Brain Struct Funct DOI 10.1007/s00429-015-1072-1

ORIGINAL ARTICLE

Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons Filipa F. Ribeiro1,2 • Raquel Neves-Tome´1,2 • Nata´lia Assaife-Lopes1,2 • Telma E. Santos3 • Rui F. M. Silva4 • Dora Brites4 • Joaquim A. Ribeiro1,2 Mo´nica M. Sousa3 • Ana M. Sebastia˜o1,2



Received: 7 January 2015 / Accepted: 27 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague–Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing Electronic supplementary material The online version of this article (doi:10.1007/s00429-015-1072-1) contains supplementary material, which is available to authorized users.

the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration. Keywords Axonal growth  BDNF  Cortical primary cultures  Microtubule dynamics  Neurite outgrowth

& Ana M. Sebastia˜o [email protected] Filipa F. Ribeiro [email protected]

Mo´nica M. Sousa [email protected] 1

Raquel Neves-Tome´ [email protected]

Instituto de Farmacologia e Neurocieˆncias, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisbon, Portugal

2

Nata´lia Assaife-Lopes [email protected]

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisbon, Portugal

3

Nerve Regeneration Group, Instituto de Biologia Molecular e Celular, IBMC, Universidade do Porto, 4150-180 Porto, Portugal

4

iMed.ULisboa, Faculdade de Farma´cia, Universidade de Lisboa, 1649-003 Lisbon, Portugal

Telma E. Santos [email protected] Rui F. M. Silva [email protected] Dora Brites [email protected] Joaquim A. Ribeiro [email protected]

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Introduction Dendritic arborization and axonal elongation play a critical role in the formation of neuronal circuits. These processes are under the tight influence of extracellular signals that in such a way play a determinant role in the functioning of the network. Adenosine is a ubiquitous neuromodulator, released by neurons and glial cells. Through activation of high affinity A1 and A2A G-protein coupled receptors (A1R and A2AR), adenosine has numerous roles in the brain, the most studied being the modulation of neuronal excitability (Sebastia˜o and Ribeiro 2009; Rombo et al. 2014) and the control of synaptic plasticity (Dias et al. 2013a). Evidences of time-dependent changes in brain A2AR mRNA and protein expression during late prenatal and early postnatal periods suggest a role for A2ARs in neuronal development (Rivkees 1995; Ade´n et al. 2000; Rebola et al. 2005). Indeed, there are several studies reporting A2AR effects upon neurite outgrowth in the neuronally differentiated PC12 cell line (Heilbronn and Zimmermann 1995; Cheng et al. 2002; Charles et al. 2003). Moreover, Canals et al. (2005) explored the effect of A2AR selective agonist, CGS 21680, in human neuroblastoma cell line SH-SY5Y and showed, for the first time, that A2ARs activation promotes neuritogenesis and differentiation in primary cultures of striatal neuronal precursors. More recently, Silva et al. (2013) reported that exposure to A2AR antagonists, including caffeine, during pregnancy and lactation in mice, delays the migration and the insertion of c-aminobutyric acid (GABA) neurons in the developing hippocampus. In adulthood, those mice displayed loss of hippocampal GABAergic neurons and have also some cognitive deficits (Silva et al. 2013). The establishment of neuronal contacts during neuronal maturation and/or regeneration depends on axonal growth, which is critical for proper circuitry formation. Whether A2ARs may influence axonal growth was not yet reported. A2ARs crosstalk with receptors for other neuromodulators (Sebastia˜o and Ribeiro 2009), including neurotrophins, as reported for the transactivation of the tropomyosin-related kinase B (TrkB) receptor (Lee and Chao 2001). In addition, A2ARs control the fast facilitatory actions of brain-derived neurotrophic factor (BDNF) at hippocampal synapses (Dio´genes et al. 2004), with consequences for synaptic plasticity (Fontinha et al. 2008), through a mechanism dependent on cAMP/PKA (Dio´genes et al. 2004; Fontinha et al. 2008) and on TrkB translocation to lipid rafts (AssaifeLopes et al. 2014). BDNF, through TrkB receptor activation, promotes neuronal survival (Thoenen et al. 1987), facilitates learning, memory and synaptic plasticity (Yamada and Nabeshima 2003; Go´mez-Palacio-Schjetnan and Escobar 2013), neurogenesis during cortical development

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(Bartkowska et al. 2007), induces the formation of primary dendrites and regulates their growth and branching (Bartrup et al. 1997; Dijkhuizen and Ghosh 2005). Considering the influence of BDNF upon neurite branching and growth and the crosstalk between A2A and TrkB receptors, we hypothesized that the action of A2ARs upon neurite outgrowth could be due to their ability to trigger and potentiate the actions of this neurotrophin. Following this hypothesis, we detected for the first time that A2ARs promote axonal elongation, as well as dendritic branching in primary cultures of cortical neurons. While the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. By further studying the effect of A2ARs activation on microtubule dynamics, we demonstrated that A2ARs activation decreases microtubule stability and increases microtubule growth speed in axonal growth cones, two molecular correlates of axonal elongation.

Materials and methods Animals Sprague–Dawley rats were acquired from Harlan Interfauna Iberia (Barcelona, Spain). All experimental procedures were in accordance with current Portuguese laws on Animal Care and with the European Union Directive (86/ 609/EEC) on the protection of animals used for experimental and other scientific purposes. All efforts were made to minimize animal suffering and to use the minimum number of animals. Reagents and drugs CGS21680 (4-[2-[[6-Amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]-benzene propanoic acid hydrochloride), ZM241385 (4-(2-[7-Amino-2-(2furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol) and U73122 (1-[6-[[(17b)-3-Methoxyestra-1,3,5(10)trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) were obtained from Tocris Cookson (Ballwin, MO, USA). Adenosine deaminase (ADA, from calf intestine 10 mg/2 mL, EC 3.5.4.4) was from Roche (Germany). LY294002 (2-(4-Morpholinyl)-8phenyl-1(4H)-benzopyran-4-one) and U0126 (1,4-Diamino2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene) were obtained from Ascent Scientific (Bristol, UK). Forskolin and Rp-cAMPS (Rp-Adenosine 30 ,50 -cyclic monophosphorothioate Triethylammonium salt hydrate) were from Sigma (St. Louis, MO, USA). The recombinant human TrkB/Fc chimera, TrkB/ Fc, was purchased from R&D systems (Minneapolis, MN,

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USA). ECL plus reagent was obtained from GE Healthcare. Bradford reagent was from Bio-Rad. All other reagents were purchased from Sigma (St. Louis, MO, USA). Antibodies The primary antibodies used in immunocytochemistry were mouse anti-MAP2 (Chemicon, Temecula, CA, USA, 1 mg/mL), anti-Tau1 (Millipore, Billerica, MA, USA), anti-NeuN (Millipore, Temecula, CA, USA), mouse monoclonal anti-acetylated tubulin (clone 6-11B-1; Sigma, St. Louis, MO, USA), rabbit anti-Tau (Synaptic Systems, Germany), rat anti-tubulin alpha (clone YL-1/2; AbD Serotec, Bio-Rad, Hercules, CA, USA). The secondary fluorescent-labelled antibodies used were Alexa Fluor 488-coupled goat anti-rabbit, Alexa Fluor 568-coupled goat anti-mouse and Alexa Fluor 568-coupled goat anti-rat (Invitrogen, Grand Island, NY, USA). For western blot, the primary antibodies used were mouse anti-A2AR (Upstate/Millipore—05-717, Darmstadt, Germany), mouse anti-TrkB (BD Biosciences, San Jose, CA, USA), rabbit anti-Phospho p44/42 MAPK (Erk1/2) (Thr202/Tyr204), -Phospho-Akt (Ser473), -PhosphoPLCc1 (Tyr783) and -p44/42 MAPK (Erk1/2) (Cell Signalling Technology, Danvers, MA, USA), mouse monoclonal anti-Akt1 (B-1) and -PLCc1 (E-12) (Santa Cruz Biotechnology, CA, USA). The secondary antibodies used were IgG anti-mouse and IgG anti-rabbit coupled to horseradish peroxidase (HRP) (Santa Cruz Biotechnology, CA, USA). Primary neuronal cultures and treatment Cortical neuronal cultures were prepared from E18 Sprague–Dawley embryos, as described by Assaife-Lopes et al. (2014), with minor modifications. Briefly, cortices were removed from embryos and dissection was performed in cold Ca2?- and Mg2?-free Hank’s balanced salt solution (HBSS) medium supplemented with 0.37 % glucose. In the same solution containing 0.25 % trypsin, the tissue was digested for 15 min at 37 °C, then washed with 10 % fetal bovine serum in HBSS to inactivate trypsin and centrifuged at 200 g at room temperature. The pellet was re-suspended in Neurobasal medium supplemented with 0.5 mM glutamine, 2 % B27 and 25 U/mL penicillin/streptomycin. Cells were dissociated, counted, and plated in 0.01 mg/mL poly-L-lysine-coated dishes at different densities. For morphometric analysis experiments, cells were plated at a low density of 4 9 104cells/mL (Vallotton et al. 2007) in a 24-well plate, and at a higher density of 6 9 105cells/mL in a 12-well plate to measure the protein levels by western blot. On day in culture (DIC) 3 (Burkhalter et al. 2007), cells were treated with the different drugs that remained in

the medium until fixation, which was 5 h after incubation (DIC 3) or at DIC 7, as specified in the ‘‘Results’’ section. Whenever the influence of a receptor ligand upon the action of another receptor ligand was tested, the first was added to the medium 20 min before the second; adenosine deaminase (ADA) (1 U/mL), an enzyme that converts extracellular adenosine into inosine, was added 1 h before the second drug, whenever specified. Inhibitors and activators of transducing pathways were added 30 min prior to the addition of receptor agonists and their effects assayed after 5 h (Dijkhuizen and Ghosh 2005). The receptor ligands used were CGS 21680 (10–100 nM), a selective A2AR agonist (Jarvis et al. 1989), ZM 241385 (50 nM), a selective A2AR antagonist (Poucher et al. 1995), and the endogenous TrkB ligand, BDNF (20 ng/mL) (Dijkhuizen and Ghosh 2005). The inhibitors used were LY294002 (a PI3K inhibitor), U73122 (a PLC inhibitor), U0126 (a MAP kinase inhibitor) and Rp-cAMPS (a blocker of cAMPmediated effects); all were tested at a final concentration of 50, 5, 10 and 100 lM, respectively (Dijkhuizen and Ghosh 2005; Lochner and Moolman 2006; Assaife-Lopes et al. 2014). The activator forskolin (an activator of adenylate cyclase to raise the levels of cAMP) was used at a final concentration of 5 lM (Cristo´va˜o-Ferreira et al. 2009; Assaife-Lopes et al. 2014). Finally, the TrkB/Fc chimera (a BDNF scavenger) was used at a final concentration of 2 lg/mL (Guo et al. 2008; Dio´genes et al. 2011; AssaifeLopes et al. 2014). Immunocytochemistry DIC 3 and DIC 7 cortical neurons grown in 24-well plates were washed with Phosphate-Buffered Saline (PBS) (NaCl 137 mM, KCl 2.1 mM, KH2PO4 1.8 mM and Na2HPO42H2O 10 mM, at pH = 7.40), and fixed with 4 % phosphate-buffered paraformaldehyde (PFA) at room temperature, for 20 min. Excess of PFA was removed by washing with PBS solution. A permeabilization step was performed for 10 min using PBS containing 0.05 % of the non-ionic detergent Triton X-100 at room temperature. This was followed by incubation with a blocking solution of PBS containing 0.25 % gelatine for 30 min, at room temperature. Cells were incubated for 1 h, at room temperature, with the monoclonal primary antibody antiMAP2 (1:200), anti-tau (1:400) and anti-tau1 (1:100) in PBS containing 0.05 % Tween 20 and 0.1 % gelatine. Washes to remove the excess of primary antibody were then performed in PBS containing 0.05 % Tween 20. Cultures were incubated for 1 h, at room temperature, with a goat anti-mouse and/or a goat anti-rabbit secondary antibody, respectively, conjugated to the fluorescent label Alexa Fluor 568 and 488 (1:500) in PBS containing 0.05 % Tween 20 and 0.1 % gelatine. For neuronal nuclei

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identification, cells were permeabilized and blocked with 6 % bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1.5 h. Incubation with the primary antibody anti-NeuN (1:100) was performed overnight at 4 °C, followed by 1 h of secondary antibody goat anti-mouse Alexa Fluor 568 (1:200). After washing in PBS containing 0.05 % Tween 20, cultures were incubated for 10 min with DAPI (40 , 60 diamidino-2-phenylindole), and coverslips mounted in Mowiol. Images of neurons were captured using a monochrome digital camera (AxioCamMR3, Zeiss) mounted on an inverted widefield fluorescence microscope (Zeiss Axiovert 200, Germany), with a 409 objective. Images were captured using the software AxioVision 4 (Carl Zeiss Imaging Systems). The pixel size in the object space was 0.25 lm and the captured images were 1388 9 1040 pixels size. Images were stored and analysed in an uncompressed 8-bit Tiff format.

total neurite length, which corresponds to the sum of all individual process lengths from a single neuron, (2) maximum neurite length, which corresponds to the length of the longest process in each neuron, (3) number of branch points, which corresponds to the total number of ramification points in all processes from a single neuron, and (4) number of roots, which corresponds to the number of primary neurites, i.e. the ones that originate from the cell body (Fig. 1b). To analyse neurons with the HCA-Vision software, cells have to be plated at low density, with a range of 1–5 9 104 cells/mL, to minimize cell–cell contact (Vallotton et al. 2007), thus allowing the identification of individual cells. Therefore, in this work, cells were plated at 4 9 104 cells/mL. As a result, most of the cells analysed were isolated neurons, with no contacts, or with a minimum contact (Fig. 1) between them, minimizing neurite overlap and subjective decisions on neurite identification in each neuron.

Morphometric analysis

Propidium iodide assay

The HCA-Vision software module for neurite outgrowth was used since it is a powerful tool for the analysis of neurite structure in fluorescence images (Vallotton et al. 2007). Through HCA-Vision, it is possible to analyse images in a fully automated manner regarding the segmentation of neuron bodies and neurites, which include all neuronal processes, therefore the axon and the dendrites. Analysis is performed in three steps (Fig. 1a), namely cell body detection (Fig. 1a1), neurite detection (Fig. 1a2), and neurite analysis (Fig. 1a3); optimal parameters of each wizard are chosen by the user, so that the software can generate neurite traces, segment the cell soma and associate neurites with their respective soma, with a consequent report of a virtual image of each neuron. Briefly, in the cell body detection step, the images are treated to suppress the noise, correct the background, and eliminate cell debris based on the small size. Then, the second step is neurite detection, in which linear features are identified by classifying pixels as belonging to a linear feature based on its intensity. Pixels with high intensity are connected and the connected components smaller than a user-defined threshold are removed. Gaps in the skeleton are closed. Finally, the neurite analysis step, apart from removing very short lateral branches that result from thinning artefacts, combines the result of the cell body detection and of the neurite detection to produce a virtual image of the neuronal cell. Table 1 indicates the set of parameters used in the three stages of image analysis. This process allows quantitative morphometric measurements and statistics, which are reported on a per-cell basis (Vallotton et al. 2007). Quantitative measurements considered relevant to assess neurite outgrowth in this work were (1)

To quantify neuronal death, 3 lg/mL of propidium iodide (PI; 3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide; Sigma) was added to DIC7 primary cortical cultures, using a protocol previously used in the Unit (Xapelli et al. 2008; Valadas et al. 2012). As a polar compound, PI only enters in dead or dying cells through the damaged cell membrane and binds to the DNA, emitting light at a wavelength of 608 nm after excitation with a 540 nm wavelength. After 30 min of incubation with PI at 37 °C, cells were fixed with PFA 4 % for 20 min and then washed with PBS solution as described above. Incubation for 10 min with DAPI was followed, and coverslips were mounted in Mowiol. Images of neurons were captured using a monochrome digital camera (AxioCamMR3, Zeiss) mounted on an inverted widefield fluorescence microscope (Zeiss Axiovert 200, Germany), with a 209 objective. Ten arbitrary photographs from each coverslip were taken and an average of 1100 cells was counted per condition in each experiment. Images were captured using the software AxioVision 4 (Carl Zeiss Imaging Systems). The pixel size in the object space was 0.20 lm and the captured images were 1388 9 1040 pixels size. Images were stored and analysed in an uncompressed 8-bit Tiff format. After cell counting using ImageJ software, cell death was presented as the ratio between the counted number of PI-positive cells and the DAPI-positive (total) cells.

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Western blot Western Blot assays were used to assess the presence of A2ARs and TrkB receptors in primary neurons throughout

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Fig. 1 Exemplification of the morphometric analysis using HCAVision software. a Representative fluorescence image analysis of two DIC 7 cultured cortical neurons by HCA-Vision software. Images with two channels (one for nucleus and another for cell structure) were taken with an inverted widefield fluorescence microscope (Zeiss Axiovert 200, Germany), with a 409 objective, and using the Zeiss filter sets 49 and 15 for detection of DAPI and Alexa Fluor 568 conjugated secondary antibody, respectively, and were analysed by HCA-Vision software. A three-step analysis was performed to obtain a virtual image of these two neurons: neuron body detection (a1), neurite detection (a2), and neurite analysis, where detected neurites in (a2) are associated to neuron bodies already detected in (a1), forming

thus two virtual neuronal images (a3). b Virtual images of two neurons obtained by HCA-Vision software; using a virtual image (b1), the software quantifies the following morphological parameters: total neurite length (b2), maximal neurite length (b3), number of branch points (b4) and number of roots (b5). From these two virtual neuronal images, total neurite length is 374.86 and 339.55 lm for the green and pink neurons (b2), respectively. In the case of maximal neurite length, green and pink neurons have, respectively, 62.49 and 69.68 lm (b3). The number of branch points measured for green and pink neurons were 18 and 14 (b4), and the number of roots 5 and 8 (b5), respectively. Scale bar [for (a) and (b)] = 20 lm

Table 1 Set of parameters used in the three stages of the image analysis by HCA-Vision software (adapted from Vallotton et al. 2007) Cell body detection

Neurite detection

Neurite analysis

Smoothing (Gaussian filter: 6 pixels)

Smoothing (Gaussian filter: 8 pixels)

Debard small branches (2 pixels)

Background correction (morphological top hat: 99 pixels)

Non-maximum suppression (contrast = 1.31)

Thicken neuron bodies (7 pixels)

Suppression of neurites (morphological opening: 7 pixels)

Removal of small objects (2 pixels)

Remove small trees (2 pixels)

Intensity thresholding (0.87)

Gap closing (distance: 8 pixels, quality = 0.94)

Nucleus channel, nucleus thresholding sensitivity (0.65) Object selection, based on area (700 pixels)

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95–100 °C for 5 min. Protein running was performed in SDS-PAGE gels (10 % for resolving and 5 % for stacking gels), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 h at room temperature with 5 % non-fat milk, or 5 % bovine serum albumin (BSA) in TBS-T (Tris buffer saline with 0.1 % Tween-20, 200 nM Tris, 1.5 M NaCl). Incubations with the primary antibodies mouse anti-A2AR (1:3000), -TrkB (1:1000), rabbit antiPhospho p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:500), -Phospho-Akt (Ser473) (1:500), -Phospho-PLCc1 (Tyr783) (1:250), and –p44/42 MAPK (Erk1/2) (1:6000), mouse monoclonal anti-Akt1 (1:500) and -PLCc1 (1:750) and rabbit polyclonal anti-b-Actin (1:10,000) were performed overnight at 4 °C, all of them diluted in 3 % BSA in TBST and 0.02 % sodium azide. The HRP-coupled secondary antibodies were diluted (1:10,000) in blocking buffer and incubated for 1 h at room temperature, with the secondary antibodies for the A2AR detection diluted at 1:2500 and for the b-Actin detection diluted at 1:20,000. Membranes were washed in TBS-T and detection of proteins was performed with ECL plus Western blotting detection reagent (GE Healthcare) using X-Ray films (Fujifilm). Optical density was determined with ImageJ software. Microtubule stability

Fig. 2 Western blot analysis of A2A and TrkB receptors in ratcultured primary cortical neurons. a Representative immunoblots which depict immunoreactive bands of the A2AR, TrkB and b-actin (loading control) proteins in rat-cultured cortical neurons obtained from total lysates of cells at different days in culture (DIC 1–DIC 7). In b and c are shown the quantitative densitometric analysis of the immunoreactivity of A2ARs (b) or TrkB (c), where 1 on the ordinate corresponds to the ratio A2AR/b-actin or TrkB/b-actin staining at DIC 1. Data are expressed as mean ± SEM from six independent cultures. Statistical significance was assessed by Dunnett’s multiple comparison test: *p \ 0.05, as compared with DIC 1 condition (first bar on the left)

the time in culture (DIC 1–7). Samples were lysed and denatured by heating at 70 °C for 30 min for A2ARs or at 95–100 °C for 5 min for TrkB receptors; b-actin was used as loading control. As shown in Fig. 2, A2ARs were already detectable at DIC 1, and their density did not significantly change (p [ 0.05) up to DIC 7. TrkB receptor levels markedly increased (p \ 0.05) from DIC 1 to DIC 3, being clearly detectable during the remaining time in culture. To evaluate overall activation of PI3K, MAPK and PLC signalling pathways, cultured neurons were lysed at different time points up to 5 h after A2AR agonist incubation. All lysates were then denatured with 59 sample buffer (350 mM Tris, 30 % glycerol, 10 % SDS, 600 mM dithiothreitol, and 0.012 % bromophenol blue, pH 6.8) at

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DIC 3 cortical neurons grown in 24-well plates were drug treated for 5 h until fixation in microtubule-stabilizing conditions with PHEM buffer (PIPES 65 mM, HEPES 25 mM, EGTA 10 mM, MgCl2 3 mM and Triton X-100 0.1 %, at pH = 6.9) with 2 % PFA at room temperature, for 20 min. A permeabilization step was performed for 5 min using PBS containing 0.2 % Triton X-100 at room temperature. This was followed by incubation with a blocking solution of PBS containing 5 % normal donkey serum (NDS) for 1 h, at room temperature. Cells were then incubated for 1 h with the mouse monoclonal primary antibody anti-acetylated tubulin (1:500), rat anti-tubulin alpha (1:500) in PBS containing 5 % NDS. Extensive washes were performed in PBS before incubation for 1 h at room temperature with both a goat anti-mouse and a goat anti-rat secondary antibodies, which were, respectively, conjugated to the fluorescent label Alexa Fluor 488 and 568 (1:1000) in PBS containing 5 % NDS. Cultured neurons were incubated for 10 min with DAPI (40 , 60 -diamidino-2-phenylindole), and coverslips were mounted in Mowiol. Images of axonal growth cones were captured in a Zeiss AxioImager Z1 widefield microscope, with a 1009 objective. The ratio of fluorescence intensities of acetylated/tyrosinated a-tubulin in growth cones was analysed using Fiji software. A minimum of 100 growth cones per condition per experiment were quantified.

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Transfection, live cell imaging and quantification of EB3 dynamics Cortical neurons from E18 Sprague–Dawley embryos were transfected with the 4D Nucleofector Amaxa system. A truncated version of EB3-GFP [a construct containing amino acids 1–200 of EB3, artificially dimerized by the addition of the leucine zipper domain of GCN4, cloned into the pEGFP-N1 vector, that efficiently accumulates at microtubule tips (Komarova et al. 2009)] was used. After transfection, cells were left in suspension for 5 min and thereafter plated in PLL-coated l-dishes (Ibidi) for 3 days. At DIC 3, cortical primary cultures were treated with 30 nM CGS 21680. Time-lapse recordings (100 frames, 1 every 2 s) were taken at 37 °C using an Andor Revolution XD Spinning Disk. Kymographs were made using a Matlab script (LAPSO) (Pereira and Maiato 2010). To calculate microtubule growth speeds, the slopes of lines drawn on kymographs from the beginning to the end of individual EB3 movements were measured. To quantify the distance of microtubule tips to the leading edge of the growth cone, EB3 fluorescence intensity was measured from the growth cone tip up to 10 lm along the growth cone using Fiji software. A minimum of 100 microtubules from at least 10 neurons were quantified. Data analysis All data are expressed as mean ± SEM from n independent cultures. Morphometric analysis in each culture was performed over 50/100 measurements taken from 50/100 individual cells, where all conditions were tested in each culture. Statistical significance was determined through the GraphPad Prism version 5.00 for Windows, GraphPad Software (San Diego California USA, http://www.graph pad.com), using one-way ANOVA followed by Bonferroni correction for multiple comparisons to compare selected pairs of columns or by Dunnett’s multiple comparison test when comparing all means with only the control mean; when only two means were compared, a Student’s t test analysis was performed. For the three statistical analysis, values of p \ 0.05 were considered to represent statistically significant differences.

Results Adenosine A2A receptor activation induces neurite outgrowth To first assess whether A2ARs influence neurite outgrowth in primary cortical neurons, the A2AR selective agonist, CGS 21680 (10–100 nM) (Jarvis et al. 1989), was applied

to the culture medium at day in culture (DIC) 3 and remained present until fixation at DIC 7. We choose DIC 3 to start drug treatment since at this time point neurons are already polarized and are starting to form dendrites (Dotti et al. 1988). Consequently, drug influences over new dendrite formation as well as over axonal and dendritic development are assessed independently of putative influences over neuronal polarization (Shelly et al. 2007). As DIC 7 neurons have the axon and dendrites already specified, and these processes have also started to branch (Dotti et al. 1988; Ziv and Smith 1996), this time point was chosen for fixation and allowed to measure alterations in the total (the sum of the individual process lengths) and maximal (the length of the longest process) neurite length, the number of primary neurites, and the number of branch points (Fig. 1, see also ‘‘Materials and methods’’). Neurons incubated with the A2AR agonist exhibited an increase in the total and maximal neurite length, as well as in the number of branch points (Fig. 3a, b1–b4; p \ 0.05, 200 neurons from four independent cultures). The effect of CGS 21680 was concentration dependent, being the maximal effect observed with CGS 21680 30 nM, which was the concentration used in most of the following experiments. At 100 nM, CGS 21680 caused a smaller (p \ 0.05) effect than that of 30 nM, probably due to receptor desensitization (Klaasse et al. 2008). In none of the concentrations tested (10–100 nM) did the A2AR agonist affect the number of roots (p [ 0.05, Fig. 3b4). Cell viability was also not affected (p [ 0.05, four independent cultures) when neurons were incubated from DIC 3 to DIC 7 in the presence of CGS 21680 (10–100 nM) (Fig. 4). The effect of CGS 21680 upon maximal neurite length and number of branch points was fully prevented in cultures incubated with the A2AR antagonist, ZM 241385 (50 nM, added 20 min before CGS 21680 and being present from DIC 3 to DIC 7) (Fig. 3c). By itself, ZM 241385 had a small but significant (p \ 0.05, 150 neurons from three independent cultures) inhibitory effect upon maximal neurite length (Fig. 3c1), being devoid of effect upon the number of branch points (Fig. 3c2). Adenosine is released by neurons and glial cells and therefore can accumulate in the extracellular medium of neurons in culture (Rosenberg et al. 2000; Fredholm et al. 2005). Consequently, we investigated if tonic activation of A2ARs by endogenous adenosine could influence neuronal outgrowth and, most importantly, whether it could be masking an effect of the exogenous agonist. To remove endogenous extracellular adenosine, adenosine deaminase (ADA, 1 U/mL), an enzyme that metabolizes adenosine to inosine (Parkinson et al. 2006) and does not penetrate in cell membranes, was added to the culture medium at DIC 3, 1 h before the addition of CGS 21680 (30 nM), and remained in the medium until fixation at DIC 7.

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Fig. 3 Adenosine A2ARs activation induces neuronal outgrowth. a Representative fluorescence digital images of cortical neurons under control conditions (left) or after 4 days (from DIC 3 to DIC 7) stimulation with CGS 21680 (right). Neuronal morphology in primary cortical cultures at DIC 7 was assessed by immunostaining the dendritic marker MAP2 (red). Nuclei are visualized by DAPI staining (blue). Virtual images of those neurons, generated to quantify neuronal morphology (total neurite length, maximal neurite length, number of branch points and number of roots) and modified by Power Point to exemplify this quantifications (white lines and dots), are shown below each control and CGS 21680 fluorescence image. b Average data for total neurite length (b1), maximal neurite length (b2), number of branch points (b3) and number of roots (b4),

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following exposure to different concentrations of A2AR agonist, CGS 21680, as indicated below each bar. c Influence of the A2AR antagonist, ZM 241380 (50 nM), or adenosine deaminase (ADA, 1 U/ mL) over the effect of CGS 21680 (30 nM) upon the maximal neurite length (c1) and the number of branch points (c2). Scale bar [for (a)] = 20 lm. Data are expressed as mean ± SEM of 150 (c1, c2) or 200 (b1–b4) neurons, taken from 4 (b) or 3 (c) independent cultures (50 neurons analysed per condition and per culture), where all conditions were tested in each culture. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05; ns (not statistically significant) as compared with control (open bar on the left) or as indicated by the horizontal lines above the columns

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the influence of BDNF upon neurite outgrowth (Dijkhuizen and Ghosh 2005). As expected, neuronal branching at DIC 3 (Fig. 5a, c) was more incipient than at DIC 7 (Fig. 3a, b), but the facilitatory effect of CGS 21680 (30 nM) upon the maximal neurite length and upon the number of branch points was already evident (p \ 0.05, 600 neurons from six independent cultures) after 5 h of incubation (Fig. 5c1, c2). The number of primary neurites, quantified as the number of roots, remained unaffected (p [ 0.05) by CGS 21680 (30 nM) (Fig. 5c3). Adenosine A2A receptor activation induces axonal elongation

Fig. 4 Cell viability and percentage of neuronal cells in primary cortical cultures is not affected by any treatment. a Bar graph depicts the number of PI-positive cells, expressed as the percentage of total cells per culture. b Bar graph depicts the number of NeuNpositive cells, expressed as the percentage of total cells per culture. Data are expressed as mean ± SEM from four independent cultures. Statistical significance was assessed by Dunnett’s multiple comparison test, as compared with control condition (first bar on the left, absence of any drug). PI propidium iodide, NeuN neuronal nuclei

Measurements of the morphological properties showed that the elimination of endogenous adenosine from the medium by ADA did not affect (p [ 0.05, 150 neurons from three independent cultures) the maximal neurite length or the number of branch points (Fig. 3c). Most important, the enhancement caused by CGS 21680 in the maximal neurite length or number of branch points was similar in the absence or presence of ADA (Fig. 3c). Data shown above allow us to conclude that prolonged (4 days) activation of A2ARs promotes maximal neurite extension and neuronal branching. We then tested whether this effect could be detected shortly after A2ARs activation. To do so, the A2AR agonist was added to the cultures at 3 DIC and the neuronal cultures were fixed after 5 h of incubation, at a time point previously used by others to test

Antibodies against microtubule-associated protein 2 (MAP2) allow to stain a protein mostly found in the somatodendritic compartment (Bernhardt and Matus 1984; Caceres et al. 1984). Although MAP2 is also found in the axon during neuronal differentiation, as the neuron matures it becomes less expressed in the distal parts of the axon, so that distal axonal staining is weak at DIC 7 (Caceres et al. 1986), resulting in the loss of its detection. An example of these limitations is demonstrated in Fig. 1 where the distal portion of the maximal neurite of the green neuron stained with anti-MAP2 antibody (Fig. 1a2) was completely lost after morphometric analysis by HCA-Vision software (Fig. 1b3). To evaluate if the enhancement in the length of the maximal neurite, observed upon A2ARs activation, corresponds to an increase in axonal elongation, we performed co-immunostaining assays for MAP2 and another MAP, the tau protein, which is present in the whole neuron, especially in the axon. The increase in the length of the maximal neurite in neurons incubated at DIC 3 with the A2AR agonist, CGS 21680 (30 nM) for 5 h, was visible and significant (p \ 0.05) either when anti-MAP2 or anti-tau antibodies were used to stain the neurons (Fig. 5a, d). As expected, the A2AR antagonist, ZM 241385 (50 nM), also prevented the effect of CGS 21680 in tau-stained neurons (Fig. 5e). During axonal growth, there is a gradient of phosphorylated tau, which decreases from the somatodendritic compartment to the terminal region of the axon (Mandell and Banker 1996). Because anti-tau-1 antibody binds to a phosphorylatable site of the tau protein when it is unphosphorylated (Mandell and Banker 1995), it is able to specifically label growing axons. We therefore performed a co-immunostaining of tau/tau-1 to better identify the axon. The maximal neurite length detected by morphometric analysis was the only process stained by the anti-tau1 antibody (Fig. 5b) and, remarkably, it was increased in the neurons incubated with CGS 21680 for 5 h at DIC 3 (Fig. 5d).

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Brain Struct Funct b Fig. 5 Adenosine A2ARs activation induces axonal elongation, an

action detected even after brief exposure to the agonist. Neuronal morphology in primary cortical cultures at 3 days in culture was assessed by immunostaining tau (green), which reaches the distal parts of the axon; the same neurons were co-immunostained for the dendritic marker MAP2 (red in a). In some cultures, the axonal structure was confirmed by co-immunostaining the specific axonal marker unphosphorylated tau (anti-tau1 antibody) (red in b). Nuclei are visualized by DAPI staining (blue). c Quantification of various parameters of neuronal morphology (MAP2 immunostaining); 600 neurons from six independent cultures (100 neurons per culture) were analysed per condition. d Comparison of the quantifications of the maximal neurite length based upon MAP2, tau or tau1 immunostaining as indicated above the columns; 150 neurons from three independent cultures (50 neurons per culture) were analysed per condition. e Blockade of the effect of CGS 21680 by the selective A2AR antagonist, ZM 241380 (50 nM) (tau immunostaining); 200 neurons per condition and from two independent cultures (100 neurons per culture) were analysed, where all the conditions were tested in each culture. Scale bar [for (a) and (b)] = 20 lm. In all cases, neurons were incubated for 5 h in the absence (-) or presence (?) of the selective A2AR agonist, CGS 21680 (30 nM). Data are expressed as mean ± SEM and statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05; ns (not statistically significant) as compared with control (absence of CGS 21680 under the same drug conditions for the same immunostaining condition) or as indicated by the horizontal lines above the columns

Altogether, the above results demonstrate that A2ARs promote the elongation of the axon in cultured cortical neurons. Elongation of the axon induced by adenosine A2A receptors relies on the activation of the PI3K, MAPK/Erk and PLC signalling pathways than on the adenylate cyclase/cAMP/PKA pathway The canonical transducing system of A2ARs is the activation of adenylate cyclase (AC) (Fredholm et al. 2001). In addition, cyclic adenosine monophosphate (cAMP) is known to play a role during neuronal differentiation, namely in axonal pathfinding (Song et al. 1997) and in axonal regeneration (Chierzi et al. 2005; Jin et al. 2009; Lau et al. 2013). So, we first hypothesized that the AC/ cAMP/PKA transducing pathway could be involved in the actions of A2ARs upon neuronal maturation. DIC 3 dissociated cortical cultures were treated with CGS 21680 for 5 h in the absence and in the presence of Rp-cAMPS (100 lM), an inactive analogue of cAMP that inhibits activation of PKA by substrate competition (Lochner and Moolman 2006). Surprisingly, the presence of Rp-cAMPS did not prevent the facilitatory effect of CGS 21680 upon maximal neurite length obtained with either anti-MAP2 or -tau antibodies (Fig. 6a). In addition, activating the PKA signalling pathway using an activator of AC, forskolin (5 lM), did not mimic the effect of CGS 21680, an absence

Fig. 6 The elongation of the axon induced by adenosine A2ARs activation is cAMP independent. a, b Quantification of the maximal neurite length following 5-h exposure to the selective A2AR agonist, CGS 21680 30 nM, at DIC 3, in the presence of an inactive analogue of cAMP, Rp-cAMPS (100 lM, a), an activator of adenylate cyclase, forskolin (5 lM, b), through MAP2 or tau immunostaining, as indicated by the horizontal line above the columns. Data are expressed as mean ± SEM from 100 (a) to 150 (b) neurons per condition, taken from 2 to 3 independent cultures, where all the conditions were tested in each culture. In all cases, presence (?) or absence (-) of each drug is indicated below each column. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05; ns (not statistically significant) as compared with control (open bar, same immunostaining) or as indicated by the horizontal lines above the columns

of effect observed with either MAP2 or tau immunostaining (Fig. 6b). We then investigated the influence of other transducing pathways upon A2AR-induced neurite elongation. We used U0126 (10 lM) as an inhibitor of MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase), LY294002 (50 lM) as an inhibitor of PI3K (phosphoinositide 3-kinase), and U73122 (5 lM) as an inhibitor of PLC (phospholipase C) (Dijkhuizen and Ghosh 2005). The concentrations of the inhibitors used were those previously reported as enough to inhibit the transducing pathways activated by BDNF (Dijkhuizen and Ghosh 2005). Because the PI3K pathway has been shown to play an essential role in neuronal survival, any loss of function of this pathway may also affect the survival of the neurons.

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Indeed, inhibition of PI3K during an early stage of dendritic formation (3–6 DIC) causes profound cell death (Kumar et al. 2005). However, as it was already shown (Dijkhuizen and Ghosh 2005), and we could confirm (data not shown), 5-h treatment of control cultures with inhibitors of the MAPK/Erk, PI3K and PLC signalling pathways did not change the viability of the cells, in contrast with their deleterious effects when present from DIC 3 to DIC 7. The blockade of MAPK/Erk, PI3K or PLC pathways with U0126 (10 lM) LY294002 (50 lM) or U73122 (5 lM) almost completely prevented the effect of CGS 21680 on the maximal neurite length (100 neurons from two independent cultures) (Fig. 7a–c). Through Western blot analysis, we could detect a slight increase in the proportion of phosphorylated Erk1/2 (pErk1/2/Erk1/2), phosphorylated Akt1 (pAkt1/Akt1) and phosphorylated PLCc1 (pPLCc1/PLCc1) in neurons incubated in the presence of CGS 21680 (30 nM) (Fig. 7d–f), being the effect predominantly observed at early time points after agonist incubation. Taken together, these data suggest an involvement of the MAPK/Erk, PI3K and PLC signalling pathways on the effect of the A2AR agonist upon axonal elongation.

Fig. 7 PI3K, MAPK/Erk and PLC signalling pathways are involvedc in A2AR-mediated axonal elongation. a–c Quantification of the maximal neurite length following 5-h exposure to the selective A2AR agonist, CGS 21680 30 nM, at DIC 3, in the presence of an inhibitor of MAPK/Erk (U0126, 10 lM, a), PI3K (LY294002, 50 lM, b) and PLC (U73122, 5 lM, c) signalling pathways, through MAP2 or tau immunostaining, as indicated by the horizontal line above the columns. Data are expressed as mean ± SEM from 100 neurons per condition, taken from two independent cultures, where all the conditions were tested in each culture. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05; ns (not statistically significant) as compared with control (open bar, same immunostaining) or as indicated by the horizontal lines above the columns. Western blot analysis of pErk1/2/Erk1/2 (d), pAkt1/Akt1 (e) and pPLCc1/PLCc1 (f) in total lysates of cells treated with CGS 21680 (30 nM) at different time points, as indicated below each bar. In the upper panels are shown representative immunoblots and in the lower panels are depicted the quantitative densitometric analysis of the immunoreactivity of phosphorylated protein normalized for total protein in the same blot; blots were probed with anti-pErk1/2 (1:500), -pAkt1 (1:500), -pPLCc1 (1:250), -Erk1/2 (1:6000), -Akt1 (1:1500) and PLCc1 (1:750); 1 represents the ratio pErk1/1/Erk1/2, pAkt1/Akt1 or pPLCc1/PLCc1 in the absence of CGS 21680; statistical significance was assessed by a Student’s t test where each time point was compared with zero time point; *p \ 0.05. All data are expressed as mean ± SEM from 9 (d), 11 (e) or 4 (f) independent cultures

Reciprocal influence of adenosine A2A receptors and TrkB BDNF receptors upon neurite outgrowth

attenuated by the presence of the A2AR antagonist (Fig. 8c). Moreover, in the presence of CGS 21680 (30 nM), BDNF (20 ng/mL) was still able to increase the number of branch points and the number of roots (Fig. 8c, d). By itself, CGS 21680 promoted an increase in the number of branch points (Fig. 8c; Fig. 3b3), but the effect of both drugs when added together was less than additive (Fig. 8c). Clearly, the A2AR agonist did not amplify the effect of BDNF upon neuronal branching. One may therefore conclude that while the predominant effect of the A2AR agonist is the elongation of the axon, an action not shared by BDNF, the prominent effect of BDNF is upon the number of roots. However, both drugs have in common an enhancement in neuronal branching, but this parameter may not be fully independent of the other two since both the increase in the number of primary neurites and the increase in the length of the axon may raise the chances of dendritic and axonal branching, and, therefore, the chances for an increase in the number of branch points. It is also worthwhile to note that the facilitatory action of CGS 21680 upon the length of the maximal neurite was attenuated by the presence of BDNF (Fig. 8b). This influence, however, does not necessarily mean an overlap of actions. Indeed, comparing neurons treated with CGS 21680 with neurons treated with BDNF, the former have fewer primary neurites, are less branched and have a longer neurite. Considering that the presence of BDNF stimulates

Our findings that MAPK/Erk, PI3K and PLC signalling pathways are required for the A2ARs-mediated facilitation of axonal elongation, together with previous evidence that the influence of BDNF upon neurite outgrowth requires similar signalling pathways (Dijkhuizen and Ghosh 2005) and that interactions between A2A/TrkB receptors have been widely reported (see Introduction), prompted the interest to evaluate if those interactions are also observed on neuronal maturation. We therefore first evaluated if the action of A2ARs upon neuronal maturation could result from the enhancement of the BDNF actions. To do so, we evaluated the influence of BDNF upon neurite outgrowth in the presence or absence of the selective A2AR ligands. As already demonstrated by others (Dijkhuizen and Ghosh 2005), our results show that BDNF induces an increase (p \ 0.05, 300 neurons from six independent cultures) in the number of branch points and in the number of roots, not affecting (p [ 0.05) the length of the maximal neurite (in Fig. 8a–d). In the presence of the A2AR antagonist, ZM 241385 (50 nM), BDNF was still able to induce an increase in the number of roots (Fig. 8d; p \ 0.05, 300 neurons from six independent cultures) and in the number of branch points (Fig. 8c; p \ 0.05, 300 neurons from six independent cultures), though the latter effect was slightly

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the formation of new primary neurites, and also neuronal branching, the length of the maximal neurite may be compromised since the neuron may develop more in terms of neurite number than in terms of neurite elongation. A

marked BDNF-induced attenuation of the facilitatory action of the A2AR agonist upon axonal elongation was also observed in neurons treated for 5 h at DIC 3 and stained with anti-tau antibody, becoming clear that CGS

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Brain Struct Funct b Fig. 8 Crosstalk between the action of both adenosine A2ARs ligands

and BDNF upon neuronal maturation. a–d A2ARs influence BDNF action upon neuronal maturation. a Representative fluorescence images of cortical neurons following incubation for 4 days (from DIC 3 to DIC 7) with/without BDNF (20 ng/mL), in the absence/ presence of either the selective A2AR antagonist, ZM 241380 (50 nM), or the agonist, CGS 21680 (30 nM). Neurons were immunostained for MAP2 (red) at DIC 7; nuclei are visualized by DAPI staining (blue). Quantitative analysis of the morphological parameters, maximal neurite length (b), number of branch points (c) and number of roots (d) were taken from images of 300 neurons per condition, from 3 to 6 independent cultures, where all the conditions were tested in each culture. Data in each panel are expressed as mean ± SEM. e, f The effect of A2ARs activation on axon elongation is prevented by the presence of exogenous BDNF. e Representative fluorescence images of cortical neurons incubated at DIC 3 for 5 h with/without the selective A2AR agonist, CGS 21680 (30 nM), and/or exogenous BDNF 20 ng/mL. Neurons were immunostained for tau (green) at DIC 3; nuclei are visualized by DAPI staining (blue). f Quantitative analysis of the maximal neurite length. Data are expressed as mean ± SEM from 200 neurons per condition, taken from two independent cultures, where all conditions were tested in each culture. In all panels with quantitative analysis, the statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05; ns (not statistically significant), as compared with control (first bar on the left, absence of any ligand) or as indicated by the horizontal bars over the columns. Scale bar [for (a) and (e)] = 20 lm

21680 in the presence of BDNF was not successful in increasing maximal neurite (Fig. 8e, f). Next, we evaluated whether axonal elongation induced by the activation of A2ARs was dependent on the accumulation of endogenous BDNF in the cultures, which would lead to tonic activation of TrkB receptors. We used a TrkB/Fc chimera, which scavenges the extracellular BDNF; the scavenger was added to the culture 30 min before the A2AR agonist, at DIC 3 for a 5-h treatment, followed by fixation and tau immunolabeling. In spite of the presence of TrkB/Fc, the A2AR agonist still induced an increase (p \ 0.05, 200 neurons from four independent cultures) in the length of the maximal neurite (Fig. 9a, b), suggesting that the influence of A2ARs upon axonal elongation is not dependent on the presence of endogenous BDNF. This could be expected since BDNF per se, when added to the culture media, was virtually devoid of effect upon maximal neurite length (Fig. 8b, f). To access whether the facilitatory action of A2ARs upon neurite branching results from an interaction with endogenous BDNF, neurons were fixed at DIC 7, since at this time point neurons are more mature and branching can be better quantified; the somatodendritic compartment was immunostained with anti-MAP2 antibody since axons from DIC 7 neurons overlap with other neurons and therefore individual neurons are hardly distinguished when immunostained with the anti-tau antibody. Hence, only dendritic branching was quantified. The increase in the

number of dendritic branch points caused by CGS 21680 (30 nM) was completely prevented by the presence of the BDNF scavenger (TrkB/Fc 2 lg/mL) (Fig. 10a, b; 200 neurons from four independent cultures). As expected, and tested as a control, the BDNF scavenger also prevented the influence of BDNF over dendritic branching (Fig. 10c). Taken together, the above results suggest that the influence of A2ARs upon dendritic arborization results from a facilitation of the action of endogenous BDNF, whereas the influence of A2ARs upon axonal elongation does not depend on BDNF. Adenosine A2A receptor activation increases microtubule dynamics and growth speed Microtubules are the major components of the axon and are composed of a- and b-tubulin heterodimers. Several posttranslational modifications of these subunits are associated with altered microtubule dynamics and stability. Posttranslational tyrosination of a-tubulin is normally associated with microtubule dynamics, whereas acetylation of atubulin is associated with microtubule stability (Janke and Kneussel 2010; Janke and Bulinski 2011). Since axonal elongation is critically dependent on microtubule dynamics, we investigated the influence of A2ARs upon this process. Neurons were co-immunostained for tyrosinated and acetylated a-tubulin (Fig. 11a) to allow quantification of the ratio between the two immunofluorescence intensities, which was used as an index of microtubule dynamics. The axonal growth cone of neurons incubated in the presence of CGS 21680 (30 nM) displayed a decreased acetylated/tyrosinated a-tubulin ratio (p \ 0.05, 300 growth cones from three independent cultures), which was prevented by the presence of A2AR antagonist, ZM 241385 (50 nM) (Fig. 11b). These results indicate that A2ARs activation increases microtubule instability, in agreement with the facilitatory influence of A2ARs upon axonal elongation. Finally, we evaluated the effect of A2ARs activation on microtubule growth speed. Neurons were transfected with pEGFP-EB3 (end-binding protein 3) and time-lapse recordings (100 frames, 1 every 2 s) at DIC3 were taken. EB3 is a microtubule plus end-tracking protein (?TIP) that accumulates at the plus ends of growing microtubules (Schuyler and Pellman 2001; Akhmanova and Steinmetz 2008) and is responsible for controlling microtubule dynamics by playing an important role in targeting a range of different proteins to the growing microtubule plus ends (Galjart 2010). Results from live imaging of pEGFP-EB3 transfected neurons (Fig. 11c, d and Online Resource 1) show that A2ARs activation induced a marked increase in microtubule growth speed (200 microtubules from two independent cultures, p \ 0.05).

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Brain Struct Funct Fig. 9 The effect of adenosine A2ARs activation on axon elongation is not dependent on the endogenous BDNF. a Representative fluorescence images of cortical neurons incubated at DIC 3 for 5 h with/ without the selective A2AR agonist, CGS 21680 30 nM, in the presence or absence of the BDNF scavenger, TrkB/Fc chimera (2 lg/mL). Neurons were immunostained for tau (green) at DIC 3; nuclei are visualized by DAPI staining (blue). Scale bar [for (a)] = 20 lm. b Quantitative analysis of the maximal neurite length. Data are expressed as mean ± SEM from 200 neurons per condition, taken from four independent cultures, where all conditions were tested in each culture. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05, as compared with control (first bar on the left, absence of any drug) or as indicated by the horizontal bars over the columns

Discussion Major findings in the present work are that A2ARs activation induces axonal elongation through a mechanism dependent on PI3K, MAPK/Erk and PLC signalling pathways, but not on cAMP/PKA or endogenous BDNF. Accordingly, A2ARs activation increases microtubule instability and axonal growth speed. Moreover, A2ARs enhance dendritic branching, which might result from their ability to boost TrkB activation by BDNF since it was lost by scavenging endogenous BDNF. Although the role of adenosine on synaptic transmission in the central nervous system (CNS) has been extensively studied, much less is known about its action upon neuronal maturation, being the few studies in neuronally differentiated PC12 cells. Evidence that enzymes leading to

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extracellular adenosine formation are essential for NGFinduced neurite outgrowth of PC12 cells (Heilbronn and Zimmermann 1995) and promote axonal growth of cultured hippocampal neurons (Dı´ez-Zaera et al. 2011) has mostly been focused upon the role of those enzymes to prevent actions of ATP upon P2X7 and P2Y receptors, which are known to inhibit axonal elongation (Dı´ez-Zaera et al. 2011; del Puerto et al. 2012). One of those studies excluded the involvement of A1Rs and A3Rs on axonal elongation but did not address the action of A2ARs (Dı´ezZaera et al. 2011). Reports regarding A2ARs actions upon neuronal maturation have been mostly related to their ability to enhance NGF-mediated neurite outgrowth (Charles et al. 2003), or to induce neurite outgrowth in a situation of hypoxia (O’Driscoll and Gorman 2005). Using the human neuroblastoma cell line SH-SY5Y and primary

Brain Struct Funct

cultures of striatal neuronal precursors, Canals et al. (2005) showed that A2ARs activation promotes neuritogenesis and differentiation, but did not address the influence of A2ARs upon axonal elongation. We now clearly distinguish two actions of A2ARs during neuronal differentiation, one upon neurite arborization, dependent on the endogenous BDNF signalling, and another, so far unknown, upon axonal elongation and microtubule dynamics. Since A2ARs are frequently coupled to Gs proteins, enhancing adenylate cyclase activity and cAMP production, and cAMP/PKA pathway has been identified as relevant for A2ARs actions in neurite outgrowth (Cheng et al. 2002; Canals et al. 2005), it was surprising that A2ARs action upon axonal elongation was insensitive to RpcAMPS and that forskolin did not mimic the effect of A2AR agonist. Our data are, however, consistent with reports showing that the promotion of dynamic instability of microtubules by Ga is independent of the cAMP/PKA pathway, resulting from enhanced GTPase activity, which leads to higher GTP/GDP turnover on tubulin and, hence, enhanced dynamic instability of microtubules necessary for neurite outgrowth (Yu et al. 2009). The enhancement of maximal neurite elongation by A2ARs activation was prevented by inhibitors of the MAPK/Erk, PLC, or Akt signalling pathways, suggesting their involvement in the A2AR-mediated action. Indeed, A2ARs activation enhanced pErk1/2, pAkt1 and pPLCc1 levels. The increase in phosphorylation of those target proteins was mostly visible within minutes after incubation, suggesting that those proteins are required in the initial steps of the transducing cascade. The overall enhancement of phosphorylation of Erk1/2, Akt1 and PLCc1 induced by the A2AR agonist in cortical neurons was mild, but high compartmentalization of these signalling molecules is expected to occur. Therefore, axonal elongation induced by A2ARs activation may recruit transducing pathways activated by TrkB receptors, but does not depend on the presence of endogenous BDNF since it was insensitive to the BDNF scavenger. The possibility that it involves transactivation of TrkB receptors, i.e. phosphorylation of TrkB receptors in the absence of endogenous BDNF, is rather unlikely because BDNF itself does not promote, and even hampers the influence of A2ARs upon axonal elongation. The finding that axonal elongation induced by A2ARs activation was fully prevented by a PI3K inhibitor agrees with what is known about the role of this pathway for axonal growth. Several groups reported that local activation of PI3K and accumulation of PIP3 at the tip of the immature neurites is essential for axon specification and elongation (Shi et al. 2003; Menager et al. 2004). PI3K is located upstream of various regulators of both actin and microtubule dynamics and activates Akt (also called protein kinase B) (Scheid and

Woodgett 2001). Downstream to Akt is glycogen synthase kinase 3 beta (GSK-3b) which plays a central role in axonal growth. Activated Akt phosphorylates GSK-3b at Ser9, inactivating its constitutive kinase activity (Grimes and Jope 2001), a process required for neuronal polarization (Jiang et al. 2005; Yoshimura et al. 2005), axon growth (Zhou et al. 2004), regeneration (Liz et al. 2014) and branching (Garrido et al. 2007). GSK-3b also regulates microtubule extension at the growth cone (Fukata et al. 2002; Cole et al. 2004; Zhou et al. 2004 Yoshimura et al. 2005). Microtubule dynamics during axonal growth is also known to be regulated by the MAPK pathway through activation of GSK3b and subsequent phosphorylation of MAP1b (Goold et al. 1999; Goold and Gordon-Weeks 2005; Trivedi et al. 2005). This may explain the blockade of the action of the A2AR agonist, CGS 21680, by the MEK inhibitor U0126. Several G-protein-coupled receptors, including A2ARs, are known to activate the MAPK pathway (Sexl et al. 1997; Gao et al. 1999; Seidel et al. 1999; Klinger et al. 2002). Finally, PLC activity was also required for axonal growth induced by A2ARs activation, since a PLC inhibitor blocked the effect of the A2AR agonist on axonal growth. PLC has been demonstrated to promote neurite extension and regulate growth cone guidance (Obara et al. 2002; Webber et al. 2005). Axonal elongation is achieved through microtubule assembly in the growth cones through a dynamic instability process, in which microtubules undergo cycles of polymerization and depolymerization (Burbank and Mitchison 2006). By transfecting neurons with EB3, we found that A2AR activation increases the speed of microtubule growth. Besides, by evaluating microtubule instability, our finding that CGS 21680 decreased the ratio of acetylated atubulin/tyrosinated a-tubulin allows us to conclude that A2ARs activation decreases microtubule stability and increases microtubule growth speed, which is central for microtubule extension and thus axonal growth. BDNF increases dendritic branching and the number of primary neurites at DIC 7, in accordance with what has been observed by Dijkhuizen and Ghosh (2005). Interestingly, the effect of BDNF upon dendritic branching, but not upon the number of primary neurites, was attenuated by the A2AR antagonist, ZM 241385, suggesting that the influence of BDNF upon neuronal branching is modulated by A2ARs. BDNF-induced spine and filopodia formation has been shown to be gated by cAMP (Ji et al. 2005) and this occurs in relatively more mature neuronal stages, such as during neuronal branching, but not in more immature stages, when the primary neurites are formed (Ji et al. 2005). The A2AR agonist, by itself, also enhanced neurite branching, without affecting the number of primary neurites. However, when comparing neurons treated with CGS 21680 with neurons

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Fig. 10 The effect of adenosine A2ARs activation on the dendritic branching is dependent on the endogenous BDNF. a Representative fluorescence images of cortical neurons following 4 days of incubation (from DIC 3 to DIC 7) with/without the selective A2AR agonist, CGS 21680 (30 nM) or BDNF (20 ng/mL), in the presence/absence of the BDNF scavenger, TrkB/Fc chimera (2 lg/mL). Neurons were immunostained for MAP2 (red) at DIC 7; nuclei are visualized by DAPI staining (blue). Scale bar [for (a)] = 20 lm. b, c Quantitative analysis of the number of branch points. Data in b illustrate the effect of CGS 21680 in the absence or presence of the BDNF scavenger;

data in c show a positive control for the ability of the BDNF scavenger to prevent BDNF-mediated actions. Data are expressed as mean ± SEM from 200 neurons per condition, taken from four independent cultures, where all conditions were tested in each culture. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons: *p \ 0.05; ns (not statistically significant), as compared with control (first bar on the left, absence of any ligand) or as indicated by the horizontal bars over the columns

treated with BDNF, the former had fewer primary neurites, were less branched and had a longer maximal neurite. In contrast, BDNF-treated neurons had more primary neurites and higher neuronal branching, in detriment of the maximal neurite length. When both drugs were used together, there was an attenuation of the effect of CGS 21680 in the maximal neurite length as well as of the effect of BDNF in the number of primary neurites, and the effects of CGS 21680 and BDNF upon neuronal branching were less than additive. Thus, in more mature neuronal stages, the enhancement of neuronal branching and the increase in

maximal neurite length, caused by A2ARs activation, counteract each other; and, as we also showed, the A2AR enhancement of neuronal branching results from an interaction with BDNF. Indeed, the action of CGS 21680 in neuronal branching, but not in maximal neurite length, was prevented in the presence of a BDNF scavenger. It therefore appears that the relative amounts of extracellular BDNF and adenosine in the dendritic and axonal compartments are key factors to shape neuronal morphology during maturation, so that A2ARs activation associated to low TrkB receptor activity favours axonal elongation,

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Fig. 11 Adenosine A2ARs activation increases microtubule dynamics. a, b A2ARs activation decreases microtubule stability. a Representative images of axonal growth cones of cortical neurons following 5-h stimulation with CGS 21680 30 nM; microtubule stability in axonal growth cones of primary cortical neurons at DIC3 is revealed by immunostaining for the acetylated a-tubulin (green) and tyrosinated a-tubulin (red). Images were taken with the AxioImager Z1 widefield microscope (Carl Zeiss, Germany), with a 1009 objective. Scale bar [for (a)] = 10 lm. b Ratio of the quantification of the immunofluorescence intensities of acetylated over tyrosinated atubulin in the presence of A2AR agonist, CGS 21680 (30 nM), and/or A2AR antagonist, ZM 241385 (50 nM). Data are expressed as mean ± SEM from 300 axonal growth cones per condition, taken from three independent cultures, where all conditions were tested in each culture (c, d) A2ARs activation increases microtubule growth speed. c Kymographs of single processes in either absence or

following exposure of primary cortical cultures to CGS 21680 (30 nM) at DIC 3. Primary cortical cultures were transfected with pEGFP-EB3 (end-binding protein 3) and time-lapse recordings (100 frames, 1 every 2 s) at DIC 3 were taken using an Andor Revolution XD Spinning Disk. Scale bar [for (c)] = 1 lm. d Quantification of microtubule growth speed (lm/min). Data are expressed as mean ± SEM from 200 microtubule plus ends, counted from at least ten neurons per condition per culture, taken from two independent cultures, where all conditions were tested in each culture. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction for multiple comparisons (b) and by an unpaired Student’s t test for comparison with control (bar on the left) (d): *p \ 0.05; ns (not statistically significant), as compared with control (first bar on the left, absence of any drug) or as indicated by the horizontal bars over the columns

while simultaneous activation of both receptors favours dendritic branching. A selective action of extracellular cues either in the dendritic or in the axonal compartments during neuronal maturation may be expected on the light of the well-known subcellular axon-dendrite asymmetry in neuronal cells (Kishi 2008). Caffeine is a naturally occurring adenosine receptor antagonist and therefore the present results may suggest an influence of caffeine during neural development. To appraise the relevance of this issue, particular caution has to be taken with the doses used in vivo, which should mimic as much as possible the daily intake of caffeine by

humans. Importantly also, caffeine actions do not result solely from A2AR antagonism since caffeine is an antagonist of different adenosine receptor subtypes. However, several studies have indeed shown that gestational exposure to caffeine may alter the normal development of the brain with cognitive consequences to the adult brain (Jua´rez-Me´ndez et al. 2006; Soellner et al. 2009; Silva et al. 2013; Mioranzza et al. 2014), by delaying migration and insertion of GABAergic neurons into the hippocampal circuitry (Silva et al. 2013), by decreasing either BDNF or TrkB receptor levels (Mioranzza et al. 2014), by affecting the different adenosine receptors expression during neural

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development (Soellner et al. 2009), or by modifying the dendritic morphology of pyramidal neurons of the rat prefrontal cortex (Jua´rez-Me´ndez et al. 2006). Further studies are required to appraise whether moderate exposure to caffeine at early developmental stages affects axonal elongation and its impact upon neuronal network structure and function.

Conclusions We identified a novel role for A2ARs as modulators of neuronal growth. A2ARs promote axonal elongation through a mechanism that requires PI3K, MAPK/Erk and PLC, but neither cAMP/PKA nor BDNF signalling pathways. Moreover, A2ARs also affect dendritic branching through an endogenous BDNF-dependent process. Axonal elongation induced by activation of A2ARs correlated with their ability to decrease the stability of microtubules and to increase the microtubule growth speed, two molecular correlates of axonal elongation. Most of the studies on the actions of adenosine in the CNS have been focused on the modulation of synaptic transmission and plasticity, either under physiological or pathological conditions (Dunwiddie and Masino 2001; Sebastia˜o and Ribeiro 2009; Dias et al. 2013a, b). Our findings that A2ARs promote axonal elongation expand this knowledge towards a trophic action of adenosine in the cortex, supporting a role of A2ARs in the structural shaping of neuronal connections during neuronal differentiation and/or regeneration. Acknowledgments This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT) Grants: PTDC/SAU-NEU/64126/2006, FCOMP-01-0124-FEDER-017455 (HMSP-ICT/0020/2010), EXPL/ BIM-MEC/0009/2013, Portugal. Filipa F Ribeiro is in receipt of a fellowship (SFRH/BD/74662/2010) from FCT. Nata´lia Assaife-Lopes was in receipt of a fellowship (SFRH/BD/21374/2005) from FCT. Telma E. Santos was supported by the project FCOMP-01-0124FEDER-021392 (PTDC/SAU-ORG/118863/2010) from FCT. We thank Regeneron Pharmaceuticals (Tarrytown, NY) for the kind gift of BDNF. Conflict of interest of interest.

The authors declare that they have no conflict

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Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.

Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons...
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