J Neuroimmune Pharmacol DOI 10.1007/s11481-013-9436-x
ORIGINAL ARTICLE
Glatiramer Acetate Protects Against Inflammatory Synaptopathy in Experimental Autoimmune Encephalomyelitis Antonietta Gentile & Silvia Rossi & Valeria Studer & Caterina Motta & Valentina De Chiara & Alessandra Musella & Helena Sepman & Diego Fresegna & Gabriele Musumeci & Giorgio Grasselli & Nabila Haji & Sagit Weiss & Liat Hayardeny & Georgia Mandolesi & Diego Centonze
Received: 29 August 2012 / Accepted: 17 January 2013 # Springer Science+Business Media New York 2013
Abstract Glutamate-mediated excitotoxicity is supposed to induce neurodegeneration in multiple sclerosis (MS). Glatiramer acetate (GA) is an immunomodulatory agent used in MS treatment with potential neuroprotective action. Aim of the present study was to investigate whether GA has effects on glutamate transmission alterations occurring in experimental autoimmune encephalomyelitis (EAE), to disclose a possible mechanism of GA-induced neuroprotection in this mouse model of MS. Single neuron electrophysiological recordings and immunofluorescence analysis of microglia activation A. Gentile : S. Rossi : V. Studer : C. Motta : V. De Chiara : A. Musella : H. Sepman : D. Fresegna : G. Musumeci : G. Grasselli : N. Haji : G. Mandolesi : D. Centonze Fondazione Santa Lucia/Centro Europeo per la Ricerca sul Cervello (CERC), Via del Fosso di Fiorano 64, 00143 Rome, Italy A. Gentile : S. Rossi : V. Studer : C. Motta : V. De Chiara : A. Musella : D. Fresegna : D. Centonze (*) Clinica Neurologica, Dipartimento di Neuroscienze, Università Tor Vergata, Via Montpellier 1, 00133 Rome, Italy e-mail:
[email protected] N. Haji National Institute of Neuroscience, University of Turin, Turin, Italy G. Grasselli Department of Neurobiology, University of Chicago, Chicago, IL 60637, USA S. Weiss : L. Hayardeny Teva Pharmaceuticals, Netanya, Israel
were performed in the striatum of EAE mice, treated or not with GA, at different stages of the disease. GA treatment was able to reverse the tumor necrosis factor-α (TNF-α)-induced alterations of striatal glutamate-mediated excitatory postsynaptic currents (EPSCs) of EAE mice. Incubation of striatal slices of control animals with lymphocytes taken from EAE mice treated with GA failed to replicate such an antiglutamatergic effect, while activated microglial cells stimulated with GA in vitro mimicked the effect of GA treatment of EAE mice. Consistently, EAE mice treated with GA had less microglial activation and less TNF-α expression than untreated EAE animals. Furthermore, direct application of GA to EAE slices replicated the in vivo protective activity of GA. Our results show that GA is neuroprotective against glutamate toxicity independently of its peripheral immunodulatory action, and through direct modulation of microglial activation and TNF-α release in the grey matter of EAE and possibly of MS brains. Keywords EAE . EPSC . GA . Microglia . Striatum . TNF-α
Introduction Although being historically considered a genuine autoimmune demyelinating disease, multiple sclerosis (MS) is nowadays viewed as a neurodegenerative disorder, characterized by axonal and neuronal damage probably resulting from sustained action of pro-inflammatory cytokines released by brain infiltrating lymphocytes and activated microglia (Trapp and
Nave 2008). Grey matter pathology in MS involves both cortical and subcortical brain areas, and is still poorly understood (Geurts and Barkhof 2008). Glutamate-mediated excitotoxicity recently emerged as one possible mechanism linking cytokine detrimental effects and neuronal synaptopathy in many neurodegenerative diseases, including MS. Moreover, it has been demonstrated that activated microglia can mediate alterations of glutamate transmission in striatal neurons of mice with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, likely through the release of tumor necrosis factor alpha (TNF-α) (Centonze et al. 2009). Among the currently available therapeutic approaches for MS treatment, Glatiramer acetate (GA, Copaxone®) represents a common first-line therapy agent, administered through daily subcutaneous injections, and is effective in reducing the progression of disability and both relapse rate and magnetic resonance imaging-defined activity (MRI) in MS patients (Comi et al. 2011). Furthermore, GA has been demonstrated able to act in the prevention and suppression of EAE induced by various encephalitogens (Arnon and Aharoni 2004). The mechanism by which GA exerts its beneficial effect in patients and animals was extensively investigated over the years in several laboratories, and particular attention was paid to its role in the first step of the disease, the immune response. In fact, GA is now recognized as an immunomodulatory agent, primarily acting by inducing a shift from proinflammatory Th1 lymphocytes to an anti-inflammatory Th2 response (Miller et al. 1998), and enhancing the suppressive properties of T regulatory cells on both EAE and MS (Hong et al. 2005; Saresella et al. 2008). Beside the robust bulk of data on the immunomodulatory action of GA, very recent studies argued a role of GA in neuroprotection, independently of its activity on lymphocytes, as indicated by the induction of the neurotrophin BDNF expression on T cells of both murine and human origin as well as in neurons (Aharoni et al. 2005a), and by increasing proliferation of neuroprogenitor cells from neuroproliferative brain areas in EAE (Aharoni et al. 2005b). Furthermore, GA reduces microglial activation in several brain areas of both EAE mice and MS patients (Aharoni et al. 2005b; Ratchford et al. 2012), as well as TNF-α release by in vitro activated microglia (Pul et al. 2011) and also acts in a T-cell independent fashion (Liu et al. 2007). Moving from these evidences, we investigated whether GA may exert its neuroprotective role in central neurons by acting on glutamatergic transmission, which is considerably potentiated in the nucleus striatum of EAE mice at various stages of the disease, and which has been demonstrated to be driven by TNF-α direct detrimental effect on synapses (Centonze et al. 2009; Rossi et al. 2011a). Moreover, we investigated the role of GA in microglia activation, supposed to be the main player in such dysregulated excitatory
synaptic transmission. By means of electrophysiological recordings on striatal medium spiny neurons (MSNs) and immunofluorescence studies here we provide novel insights into the mechanism of GA-mediated neuroprotection.
Material and methods EAE induction and clinical evaluation EAE was induced in 6–8 weeks female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) as previously described (Centonze et al. 2009; Rossi et al. 2011a, 2011b). Briefly, 200 μg myelin-oligodendrocyte glycoprotein (MOG 35–55) (>85 % purity, Espikem, Florence, Italy) was administrated by three sub-cutaneous injections of 100 μl of incomplete Freund’s adjuvant containing 8 mg/ml Mycobacterium tuberculosis (strain H37Ra, Difco, Lawrence, KS, USA) (CFA, complete Freund’s adjuvant). A 500 ng dose of pertussis toxin (Sigma, St. Louis, MO, USA) was administrated by intravenous injection on the day of the immunization and 2 days later. Control mice received the same treatment, but the injected emulsion composition was CFA lacking the MOG peptide. Clinical score (0=healthy; 1=limp tail; 2=ataxia and/or paresis of hindlimbs; 3=paralysis of hindlimbs and/or paresis of forelimbs; 4=tetraparalysis; 5=moribund or death) was recorded daily. All efforts were made to minimize animal suffering and to reduce the number of mice used, in accordance with the European Communities Council Directive of 24 November, 1986 (86/609/EEC). Glatiramer acetate administration Mice received a preventing treatment with GA, known to be effective in preventing the development of the disease (Aharoni et al. 2011; Weber et al. 2007). GA (stock n° 24290/110 Teva, Israel) was dissolved in sterile saline (0.9 % NaCl) and administered (150 μg/100 μl) subcutaneously for 7 consecutive days, starting 1 week before the immunization protocol, according to already published administration protocol (Weber et al. 2007). Control CFA and EAE mice received 100 μl of sterile saline (vehicle). Electrophysiology EAE mice were killed at the peak of the acute phase (21– 25 days post immunization, dpi) and in the chronic stage (45–50 dpi) by cervical dislocation under halothane anesthesia. Corticostriatal coronal slices (200 μm) were prepared from fresh tissue blocks of the brain by means of a vibratome (Centonze et al. 2009; Rossi et al. 2011b). A single slice was then transferred to a recording chamber and
submerged in a continuously flowing artificial CSF (ACSF) (34 °C, 2–3 ml/min) gassed with 95 % O2–5 % CO2. The composition of the control ACSF was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, and 25 NaHCO3. Recording pipettes were advanced towards individual striatal cells in the slice under positive pressure and visual control (WinVision 2000, Delta Sistemi, Italy) and, on contact, tight GΩ seals were made by applying negative pressure. The membrane patch was then ruptured by suction and membrane current and potential monitored using an Axopatch 1D patch clamp amplifier (Molecular Devices, Foster City, CA, USA). Whole-cell access resistances measured in voltage clamp were in the range of 5–20 MΩ. Whole-cell patch clamp recordings were made with borosilicate glass pipettes (1.8 mm o.d.; 2–3 MΩ), in voltage-clamp mode, at the holding potential (HP) of −80 mV. To study glutamate-mediated spontaneous excitatory postsynaptic currents (sEPSCs), the recording pipettes were filled with internal solution of the following composition (mM): K-gluconate (125), NaCl (10), CaCl2, (1.0), MgCl2 (2.0), 1,2-bis (2-aminophenoxy) ethane-N,N,N, N-tetraacetic acid (BAPTA; 0.5), N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) (HEPES; 19), guanosine triphosphate (GTP; 0.3), and Mg-adenosine triphosphate (MgATP; 1.0), adjusted to pH7.3 with KOH. Bicuculline (10 μM) was added to the perfusing solution to block GABAA-mediated transmission. Synaptic events were stored by using PCLAMP 9 (Axon Instruments) and analyzed offline on a personal computer with Mini Analysis 5.1 (Synaptosoft, Leonia, NJ, USA) software. The detection threshold of sEPSCs was set at twice the baseline noise. The fact that no false events would be identified was confirmed by visual inspection for each experiment. Offline analysis was performed on spontaneous synaptic events recorded during fixed time epochs (1–2 min), sampled every 2–3 min (5–12 samplings) (Centonze et al. 2009; Rossi et al. 2011b). Only cells that exhibited stable frequencies in control (less than 20 % changes during the control samplings) were taken into account. For sEPSCs kinetic analysis, events with peak amplitude between 10 and 50 pA were grouped, aligned by half-rise time, and normalized by peak amplitude. In each cell, all events between 10 and 50 pA were averaged to obtain rise times, decay times, and half widths (Centonze et al. 2009). One to nine cells per animal were recorded. For each type of experiment and time point, at least four distinct animals were employed from each experimental group. Throughout the text “n” in electrophysiological experiments refers to the number of cells. Drugs were applied by dissolving them to the desired final concentration in the bathing ACSF. Drugs were (in μM): CNQX (10), MK-801 (30) (Tocris Cookson, Bristol, UK), Bicuculline (10) (SigmaRBI, St. Louis, USA). GA was applied to the bath at a final concentration of 10 μg/ml for 2 h.
Immunohistochemistry and microscopy Mice (21 dpi) were deeply anesthetized and intracardially perfused with ice-cold 4 % paraformaldehyde. Brains were post-fixed for 2 h and equilibrated with 30 % sucrose at least one overnight. Thirty micrometer-thick coronal sections were permeabilized in PBS with Triton-X 0.25 % (Tx-PBS). All following incubations were performed in Tx-PBS. Sections were pre-incubated with 10 % normal donkey serum solution for 1 h at RT and incubated with the primary antibody overnight at +4 °C, then, after washing, they were incubated with secondary antibodies for 2 h at RT, rinsed and DAPI counterstained. Primary antibodies were used as following: rabbit antiIba1 (1:500, Wako); mouse anti-TNF-α (1:500, Abcam). Secondary antibodies used were: Cy3-conjugated donkey anti-rabbit (1:200, Jackson), Alexa-Fluor 488-conjugated donkey anti-mouse (1:200, Molecular Probes). Sections were mounted with Vecta-shield (Vector Labs, USA) on poly-Llysine-coated slides, air-dried and coverslipped. Images were acquired using a LSM5 Zeiss confocal laser-scanner microscope (Zeiss, Göttingen, Germany). For the analysis of microglia density and morphology we acquired immunostaining stacks of the striatum by using a 40× oil objective (zoom 0.5×, pixel resolution 1,024×1,024, 5.89 μm Z-stack, pinhole of 1 airy units); the stacks were zprojected and exported in TIFF file format by NIH ImageJ software (http://rsb.info.nih.gov/ij/). Iba1-positive cells were counted manually on the z-projections and divided by the surface of striatal sections (microglial density). Microglial surface was measured by Iba1 staining, maintaining the same threshold between the groups. Measurements were repeated for 7–9 images from 5 sections (n=4 mice per group). For the quantification of TNF-α density in microglial cells, a 40× oil objective (pinhole of 1 airy unit) was used (zoom: 1×, z-step: 0.49 μm) and the entire body of the cell was included in the stack according to Iba1 staining. From each image 2–3 cells were considered for co-localization area analysis. The overlapping signal was detected by a colocalization software (ImageJ). All measurements were performed on at least 5 images acquired from at least 5 serial sections per animal (n=4 mice per group): in the text “n” refers to the number of analyzed cells. CD3+ cells isolation from spleen of immunized mice 7–8 dpi (presymptomatic) and 21–25 dpi (symptomatic) mice from vehicle-receiving EAE (n=3), GA-treated EAE (n=3), and relative control vehicle-receiving CFA (n=3) groups were killed through cervical dislocation and the spleens were removed and stored in sterile PBS. After mechanical dissociation of the tissue, the cell suspension was passed through a 40 μm cell strainer (BD, Falcon) to remove cell debris and centrifuged. The cell suspension
obtained was subjected to magnetic cell sorting separation (CD3 microbeads kit, Miltenyi Biotec) in order to obtain pure lymphocytes population. Then, 5×103 pure cells were incubated with striatal slices for 30–60 min, prior to electrophysiological recordings. BV2 microglial cell lines activation and GA treatment The BV2 immortalized murine microglial cell line was stimulated with Th1-specific pro-inflammatory cytokines, TNF-α (200U/ml), interferon-γ (IFN-γ, 500 U/ml) and Interleukin 1-β (Il 1-β, 100 U/ml) for 24 h, as already published (Centonze et al. 2009), in the presence or absence of GA (62.5 μg/ml). This concentration has been already tested in primary rat microglia and found to be not cytotoxic (Pul et al. 2011). After the incubation time, the cells were harvested and trypan blue exclusion assay was performed to confirm lack of cytotoxicity of the treatment. Then, 0,5×106 to 1×106 cells were put on single striatal slices and incubated for 30–60 min for electrophysiological recordings. TNF-α ELISA measurement 24 h conditioned medium was harvested and centrifuged at 1,200 rpm in microfuge in order to remove any dead or detached cells. The medium was then aliquoted and stored at −80 °C until use. TNF-α ELISA was performed according to manifacturer’s guide (E-Bioscience). Conditioned medium from control not activated, Th1 activated and GA+Th1 activated cells (n=4 for each condition) were diluted 1:10 in diluent buffer and run in the same assay. Quantification of cytokine content was made according to standard curve in the linear range (from 8 to 250 pg/ml) and adjusted for the dilution factor. Statistical analysis For each type of experiment and time point, at least five mice each group were employed, unless otherwise specified. Throughout the text “n” refers to the number of animals, except for electrophysiological experiments. Data were presented as the mean ± S.E.M. The significance level was established at p
