Wikisource:WikiProject Open Access/Programmatic import from PubMed Central/Prophylactic versus Therapeutic Fingolimod Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis

by Tommaso Bonfiglio; Guendalina Olivero; Elisa Merega; Silvia Di Prisco; Cristina Padolecchia; Massimo Grilli; Marco Milanese; Lorenzo Di Cesare Mannelli; Carla Ghelardini; Giambattista Bonanno; Mario Marchi; Anna Pittaluga

Abstract

Fingolimod, the first oral, disease-modifying therapy for MS, has been recently proposed to modulate glutamate transmission in the central nervous system (CNS) of mice suffering from Experimental Autoimmune Encephalomyelitis (EAE) and in MS patients. Our study aims at investigating whether oral fingolimod recovers presynaptic defects that occur at different stages of disease in the CNS of EAE mice. In vivo prophylactic (0.3 mg/kg for 14 days, from the 7^th day post immunization, d.p.i, the drug dissolved in the drinking water) fingolimod significantly reduced the clinical symptoms and the anxiety-related behaviour in EAE mice. Spinal cord inflammation, demyelination and glial cell activation are markers of EAE progression. These signs were ameliorated following oral fingolimod administration. Glutamate exocytosis was shown to be impaired in cortical and spinal cord terminals isolated from EAE mice at 21 ± 1 d.p.i., while GABA alteration emerged only at the spinal cord level. Prophylactic fingolimod recovered these presynaptic defects, restoring altered glutamate and GABA release efficiency. The beneficial effect occurred in a dose-dependent, region-specific manner, since lower (0.1–0.03 mg/kg) doses restored, although to a different extent, synaptic defects in cortical but not spinal cord terminals. A delayed reduction of glutamate, but not of GABA, exocytosis was observed in hippocampal terminals of EAE mice at 35 d.p.i. Therapeutic (0.3 mg/kg, from 21 d.p.i. for 14 days) fingolimod restored glutamate exocytosis in the cortex and in the hippocampus of EAE mice at 35 ± 1 d.p.i. but not in the spinal cord, where also GABAergic defects remained unmodified. These results improve our knowledge of the molecular events accounting for the beneficial effects elicited by fingolimod in demyelinating disorders.

Introduction

Multiple sclerosis (MS) is mediated by an immune attack directed at myelin, which leads to a progressively degenerating disorder of the central nervous system (CNS). Although immunological mechanisms are responsible for the majority of the cascade of events leading to MS, pathogenetic events involving neurons and astrocytes have been recently implicated in the pathogenesis of this disease [^[1]–^[2]].

More precisely, recent work has targeted glutamate and GABA transmission at chemical synapsis in the CNS of EAE mice and MS patients. Central glutamatergic and GABAergic neuronal defects were proposed to determine synaptic pathology in MS patients and in animals affected by Experimental Autoimmune Encephalomyelitis (EAE). These impairments might account for a reduced ability of CNS to cope with central neuro-injuries. In particular, the endogenous bioavailability of glutamate in the cerebrospinal fluid of MS patients as well as EAE mice was altered with respect to healthy individuals [^[3]]. Glutamate release efficiency was augmented in the striatum and the spinal cord [^[4],^[5]–^[6]], but was significantly reduced in cortical nerve endings of EAE animals [^[7],^[8],^[9],^[10]]. A decrease in the inhibitory amino acid GABA and its synthesising enzyme GABA decarboxylase was also measured in the spinal cord of EAE guinea pigs and cerebrospinal fluid of MS patients [^[11],^[12]] and a loss of inhibitory interneurons was detected in the brain of EAE mice [^[13]].

Fingolimod (FTY720, Gilenya^®) was the first oral, disease-modifying therapy for MS [^[14]]. It reduces relapses, disability progression, and brain atrophy in patients suffering from the relapsing-remitting form of MS [^[15]]. Fingolimod is a pro-drug that is rapidly phosphorylated to the active compound fingolimod-phosphate. By binding at the sphingosine-1-phosphate receptors (S1PR), the drug prevents the egress of lymphocytes and exerts central effects including neuroprotection and remyelination [^[16],^[17]]. Besides its immunomodulatory activity, fingolimod is beneficial to central glutamate transmission in the CNS of EAE mice [^[18]]. Inasmuch, it restores glutamate-mediated intercortical excitability in MS patients suffering from the relapsing-remitting form of disease [^[19]]. In an attempt to provide further evidence supporting the beneficial effect of fingolimod in CNS, we investigated whether the continuous in vivo administration of this drug could ameliorate glutamate and GABA presynaptic abnormalities in selected CNS regions of EAE mice. Fingolimod was dissolved in the mice drinking water for 14 days, starting either from an early asymptomatic stage of the disease (prophylactic administration), or after the acute stage of disease (therapeutic administration). Depending on the posology adopted, fingolimod ameliorated in a region-dependent manner glutamate and GABA release efficiency in the CNS of EAE mice. Both prophylactic and therapeutic administration restored glutamate release efficiency in the cortex and in the hippocampus, while prophylactic, but not therapeutic, fingolimod was beneficial at the spinal cord level.

Materials and Methods

Animals

Mice (female, strain C57BL/6J) were obtained from Charles River (Calco, Italy) and were housed in the animal facility of the Department of Pharmacy, Section of Pharmacology and Toxicology, School of Medical and Pharmaceutical Sciences, University of Genoa (authorization n. 484 of 2004, June, 8^th). The experimental procedures were in strict accordance with the European legislation (Directive 2010/63/EU for animal experiments), with the ARRIVE guidelines, and they were approved by the Committee on the Ethics of Animal Experiments of the University of Genoa and by the Italian Ministry of Health (DDL 26/2014 and previous legislation; permit number 50/2011-B and number 612/2015-PR). All efforts were made to minimize animal suffering and the number of animals necessary to produce reliable results.

EAE induction and clinical score

For EAE induction, mice (female, strain C57BL/6J, 18–20 g, 6–8 weeks) were immunized accordingly to a standard protocol [^[20]], with minor modifications. Briefly, animals were subcutaneously injected with incomplete Freund’s adjuvant containing 4 mg/ml Mycobacterium tuberculosis (strain H37Ra) and 200 μg of the MOG_35–55 peptide. Immunization with MOG_35–55 was followed by i.p. administration of 250 ng of Pertussis toxin on day 0 and after 48 h. Clinical scores (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) were recorded daily. MOG_35-55(+) EAE mice were killed by decapitation at 21 ± 1 or 35 ± 1 days post immunization (d.p.i.) as indicated. Control, non-immunized mice received the same treatment in the absence of the myelin antigen [MOG_35-55(-) mice].

Drug treatments

Mice were randomly assigned to the following groups: control, EAE, fingolimod-treated control, and fingolimod-treated EAE mice. Fingolimod, supplied by Novartis Pharma AG, was given orally, dissolved in the drinking water (concentration as indicated in the text). Recent studies demonstrated that the administration of fingolimod to EAE mice in the drinking water results in beneficial responses that are comparable to those observed following intraperitoneal injection ([^[21]] and references therein) or gavage [^[22]]. This route of administration was therefore adopted in order to minimize the daily handling and stress associated with the other methods of drug delivery. After a three-day trial to determine the amount of water consumed by each group of mice, mice were treated with fingolimod. The amount of drug dissolved in the water was adjusted daily to assure the correct dosage. When studying the effect of the prophylactic administration of fingolimod, the drug was added at the drinking water starting from 7 days post immunization (d.p.i.); that represents an asymptomatic stage of disease, for 14 days, till 21 d.p.i. We refer to this treatment as the “prophylactic fingolimod” treatment. When studying the effect of the administration of fingolimod starting from the acute stage of disease, the drug was added at the drinking water starting from 21 d.p.i.; that represents the stage of disease characterized by the most severe clinical symptoms. The drug was administered for 14 days, till 35 d.p.i. We refer to this treatment as the “therapeutic fingolimod” treatment. Either prophylactic or therapeutic fingolimod administration did not modify per se the daily water intake. The incidence of disease in the EAE mice enrolled in the study concerning therapeutic fingolimod was 100%, as only sick animals were included in the fingolimod untreated and treated EAE mice groups. As to the prophylactic treatment, drug administration started before the onset of the clinical symptoms, such that the disease incidence in these groups could not be predicted. However, in these studies, the untreated control EAE mice showed mild (i.e., mild to severe tail weakness, clinical score = 1.5) to serious (ataxia and/or paresis of hindlimbs, clinical score of about 2–2.5) clinical symptoms. Sick animals were not included in the study. Since the functional scores are not normally distributed data, the non parametric Wilcoxon analysis was applied to test the effect of the fingolimod treatment on functional recovery in EAE mice.

Behavioural studies

Light dark box test

The light-dark box consists of a lighted and dark compartments (each comprising 35 cm x 30 cm x 21 cm). Animals were placed in the centre of the light zone, and they were allowed to explore the box for 10 min. Anxiety was analyzed in the light-dark box, by monitoring the time spent in the lighted compartment as well as by quantifying the number of crossings from the lighted to the dark side of the field [^[8],^[23]].

Open field test

The open field consists of a square arena (34 x 34 cm with 24 cm walls) with walls made of translucent plastic. White lines were drawn on the floor of the box and divided into 9 squares. Mice were placed in the middle of the open field and were allowed to explore the field freely for 6 min. Thigmotaxis (the tendency to stay on the periphery of the open field) was quantified as time spent in the periphery (seconds, sec.) to evaluate anxiety-related behaviour [^[24]].

Histological analysis

Luxol Fast Blue

Twenty-one days after EAE induction, mice were sacrificed and the spinal cord (the lumbar part) was extracted. Formalin-fixed paraffin sections (7 μm) were prepared. Following standard dewaxing and rehydration, tissue sections were immersed overnight in 0.1% Luxol Fast Blue solution) at 56–60°C. Sections were rinsed in deionized water. Differentiation was initiated with immersion in 0.05% aqueous lithium carbonate for 15 sec followed by differentiation in multiple immersions in fresh 70% ethanol until grey and white matter could be distinguished, and nuclei decolorized. After washing in deionized water, sections were immersed in 0.8% Periodic Acid for 10 min and then rinsed in distilled water. Sections were incubated with Schiff ‘s reagent for 20 min and rinsed in distilled water for 15 min, and then dehydrated in 50 and 100% ethanol and coverslipped. Sections were examined using an Olympus BX40 microscope (Olympus, Milan, Italy) and photographed using a digital camera Olympus DP50 (Olympus, Milan, Italy).

Hematoxylin/Eosin

Hematoxylin has a deep blue-purple colour and stains nucleic acids. Eosin is pink and stains proteins non-specifically. In particular, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Twenty-one days after EAE induction, mice were sacrificed and spinal cord was extracted. Formalin-fixed paraffin sections (7 μm) were collected. Following standard dewaxing and rehydration, tissue sections were immersed in eosin solution (Bio-optica, Milan, Italy) for 3 min. After 5–6 min running water, sections were immersed in hematoxylin solution (Bio-optica, Milan, Italy) for 3 min. After 5–6 min running water, sections were dehydrated and coverslipped. Sections were examined using an Olympus BX40 microscope (Olympus, Milan, Italy) and photographed using a digital camera Olympus DP50 (Olympus, Milan, Italy).

Immunofluorescence staining

Twenty-one days after EAE induction, mice were sacrificed, the L4/L5 segments of the spinal cord were exposed from the lumbovertebral column via laminectomy and identified by tracing the dorsal roots from their respective dorsal root ganglia. Formalin-fixed cryostat sections (10 μm) were washed 3 × phosphate-buffered saline (PBS), 0.3% Triton X-100 for 5 min and then were incubated, at room temperature, for 1 h in blocking solution (PBS, 0.3% Triton X-100, 5% albumin bovine serum; PBST). Slices were incubated overnight at 4°C in PBST containing rabbit primary antisera. The primary antibody used were directed against cluster of differentiation 3 (CD3; rabbit, 1:100 dilution) for T-cell co-receptor staining, chemokine (C-C motif) ligand 5 (CCL5; rabbit, 1:100 dilution) for RANTES (Regulated upon Activation Normal T cell Expressed and Secreted) staining, ionized calcium binding adapter molecule 1 (Iba1; rabbit, 1:1000 dilution) for microglial staining, glial fibrillary acidic protein (GFAP; rabbit, 1:1000 dilution) for astrocytes staining. The following day, slides were washed 3 × PBS, 0.3% Triton X-100 for 5 minutes and then sections were incubated in goat anti-rabbit immune globulin G (IgG) secondary antibody labelled with Alexa Fluor 568 (1:500 dilution) and 4',6-diamidino-2-phenylindole (DAPI; 1:2000 dilution), a nuclei marker, in PBST at room temperature for 2 h, in the dark. After 3 × PBS 0.3% Triton X-100 wash for 10 min, slices were mounted using ProLong Gold as a mounting medium. Staining procedures were in accord with a previously published method [^[25]]. Negative control sections (no exposure to the primary antisera) were processed concurrently, in order to exclude the presence of non-specific immunofluorescent staining or cross-immunostaining.

Images were acquired by using an Olympus BX63 microscope equipped with an Olympus XM10 camera and coupled to CellSense Dimension Software (Olympus, Milan, Italy). Quantitative analysis of CD3-, Iba1- and GFAP-positive cells was performed by collecting three independent fields through a 20X 0.40NA objective of each mouse spinal cord. Positive cells were counted using the “cell counter” plugin of ImageJ (NIH, Bethesda, MD, USA). Quantitative analysis of CCL5 positive area was performed by collecting three independent fields through a 20X 0.40NA objective of each mouse spinal cord. CCL-5 positive areas were measured using the “threshold” function of ImageJ; data were expressed as the fluorescent area (μm²)/mm² of section.

Release studies

Purified synaptosomes were prepared by homogenizing the cortex, the hippocampi and the spinal cord [^[26]] of control and EAE mice in 10 volumes of 0.32 M sucrose, buffered to pH 7.4 with Tris-(hydroxymethyl)-amino methane [Tris, final concentration (f.c.) 0.01 M] [^[27]]. The homogenate was centrifuged at 1,000 x g for 5 min, and the supernatant was stratified on a discontinuous Percoll gradient (2%, 6%, 10% and 20% v/v in Tris-buffered sucrose) and centrifuged at 33,500 x g for 5 min. The layer between 10% and 20% Percoll (synaptosomal fraction) was collected and washed by centrifugation [^[28]]. The synaptosomal pellets were resuspended in a physiological solution with the following composition (mM): NaCl, 140; KCl, 3; MgSO₄, 1.2; CaCl₂, 1.2; NaH₂PO₄, 1.2; NaHCO₃, 5; HEPES, 10; glucose, 10; pH 7.2–7.4.

Synaptosomes were incubated for 15 min a 37°C in a rotary water bath to equilibrate the system and identical portions of the synaptosomal suspensions were layered on microporous filters at the bottom of parallel chambers in a Superfusion System ([^[29]]; Ugo Basile, Comerio, Varese, Italy; [^[30]]) and maintained at 37°C. Synaptosomes were transiently (90 sec) exposed, at t = 39 min, to high KCl containing medium (12 mM or 15 mM, as appropriate, NaCl substituting for an equimolar concentration of KCl). Fractions were collected as follow: two 3-min fractions (basal release), one before (t = 36–39 min, b1) and one after (t = 45–48 min, b2) a 6-min fraction (t = 39–45 min; evoked release). Superfusion was always performed with media containing 50 μM amino-oxyacetic acid to avoid metabolism of GABA. Fractions collected were analysed for endogenous glutamate and GABA contents (see below). Synaptosomal protein contents were determined with BCA kit. The amount of endogenous amino acid from synaptosomes in each superfusate fractions was expressed as picomoles per milligram of protein (pmol/mg protein). The K⁺-induced overflow of endogenous glutamate and GABA from synaptosomes was estimated by subtracting the neurotransmitter content into the first and the third fractions collected (basal release, b1 and b3) from that in the 6-min fraction collected during and after the depolarization pulse (evoked release, b2).

Endogenous amino acid determination

Collected fractions were analyzed for the endogenous neurotransmitter content. Endogenous glutamate and GABA were measured by high-performance liquid chromatography analysis after precolumn derivatization with o-phthalaldehyde and separation on a C18 reverse-phase chromatographic column (10×4.6 mm, 3 μm; at 30°C; Chrompack, Middleburg, The Netherlands) coupled with fluorimetric detection (excitation wavelength, 350 nm; emission wavelength, 450 nm). Buffers and the gradient program were described elsewhere [^[31]]. Homoserine was used as internal standard.

Calculations and statistical analysis

Analysis of variance was performed by anova followed by Dunnett’s test or Newman Keuls multiple-comparisons test as appropriate; direct comparisons were performed by Student’s t-test. The statistical difference between the untreated and treated EAE mice cumulative score was analyzed by applying the non-parametric Wilcoxon analysis. Data were considered significant for p < 0.05 at least.

Reagents

Pertussis toxin and the Freund’s incomplete adjuvant, Solvent Blue 38, Periodic Acid were acquired from Sigma-Aldrich (Saint Louis, MO, USA). Pierce^™ BCA Protein Assay Kit was from Thermo Fisher Scientific 168 Third Avenue Waltham, MA USA 02451. Myelin oligodendrocyte glycoprotein (MOG) was purchased from Espikem (Florence, Italy). Mycobacterium tuberculosis (H37Ra) was obtained from DIFCO BACTO Microbiology (Lawrence, KA, USA). Fingolimod was supplied by Novartis Pharma AG, Basel, Switzerland. The anti-rabbit Alexa Fluor 568 was acquired from Molecular Probes, Invitrogen (Carlsbad, CA, USA). Schiff ‘s reagent is from Bio-optica (Milan, Italy). Primary antibody against CD3 was from SP Clone (Pleasanton, CA, USA). Primary antibody against CCL5 was from Abcam (Cambridge, UK) ionized calcium binding adapter molecule 1 (Iba1; rabbit, was from Wako (Richmond, VA, USA). Primary antibody against Anti Iba1; rabbit, was from Wako (Richmond, VA, USA). Primary antibody against glial fibrillary acidic protein (GFAP; rabbit) was from DAKO, (Carpinteria, CA, USA). Primary antibody against cluster of differentiation 3 (CD3; rabbit) was from SP Clone (Pleasanton, CA, USA). DAPI (4',6-diamidin-2-fenilindolo) and ProLong Gold were from Life Technologies-Thermo scientific, (Rockford, IL, USA).

Results

Prophylactic fingolimod reduces the severity of clinical signs and anxiety-related behaviour in EAE mice

Control [non-immunized, MOG_35-55 (-)] and EAE [immunized, MOG_35-55 (+)] female C57JBl mice were randomly assigned to the following groups: fingolimod-untreated control, fingolimod (0.3 mg/kg)-treated control, fingolimod-untreated EAE, or fingolimod (0.3 mg/kg)-treated EAE mice. Clinical signs in fingolimod-untreated EAE mice became evident at 12 ± 1 d.p.i. and developed a rapid worsening that peaked around 20 ± 1 d.p.i. (clinical score = 2.12 ± 0.29, n = 22 animals), when a chronic phase was reached (Fig 1)