultures at the same experimental condition
Fig. 7 S100B concentration in the extracellular medium rises from astrocyte and neuron monocultures to neuron-astrocyte co-cultures, increases by UCB addition, and is only prevented by GUDCA in astrocytes. Pure astrocytes and neurons, as well as neuron-astrocyte co-cultures, were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments, both cultures were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB during 48 h. In parallel experiments cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. After incubation, the extracellular medium free from cellular debris was used to assess the levels of S100B by ELISA, as described in the ‘‘Methods’’ section. Results are expressed as mean ± SEM. *p \ 0.05 and **p \ 0.01 versus respective control; #p \ 0.05 and ##p \ 0.01 versus co-cultures at the same experimental condition; §p \ 0.05 versus UCB alone
neurotoxicity we observed in the indirect cultures with astrocytes. In addition, we also speculate that the GUDCA pro-survival properties are not caused by a down-regulation of such cytokine.
123
10
Neurotox Res (2014) 26:1–15
S100B Secretion Increases in the Co-cultures as Compared to Monocultures, and is Highly Enhanced by UCB Addition, While GUDCA Only Shows Preventive Efficacy in UCB-treated Astrocytes S100B protein is a widely used indicator of glial activation and/or death in several conditions of brain injury (Gonc¸alves et al. 2008; Yardan et al. 2011). So, we hypothesized that activation of astrocytes by UCB through the release of S100B could be the trigger of UCB-induced apoptosis, loss of cell viability and neuritic tree collapse in neurons cocultured with astrocytes observed in the previous experiments. Thus, we decided to evaluate the production of S100B by both astrocyte and neuron monocultures in the presence of UCB (as we did for TNF-a assessment), as well as the one produced by the co-cultures, either in the absence or in the presence of GUDCA. Interestingly, we have observed that the release of S100B greatly increased in co-cultures as compared with pure cell cultures (p \ 0.01 and p \ 0.05, respectively) (Fig. 7). Furthermore, we found that exposure of pure astrocytes to 50 lM UCB for 48 h induced a small but significant increase in extracellular S100B content (from 1.7 to 2.1 ng/ mL, p \ 0.05) not observed in neurons. No influence by UCB or GUDCA was noticed on the S100B secretion by neurons. Contrasting effects were obtained in co-cultures where the UCB-induced extracellular content in S100B almost doubled (from 3.7 to 6.6 ng/mL, p \ 0.01) and co-incubation with GUDCA was ineffective. Thus, as observed for the cytokine, the neuroprotection exerted by GUDCA does not involve any potential inhibition of the S100B secretion by this bile acid. We may then assume that S100B is a determinant of the neuronal dysfunction by UCB in a more complex cellular system where astrocyte activation by UCB is also enhanced by the neuronal environment and by the cross-talk dysregulation that may occur. NO Production in Co-cultures is Highly Decreased as Compared to Astrocyte Monocultures, Although Significantly Increased by UCB, and is not Prevented by GUDCA Abnormal activation of astrocytes with excessive NO production may be implicated in neuronal degeneration (Ihara et al. 2012; Dawson and Dawson 1996) and UCB has revealed to trigger NO release from both astrocytes and neurons, although at a greater amount in the glial cells (Falca˜o et al. 2013). Here we also observed an increased NO content in the astrocyte extracellular media as compared with that of neurons. Indeed, we obtained concentrations of at least threefolds higher (near 13 lM) than those released by neurons (close to 4 lM) (Fig. 8). In
123
Fig. 8 Nitrite concentration in the extracellular medium decreases from astrocyte monocultures to neuron monocultures and to neuronastrocyte co-cultures, the only ones where NO generation was induced by UCB, but not protected by GUDCA. Astrocyte and neuron monocultures, as well as neuron-astrocyte co-cultures, were incubated in the absence (Control) or in the presence of 50 lM UCB (plus 100 lM HSA) for 48 h. In another set of experiments, both cultures were pre-treated for 1 h with 50 lM GUDCA (plus 100 lM HSA) prior to the addition of 50 lM UCB for 48 h. In parallel experiments, cells were treated with 50 lM GUDCA (plus 100 lM HSA) for 48 h. After incubation the extracellular medium free from cellular debris was used to assess the levels of nitrites by the Griess reagent, as described in the ‘‘Methods’’ section. Results are expressed as mean ± SEM. **p \ 0.01 versus respective control; ##p \ 0.01 versus co-cultures at the same experimental condition
addition, neither UCB nor GUDCA modified NO production by cell monocultures. Curiously, the neuron-astrocyte intercellular signaling was able to prevent its generation by astrocytes (Fernandez-Fernandez et al. 2012). Most important, basal NO levels in co-cultures at the levels observed in isolated neurons significantly increase in the presence of UCB (from 3.2 to 5.4 lM, p \ 0.01). Although this increase may concur to the higher susceptibility of neurons to UCB cytotoxicity in the co-culture system, coincubation with GUDCA was not able to halt the UCBinduced increase of NO production in such model. We may then assume that GUDCA does not prevent the release of soluble factors by UCB-activated astrocytes in co-cultures, but by protecting damage at the level of the cell and mitochondrial membranes may increase the neuronal resistance to the cytotoxic effects of UCB and to the astrocyte-mediated soluble factors.
Discussion To investigate whether communication between astrocytes and neurons protect or aggravate UCB-induced neurodegeneration, we have used both cells either as monocultures or co-cultures submitted to a preconditioning period of 24 h before the exposure to UCB for 48 h. In a previous study (Falca˜o et al. 2013) we unexpectedly observed that astrocytes in the presence of neurons sustain cell
Neurotox Res (2014) 26:1–15
homeostasis through its trophic influence, thus increasing neuronal resistance to the toxic effect of UCB. It is well known that astrocyte activation exerts neuroprotection when it is not excessive (Li et al. 2009), which was showed to depend on both the extension and duration of the damage (Ait-Ikhlef et al. 2000; Brown and Bal-Price 2003). We therefore decided to allow first the free communication between neurons and astrocytes in the co-culture system before adding UCB, and extended the incubation period with UCB to 48 h. In addition we explored the potential beneficial effects of GUDCA in this new experimental model, since our precedent in vitro work has demonstrated that its anti-apoptotic, anti-inflammatory and anti-oxidant properties preserved nerve cells from UCB injury (Vaz et al. 2010; Silva et al. 2012; Fernandes et al. 2007; Brito et al. 2008a). Data obtained suggest that UCB exerts a greater toxic effect (decreased cell viability and neuritic atrophy) when astrocytes and neurons are co-cultivated. An increased expression of Mrp1 and elevated secretion of both TNF-a and S100B were observed in co-cultures independently of homeostasis be established prior to incubation with UCB or not. NO plays a role in UCB toxicity (Brito et al. 2010). The net decrease in NO now observed in co-cultures suggests that intercellular signaling triggers key metabolic changes aimed at preventing the propagation of neurodegeneration, as previously suggested (Bolan˜os and Almeida 1999). In contrast to our previous results (Falca˜o et al. 2013), we have now observed that the preconditioning period, by allowing the cross-talk between both cells and the establishment of the homeostatic conditions prior to UCB treatment, favors UCB-induced increase of NO production in this co-culture model. The differences observed when communication between neurons and astrocytes was established concomitantly with UCB exposure as in the first model (Falca˜o et al. 2013), and the present one where bidirectional signaling recognition preceded the exposure to UCB, highlight the either beneficial or detrimental effect, respectively, that astrocytes may assume in the presence of neurons, accordingly to the environmental conditions. Astrocytes can defend neurons from stressors by transiently up-regulating the release of neuroprotective molecules that reestablish homeostasis (Tian et al. 2012). However they can also be harmful to neurons if the time of exposure to a neurotoxin elongates and the environmental milieu changes (Farina et al. 2007; Tian et al. 2012). In our case, we are keen to conclude that the crucial influence of such difference was determined mainly by the preconditioning effect. In line with this conclusion is the observation that in the presence of this preconditioning period and using a 24 h UCB incubation, instead of the adopted 48 h, we have observed an increase in the UCB-induced neuronal dysfunction in
11
the co-culture system, although less markedly (results not shown). An increased UCB-induced neuronal apoptosis and loss of cell viability (Falca˜o et al. 2006) together with the reduction in neurite extension and ramification (Falca˜o et al. 2007b; Fernandes et al. 2009) have been reported. However, this is the first time that increased effects in neuron-astrocyte co-cultures are reported particularly since a protection was demonstrated in the absence of cell preconditioning before exposure to UCB (Falca˜o et al. 2013). Astrocytes were shown to regulate the synthesis and/or intracellular distribution of MAP-2, the morphology of neurons (Chamak et al. 1987), and to promote their survival (Drukarch et al. 1997). Astrocytes also protect neurons from extracellular reactive oxygen (Drukarch et al. 1998) and nitrogen species (Tanaka et al. 1999), as well as from ethanol-induced oxidative stress and apoptotic death (Watts et al. 2005). Therefore, it is not surprising that alterations in astrocyte functionality have been indicated to be involved in the pathogenesis of an increasing number of CNS diseases (De Keyser et al. 2008). Since UCB triggers astrocyte reactivity (Fernandes et al. 2004, 2006; Falca˜o et al. 2005), we assume that this reactivity negatively influences UCB injury to neurons. Indeed, in the animal model of unconjugated hyperbilirubinemia, the Gunn rat, a cerebellar hypoplasia accompanied by a dramatic loss of Purkinje neurons and of cerebellar granular neurons (Lin et al. 2005) was observed, while others verified astrocyte hypertrophy in the affected cerebellar lobes (Mikoshiba et al. 1980) and increased GFAP staining in the cerebellum (O’Callaghan and Miller 1985), reflecting astrocytosis, a feature of astrocyte reactivity. Reactive astrocytes were indicated to have influence on neuritic outgrowth (Lefranc¸ois et al. 1997), as it seems to have occurred in the present model after treatment with UCB. We believe that our co-culture system is a convenient and powerful approach to study the contribution of UCB-induced activation of astrocytes to neuronal dysfunction, although several questions may remain in linking the present in vitro with the in vivo results. An increased susceptibility of neurons to UCB in the presence of astrocytes may be due to a down-regulation of Mrp1, which protects cells from intracellular UCB accumulation (Calligaris et al. 2006; Cekic et al. 2003). We did not find any variation in Mrp1 expression by UCB treatment in both pure neurons and co-cultures after preconditioning, indicating a lack of modulation of the ABC transporter, a result corroborated by studies in neurospheres and in differentiating neurons (data not published). Nevertheless, it is worthwhile to point out that the basal levels of Mrp1 increase by co-culturing neurons with astrocytes, suggesting that the expression and the release of soluble factors might have contributed to its up-regulation,
123
12
a finding also observed in the absence of preconditioning (Falca˜o et al. 2013). Our results confirm that TNF-a is released by astrocytes in concentrations that at least double the ones by neurons (Falca˜o et al. 2006). Curiously, the summing effect now observed in co-cultures was not found in the previous model without the preconditioning step (Falca˜o et al. 2013). It is conceivable that an exposure to higher TNF-a levels when astrocytes are present in the co-culture system is linked to the increased expression of neuronal Mrp1 since TNF-a was shown to up-regulate Mrp1 (Ronaldson et al. 2010), as observed in cells treated with lipopolysaccharide (Cherrington et al. 2004). These events may have accounted for the increased levels of S100B in the co-culture extracellular medium similarly to what was reported for astrocytes incubated with TNF-a (Edwards and Robinson 2006). In contrast with the non-preconditioning condition, UCB triggered the release of increased amounts of S100B, an effect that was shown to contribute to neurodegeneration (MalchiodiAlbedi et al. 2001). The UCB-induced release of S100B is consistent with data showing that the serum levels of S100B correlate with total bilirubin concentrations and the appearance of early-phase UCB encephalopathy (Okumus et al. 2008). Similarly to other brain injuries (Mrak and Griffinbc 2001; Rothermundt et al. 2003) we believe that S100B may be implicated in the increased impairment of neuritic arborization and cell demise that occurred in the presence of astrocytes and UCB. It was reported that S100B does not stimulate NO production or influence neuronal viability in the absence of astrocytes, but that NO generation by astrocytes mediates neuronal cell death in astrocyte-neuron co-cultures (Hu et al. 1997). In the present work we have observed a higher generation of NO by astrocytes as compared to neurons, but no cell reactivity to UCB was obtained in both monocultures. Nevertheless, the neuronal NO transmitter significantly increased in the extracellular media of the cocultures and may relate to nitrosative stress, neuronal damage, and neuritic impairment (Vaz et al. 2011b; Wang et al. 2006; Silva et al. 2012). This finding, not observed in the absence of preconditioning (Falca˜o et al. 2013), where astrocytes provide appropriated feedback regulation on neuronal activity and possibly dampen UCB neurotoxicity, may here result from UCB-induced astrocyte malfunction and subsequent inability to stabilize the neuronal function (Amiri et al. 2012). We further hypothesize that excitotoxicity mediated by glutamate in response to NO production (Bal-Price et al. 2002) is not involved in the increased detrimental effects produced by UCB in co-cultures, once similar extracellular glutamate levels were obtained in all the UCB-treated conditions (data not shown). Astrocyte activation by UCB was previously
123
Neurotox Res (2014) 26:1–15
demonstrated (Fernandes et al. 2004, 2006; Falca˜o et al. 2005, 2006) and GUDCA showed to modulate this reactivity (Fernandes et al. 2007). Furthermore, this bile acid has shown to be neuroprotective in conditions of oxidative and nitrosative stress by UCB (Brito et al. 2008a; Vaz et al. 2010; Silva et al. 2012). The demonstration that GUDCA maintains its neuroprotective properties in the co-culture system exposed to conditions mimicking BIND is interesting and points to its therapeutical indications. However, such properties were not associated with the modulation of TNF-a, NO or S100B secretion into the extracellular medium, suggesting that its mode of action is possibly downstream these signaling molecules, by preventing mitochondrial dysfunction (Vaz et al. 2010) or nuclear factor-jB activation (Fernandes et al. 2007). In conclusion, we believe that the co-culture model and the experimental conditions used in the present study mimic the effects produced by severe hyperbilirubinemia in the brain parenchyma and reproduce the dysregulated neuron-astrocyte cross-talk. The main impairments observed include the increased production of S100B and NO in conditions that led to reduced neurite extension and ramification, and culminated in increased apoptosis and neuronal demise. Together with our previous work (Falca˜o et al. 2013) we are convinced that both models may be useful to understand regulatory and dysregulatory mechanisms/signals in other types of neurological and immunological processes in the brain, which may help the diagnosis and prevention of various neurological diseases. Finally, our data support the conclusion that astrocytes are a proximal target for the treatment of BIND and that GUDCA, by preventing UCB-induced neurotoxicity even in the presence of astrocytes, may be a resourceful molecule to be used whenever classical treatment to neonatal jaundice is not successful. Acknowledgments This work was supported by FEDER (COMPETE Programme) and by National funds (FCT—Fundac¸a˜o para a Cieˆncia e a Tecnologia—Projects PTDC/SAU-NEU/64385/2006 to D. B. and PEst-OE/SAU/UI4013/2011 to iMed.UL). A. S. F. holds a a post doctoral research position (C2007-FFUL/UBMBE/02/2011) granted by FCT. The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest The authors declare that there are no actual or potential conflicts of interest.
References Ahlfors CE, Wennberg RP, Ostrow JD, Tiribelli C (2009) Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin Chem 55(7):1288–1299
Neurotox Res (2014) 26:1–15 Ait-Ikhlef A, Hantaz-Ambroise D, Henderson CE, Rieger F (2000) Influence of factors secreted by wobbler astrocytes on neuronal and motoneuronal survival. J Neurosci Res 59(1):100–106 Allaman I, Gavillet M, Belanger M, Laroche T, Viertl D, Lashuel HA, Magistretti PJ (2010) Amyloid-b aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability. J Neurosci 30(9):3326–3338 Allaman I, Belanger M, Magistretti PJ (2011) Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci 34(2):76–87 Amiri M, Bahrami F, Janahmadi M (2012) Functional contributions of astrocytes in synchronization of a neuronal network model. J Theor Biol 292:60–70 Anderl JL, Redpath S, Ball AJ (2009) A neuronal and astrocyte coculture assay for high content analysis of neurotoxicity. J Vis Exp 27:1173–1180 Bal-Price A, Moneer Z, Brown GC (2002) Nitric oxide induces rapid, calcium-dependent release of vesicular glutamate and ATP from cultured rat astrocytes. Glia 40(3):312–323 Blondeau JP, Beslin A, Chantoux F, Francon J (1993) Triiodothyronine is a high-affinity inhibitor of amino acid transport system L1 in cultured astrocytes. J Neurochem 60(4):1407–1413 Bolan˜os JP, Almeida A (1999) Roles of nitric oxide in brain hypoxiaischemia. Biochim Biophys Acta 1411(2–3):415–436 Brites D (2012) The evolving landscape of neurotoxicity by unconjugated bilirubin: role of glial cells and inflammation. Front Pharmacol 3:88 Brites D, Rodrigues CMP, Oliveira N, Cardoso M, Graca LM (1998) Correction of maternal serum bile acid profile during ursodeoxycholic acid therapy in cholestasis of pregnancy. J Hepatol 28(1):91–98 Brito MA, Lima S, Fernandes A, Falca˜o AS, Silva RFM, Butterfield DA, Brites D (2008a) Bilirubin injury to neurons: contribution of oxidative stress and rescue by glycoursodeoxycholic acid. Neurotoxicology 29(2):259–269 Brito MA, Rosa AI, Falca˜o AS, Fernandes A, Silva RFM, Butterfield DA, Brites D (2008b) Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells. Neurobiol Dis 29(1):30–40 Brito MA, Vaz AR, Silva SL, Falca˜o AS, Fernandes A, Silva RFM, Brites D (2010) N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity. Mol Med 16(9–10):372–380 Brown DR (1999) Neurons depend on astrocytes in a coculture system for protection from glutamate toxicity. Mol Cell Neurosci 13(5):379–389 Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27(3):325–355 Butt AM (2011) ATP: a ubiquitous gliotransmitter integrating neuronglial networks. Semin Cell Dev Biol 22(2):205–213 Calligaris S, Cekic D, Roca-Burgos L, Gerin F, Mazzone G, Ostrow JD, Tiribelli C (2006) Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity. FEBS Lett 580(5):1355–1359 Cekic D, Bellarosa C, Garcia-Mediavilla MV, Rigato I, Pascolo L, Ostrow JD, Tiribelli C (2003) Upregulation in the expression of multidrug resistance protein Mrp1 mRNA and protein by increased bilirubin production in rat. Biochem Biophys Res Commun 311(4):891–896 Chamak B, Fellous A, Glowinski J, Prochiantz A (1987) MAP2 expression and neuritic outgrowth and branching are coregulated through region-specific neuro-astroglial interactions. J Neurosci 7(10):3163–3170 Cherrington NJ, Slitt AL, Li N, Klaassen CD (2004) Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats. Drug Metab Dispos 32(7):734–741
13 Dawson VL, Dawson TM (1996) Nitric oxide in neuronal degeneration. Proc Soc Exp Biol Med 211(1):33–40 De Keyser J, Mostert JP, Koch MW (2008) Dysfunctional astrocytes as key players in the pathogenesis of central nervous system disorders. J Neurol Sci 267(1–2):3–16 Domenici MR, Paradisi S, Sacchetti B, Gaudi S, Balduzzi M, Bernardo A, Ajmone-Cat MA, Minghetti L, Malchiodi-Albedi F (2002) The presence of astrocytes enhances beta amyloidinduced neurotoxicity in hippocampal cell cultures. J Physiol Paris 96(3–4):313–316 Drukarch B, Schepens E, Jongenelen CA, Stoof JC, Langeveld CH (1997) Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency. Brain Res 770(1–2):123–130 Drukarch B, Schepens E, Stoof JC, Langeveld CH, Van Muiswinkel FL (1998) Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species. Free Radic Biol Med 25(2):217–220 Edwards MM, Robinson SR (2006) TNF alpha affects the expression of GFAP and S100B: implications for Alzheimer’s disease. J Neural Transm 113(11):1709–1715 Falca˜o AS, Fernandes A, Brito MA, Silva RFM, Brites D (2005) Bilirubin-induced inflammatory response, glutamate release, and cell death in rat cortical astrocytes are enhanced in younger cells. Neurobiol Dis 20(2):199–206 Falca˜o AS, Fernandes A, Brito MA, Silva RFM, Brites D (2006) Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state. Acta Neuropathol 112(1):95–105 Falca˜o AS, Bellarosa C, Fernandes A, Brito MA, Silva RFM, Tiribelli C, Brites D (2007a) Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin. Neuroscience 144(3):878–888 Falca˜o AS, Silva RFM, Pancadas S, Fernandes A, Brito MA, Brites D (2007b) Apoptosis and impairment of neurite network by short exposure of immature rat cortical neurons to unconjugated bilirubin increase with cell differentiation and are additionally enhanced by an inflammatory stimulus. J Neurosci Res 85(6):1229–1239 Falca˜o AS, Silva RFM, Vaz AR, Silva SL, Fernandes A, Brites D (2013) Cross-talk between neurons and astrocytes in response to bilirubin: early beneficial effects. Neurochem Res 38(3):644–659 Farina C, Aloisi F, Meinl E (2007) Astrocytes are active players in cerebral innate immunity. Trends Immunol 28(3):138–145 Fernandes A, Brites D (2009) Contribution of inflammatory processes to nerve cell toxicity by bilirubin and efficacy of potential therapeutic agents. Curr Pharm Des 15(25):2915–2926 Fernandes A, Silva RFM, Falca˜o AS, Brito MA, Brites D (2004) Cytokine production, glutamate release and cell death in rat cultured astrocytes treated with unconjugated bilirubin and LPS. J Neuroimmunol 153(1–2):64–75 Fernandes A, Falca˜o AS, Silva RFM, Gordo AC, Gama MJ, Brito MA, Brites D (2006) Inflammatory signalling pathways involved in astroglial activation by unconjugated bilirubin. J Neurochem 96(6):1667–1679 Fernandes A, Vaz AR, Falca˜o AS, Silva RFM, Brito MA, Brites D (2007) Glycoursodeoxycholic acid and interleukin-10 modulate the reactivity of rat cortical astrocytes to unconjugated bilirubin. J Neuropathol Exp Neurol 66(9):789–798 Fernandes A, Falca˜o AS, Abranches E, Bekman E, Henrique D, Lanier LM, Brites D (2009) Bilirubin as a determinant for altered neurogenesis, neuritogenesis, and synaptogenesis. Dev Neurobiol 69(9):568–582 Fernandes A, Barateiro A, Falca˜o AS, Silva SL, Vaz AR, Brito MA, Silva RFM, Brites D (2011) Astrocyte reactivity to unconjugated bilirubin requires TNF-alpha and IL-1beta receptor signaling pathways. Glia 59(1):14–25
123
14 Fernandez-Fernandez S, Almeida A, Bolan˜os JP (2012) Antioxidant and bioenergetic coupling between neurons and astrocytes. Biochem J 443(1):3–11 Fernetti C, Pascolo L, Podda E, Gennaro R, Stebel M, Tiribelli C (2001) Preparation of an antibody recognizing both human and rodent MRP1. Biochem Biophys Res Commun 288(4):1064–1068 Gennuso F, Fernetti C, Tirolo C, Testa N, L’Episcopo F, Caniglia S, Morale MC, Ostrow JD, Pascolo L, Tiribelli C, Marchetti B (2004) Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistanceassociated protein 1 (Mrp1). Proc Natl Acad Sci USA 101(8):2470–2475 Gonc¸alves CA, Leite MC, Nardin P (2008) Biological and methodological features of the measurement of S100B, a putative marker of brain injury. Clin Biochem 41(10–11):755–763 Hansen TW (2002) Mechanisms of bilirubin toxicity: clinical implications. Clin Perinatol 29(4):765–778 viii Hu J, Ferreira A, Van Eldik LJ (1997) S100beta induces neuronal cell death through nitric oxide release from astrocytes. J Neurochem 69(6):2294–2301 Ihara H, Yamamoto H, Ida T, Tsutsuki H, Sakamoto T, Fujita T, Okada T, Kozaki S (2012) Inhibition of nitric oxide production and inducible nitric oxide synthase expression by a polymethoxyflavone from young fruits of Citrus unshiu in rat primary astrocytes. Biosci Biotechnol Biochem 76(10):1843–1848 Jones EV, Cook D, Murai KK (2012) A neuron-astrocyte co-culture system to investigate astrocyte-secreted factors in mouse neuronal development. Methods Mol Biol 814:341–352 Kaplan M, Hammerman C (2004) Understanding and preventing severe neonatal hyperbilirubinemia: is bilirubin neurotoxity really a concern in the developed world? Clin Perinatol 31(3):555–575 Kaplan M, Hammerman C (2005) Understanding severe hyperbilirubinemia and preventing kernicterus: adjuncts in the interpretation of neonatal serum bilirubin. Clin Chim Acta 356(1–2):9–21 Kirchhoff F, Dringen R, Giaume C (2001) Pathways of neuronastrocyte interactions and their possible role in neuroprotection. Eur Arch Psychiatry Clin Neurosci 251(4):159–169 Laird MD, Vender JR, Dhandapani KM (2008) Opposing roles for reactive astrocytes following traumatic brain injury. Neurosignals 16(2–3):154–164 Lazaridis KN, Gores GJ, Lindor KD (2001) Ursodeoxycholic acid ‘mechanisms of action and clinical use in hepatobiliary disorders’. J Hepatol 35(1):134–146 Lefranc¸ois T, Fages C, Peschanski M, Tardy M (1997) Neuritic outgrowth associated with astroglial phenotypic changes induced by antisense glial fibrillary acidic protein (GFAP) mRNA in injured neuron-astrocyte cocultures. J Neurosci 17(11):4121–4128 Leite MC, Galland F, Brolese G, Guerra MC, Bortolotto JW, Freitas R, Almeida LM, Gottfried C, Goncalves CA (2008) A simple, sensitive and widely applicable ELISA for S100B: methodological features of the measurement of this glial protein. J Neurosci Methods 169(1):93–99 Li XZ, Bai LM, Yang YP, Luo WF, Hu WD, Chen JP, Mao CJ, Liu CF (2009) Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation. Neurosci Res 65(3):252–258 Lin S, Wei X, Bales KR, Paul AB, Ma Z, Yan G, Paul SM, Du Y (2005) Minocycline blocks bilirubin neurotoxicity and prevents hyperbilirubinemia-induced cerebellar hypoplasia in the Gunn rat. Eur J Neurosci 22(1):21–27 Malchiodi-Albedi F, Domenici MR, Paradisi S, Bernardo A, AjmoneCat MA, Minghetti L (2001) Astrocytes contribute to neuronal impairment in bA toxicity increasing apoptosis in rat hippocampal neurons. Glia 34(1):68–72
123
Neurotox Res (2014) 26:1–15 McDonagh AF (1979) Bile pigments: bilatrienes and 5,15 biladienes. In: Dolphin D (ed) The porphyrins. Academic Press, New York, pp 294–491 Meeks JP, Mennerick S (2003) Feeding hungry neurons: astrocytes deliver food for thought. Neuron 37(2):187–189 Mikoshiba K, Kohsaka S, Takamatsu K, Tsukada Y (1980) Cerebellar hypoplasia in the Gunn rat with hereditary hyperbilirubinemia: immunohistochemical and neurochemical studies. J Neurochem 35(6):1309–1318 Miller JA, Trout BR, Sullivan KA, Bialecki RA, Roberts RA, Tjalkens RB (2011) Low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine causes inflammatory activation of astrocytes in nuclear factor-kappaB reporter mice prior to loss of dopaminergic neurons. J Neurosci Res 89(3):406–417 Mrak RE, Griffinbc WS (2001) The role of activated astrocytes and of the neurotrophic cytokine S100B in the pathogenesis of Alzheimer’s disease. Neurobiol Aging 22(6):915–922 O’Callaghan JP, Miller DB (1985) Cerebellar hypoplasia in the Gunn rat is associated with quantitative changes in neurotypic and gliotypic proteins. J Pharmacol Exp Ther 234(2):522–533 Okumus N, Turkyilmaz C, Onal EE, Atalay Y, Serdaroglu A, Elbeg S, Koc E, Deda G, Cansu A, Gunduz B (2008) Tau and S100B proteins as biochemical markers of bilirubin-induced neurotoxicity in term neonates. Pediatr Neurol 39(4):245–252 Ostrow JD, Pascolo L, Tiribelli C (2002) Mechanisms of bilirubin neurotoxicity. Hepatology 35(5):1277–1280 Palmela I, Sasaki H, Cardoso FL, Moutinho M, Kim KS, Brites D, Brito MA (2012) Time-dependent dual effects of high levels of unconjugated bilirubin on the human blood-brain barrier lining. Front Cell Neurosci 6:22 Podda M, Ghezzi C, Battezzati PM, Crosignani A, Zuin M, Roda A (1990) Effects of ursodeoxycholic acid and taurine on serum liver enzymes and bile acids in chronic hepatitis. Gastroenterology 98(4):1044–1050 Poupon RE, Poupon R, Balkau B (1994) Ursodiol for the long-term treatment of primary biliary cirrhosis. The UDCA-PBC Study Group. N Engl J Med 330(19):1342–1347 Rigato I, Pascolo L, Fernetti C, Ostrow JD, Tiribelli C (2004) The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin. Biochem J 383(Pt 2):335–341 Ronaldson PT, Ashraf T, Bendayan R (2010) Regulation of multidrug resistance protein 1 by tumor necrosis factor a in cultured glial cells: involvement of nuclear factor-jB and c-Jun N-terminal kinase signaling pathways. Mol Pharmacol 77(4):644–659 Rothermundt M, Peters M, Prehn JH, Arolt V (2003) S100B in brain damage and neurodegeneration. Microsc Res Tech 60(6):614–632 Rubaltelli FF, Griffith PF (1992) Management of neonatal hyperbilirubinemia and prevention of kernicterus. Drugs 43(6):864–872 Rudolph G, Kloeters-Plachky P, Sauer P, Stiehl A (2002) Intestinal absorption and biliary secretion of ursodeoxycholic acid and its taurine conjugate. Eur J Clin Invest 32(8):575–580 Sidoryk-Wegrzynowicz M, Wegrzynowicz M, Lee E, Bowman AB, Aschner M (2011) Role of astrocytes in brain function and disease. Toxicol Pathol 39(1):115–123 Silva R, Mata LR, Gulbenkian S, Brito MA, Tiribelli C, Brites D (1999) Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and pH. Biochem Biophys Res Commun 265(1):67–72 Silva RFM, Rodrigues CMP, Brites D (2001) Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid. J Hepatol 34(3):402–408 Silva RFM, Rodrigues CMP, Brites D (2002) Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin. Pediatr Res 51(4):535–541
Neurotox Res (2014) 26:1–15 Silva SL, Oso´rio C, Vaz AR, Barateiro A, Falca˜o AS, Silva RFM, Brites D (2011) Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin. J Neurochem 117(3): 412–424 Silva SL, Vaz AR, Dio´genes MJ, van Rooijen N, Sebastia˜o AM, Fernandes A, Silva RFM, Brites D (2012) Neuritic growth impairment and cell death by unconjugated bilirubin is mediated by NO and glutamate, modulated by microglia, and prevented by glycoursodeoxycholic acid and interleukin-10. Neuropharmacology 62(7):2398–2408 Simoni P, Cerre C, Cipolla A, Polimeni C, Pistillo A, Ceschel G, Roda E, Roda A (1995) Bioavailability study of a new, sinking, enteric-coated ursodeoxycholic acid formulation. Pharmacol Res 31(2):115–119 Tanaka J, Toku K, Zhang B, Ishihara K, Sakanaka M, Maeda N (1999) Astrocytes prevent neuronal death induced by reactive oxygen and nitrogen species. Glia 28(2):85–96 Tian L, Ma L, Kaarela T, Li Z (2012) Neuroimmune crosstalk in the central nervous system and its significance for neurological diseases. J Neuroinflammation 9:155 Vaz AR, Delgado-Esteban M, Brito MA, Bolan˜os JP, Brites D, Almeida A (2010) Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical
15 neurons: assessment of the protective effects of glycoursodeoxycholic acid. J Neurochem 112(1):56–65 Vaz AR, Silva SL, Barateiro A, Falca˜o AS, Fernandes A, Brito MA, Brites D (2011a) Selective vulnerability of rat brain regions to unconjugated bilirubin. Mol Cell Neurosci 48(1):82–93 Vaz AR, Silva SL, Barateiro A, Fernandes A, Falca˜o AS, Brito MA, Brites D (2011b) Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin. Exp Neurol 229(2):381–390 Viviani B (2006) Preparation and coculture of neurons and glial cells. Curr Protoc Cell Biol Chap. 2:Unit 2.7. doi: 10.1002/ 0471143030.cb0207s32 Wang JY, Wen LL, Huang YN, Chen YT, Ku MC (2006) Dual effects of antioxidants in neurodegeneration: direct neuroprotection against oxidative stress and indirect protection via suppression of glia-mediated inflammation. Curr Pharm Des 12(27):3521–3533 Watts LT, Rathinam ML, Schenker S, Henderson GI (2005) Astrocytes protect neurons from ethanol-induced oxidative stress and apoptotic death. J Neurosci Res 80(5):655–666 Yardan T, Erenler AK, Baydin A, Aydin K, Cokluk C (2011) Usefulness of S100B protein in neurological disorders. J Pak Med Assoc 61(3):276–281
123
