Abstract

Although Multiple Sclerosis (MS) is regarded as a white matter disease, the incidence of demyelination and axonal injury is prominent also in gray matter. In MS, extracellular adenosine triphosphate (ATP) is an important mediator of central nervous system pathology via its ability to cause oligodendrocyte excitotoxicity. We have analyzed the distribution pattern of all ionotropic P2X and metabotropic P2Y receptors for ATP in postmortem samples of the cerebral cortex from healthy human subjects as well as MS patients. We focus particularly on the P2Y12 subtype that is highly enriched in oligodendrocytes. We correlate the expression of this receptor to the extent of gray matter demyelination and pathological alterations occurring during secondary progressive MS. Using triple immunofluorescence and confocal analysis, we show that in sections of cerebral cortex from postmortem MS brains, the P2Y12 protein is present in myelin and interlaminar astrocytes but absent from protoplasmic astrocytes residing in the deeper cortical layers, from microglia/macrophages, and from intact demyelinated axons. We report that a decreased P2Y12 receptor immunoreactivity in proximity to the lesions is directly correlated with the extent of demyelination found in all types of gray matter cortical plaques (I–III) and subcortical white matter. Our study provides further insights into the pathogenetic features of MS and suggests that the loss of purinergic P2Y12 receptors might be detrimental to tissue integrity.

Introduction

Multiple sclerosis (MS) is thought to be initiated by an acute autoimmune inflammatory reaction to myelin components and then to progress into a chronic phase in which oligodendrocytes, myelin, and axons degenerate (reviewed in Lassmann 1998; Compston and Coles 2002; Hauser and Oksenberg 2006; Stadelmann and Brück 2008). MS lesions are abundant in the cerebral cortex (Dawson 1916; Brownell and Hughes 1962; Lumsden 1970; Magliozzi et al. 2007; Lassmann and Lucchinetti 2008; reviewed in Lassmann 2007), where they constitute a significant proportion of the overall pathology of the brain, with a particularly high prevalence of plaques being observed in progressive stages of the disease (Kidd et al. 1999; Bø et al. 2003; Kutzelnigg et al. 2005; Stadelmann and Brück 2008). Although MS is still regarded as a white matter disease, the incidence of demyelination and oligodendrocyte or neuron/axon injury is prominent and widespread in gray matter (Peterson et al. 2001; Vercellino et al. 2005; Wegner et al. 2006; Magliozzi et al. 2007; Pirko et al. 2007; Gilmore et al. 2009; reviewed in Bö et al. 2006; Geurts 2008; Geurts and Barkhof 2008). In addition to changes to oligodendrocytes and neurons, current knowledge also emphasizes an important role for astrocytes and microglia (reviewed in He and Sun 2007). Astrocytes can promote inflammation, damage to oligodendrocytes and axons, formation of the glial scar (Holley et al. 2003) but, at the same time, can support migration, proliferation, and differentiation of oligodendrocyte progenitors (Williams et al. 2007). Likewise, microglia may not only play an essential primary role in MS pathogenesis but also restores the damaged tissue (reviewed in Block and Hong 2005; Gay 2007; Muzio et al. 2007; Sanders and De Keyser 2007). As a result, all glial cells are likely to play important roles in both the destructive and restorative phases of MS. Hence, a major challenge in glial cell research and in MS is to discern the conditions and factors that might contribute to the outcome of this unsteady equilibrium, and the major aim of our work is to establish if there is a function for purinergic signaling in MS, particularly for the P2Y12 receptor subtype.

Indeed, extracellular purine/pyrimidine nucleotides are among the exogenous signals playing important roles, either destructive or protective, in neuron-to-glia and glia-to-glia communication, in the normal and injured brain (reviewed in Volonté et al. 2003; Fields and Burnstock 2006; Franke et al. 2006; Inoue et al. 2007; Apolloni et al. 2009). They activate membrane-bound P2 receptors subdivided into 7 ligand-gated ion channels (P2X receptors, reviewed in Köles et al. 2007) and 8 G-protein-coupled receptors (P2Y subtypes, reviewed in Fischer and Krügel 2007), which are ubiquitously and concurrently expressed on several different cell phenotypes (reviewed in Volonté et al. 2006; Burnstock 2007a, 2007b, 2008; Volonté, Amadio, and D’Ambrosi 2008). Oligodendrocytes express both ionotropic and metabotropic P2 receptors (Morán-Jiménez and Matute 2000; James and Butt 2002) and extracellular adenosine triphosphate (ATP) contributes to MS-associated release of interleukin-1beta and induction of cyclooxygenase-2 (Yiangou et al. 2006), via activation of the P2X7 subtype. Activation of the P2X7 receptor can moreover trigger oligodendrocyte excitotoxicity and cause in vivo lesions reminiscent of MS plaques, that is, demyelination, oligodendrocyte death, and axonal damage (Matute et al. 2007; reviewed in Matute 2008). In addition, the metabotropic P2Y12 receptor is present in vivo only in oligodendrocyte progenitor cells in rat white matter (Laitinen et al. 2001), whereas further studies in vitro established also the simultaneous expression, Ca2+ signaling and functioning of several additional P2X and P2Y subtypes (Agresti, Meomartini, Amadio, Ambrosini, Serafini, et al. 2005). We recently established in vivo the presence of the P2Y12 receptor in oligodendrocytes and myelin sheaths of rat cerebral cortex, subcortical areas, and periventricular white matter (Amadio et al. 2006). For this reason, here, we analyzed the cellular distribution of the P2Y12 protein in MS cerebral cortex, with the aim of correlating this receptor to the extent of gray matter demyelination.

Materials and Methods

Tissue Source

The tissues supplied by the UK Multiple Sclerosis Tissue Bank at Imperial College, London, were collected postmortem with fully informed consent from both donors and close relatives. Procedures for retrieval, processing, and storage have gained ethical approval from all appropriate committees. The brain tissues analyzed in this study were from 15 neuropathologically confirmed cases of MS, matched for sex and disease courses (all secondary progressive MS, SPMS) but presenting different ages (range 34–80 years), disease durations (range 11–50 years) and causes of death (see Table 1). Analysis was performed also on samples from patients who died due to nonneurological diseases. Cerebral hemispheres were fixed with 4% paraformaldehyde for about 2 weeks, coronally sliced, and blocked. Individual blocks were cryoprotected in 30% sucrose for 1 week and frozen by immersion in isopentane precooled on a bed of dry ice. Frozen tissue blocks were stored at −80 °C.

Table 1

Summary of patient details

Case Age (years) Sex Clinical diagnosis Disease duration (years) Cause of death DTPI (h) Number of sections analyzed 
MS058 51 SPMS 21 MS 15 30 
MS062 49 SPMS 19 Respiratory infection 10 22 
MS073 80 SPMS 50 Bronchopneumonia 20 24 
MS074 64 SPMS 36 Gastrointestinal bleed/obstruction, aspiration pneumonia 26 
MS076 49 SPMS 18 Chronic renal failure, heart disease 31 24 
MS079 49 SPMS 23 Bronchopneumonia, MS 37 
MS088 54 SPMS 17 Bronchopneumonia 22 26 
MS092 37 SPMS 17 MS 26 18 
MS109 60 SPMS 25 Myocardial infarct 22 21 
MS114 52 SPMS 15 Pneumonia, sepsis, pulmonary embolism 12 66 
MS125 76 SPMS 31 MS 13 116 
MS128 78 SPMS 50 Small bowel obstruction, pneumonia 22 18 
MS143 62 SPMS 18 Pulmonary embolism 13 82 
MS154 34 SPMS 11 Pneumonia 12 110 
MS163 45 SPMS Urinary tract infection, MS 28 123 
Case Age (years) Sex Clinical diagnosis Disease duration (years) Cause of death DTPI (h) Number of sections analyzed 
MS058 51 SPMS 21 MS 15 30 
MS062 49 SPMS 19 Respiratory infection 10 22 
MS073 80 SPMS 50 Bronchopneumonia 20 24 
MS074 64 SPMS 36 Gastrointestinal bleed/obstruction, aspiration pneumonia 26 
MS076 49 SPMS 18 Chronic renal failure, heart disease 31 24 
MS079 49 SPMS 23 Bronchopneumonia, MS 37 
MS088 54 SPMS 17 Bronchopneumonia 22 26 
MS092 37 SPMS 17 MS 26 18 
MS109 60 SPMS 25 Myocardial infarct 22 21 
MS114 52 SPMS 15 Pneumonia, sepsis, pulmonary embolism 12 66 
MS125 76 SPMS 31 MS 13 116 
MS128 78 SPMS 50 Small bowel obstruction, pneumonia 22 18 
MS143 62 SPMS 18 Pulmonary embolism 13 82 
MS154 34 SPMS 11 Pneumonia 12 110 
MS163 45 SPMS Urinary tract infection, MS 28 123 

Note: DTPI, death-tissue preservation interval.

Lesion Detection and Classification

Cryostat sections (30–40 μm thick) were either stained with Luxol fast blue and cresyl fast violet (Kluver–Barrera staining), in order to detect white matter lesions and their cellularity, or subjected to immunohistochemistry for myelin basic protein (MBP), in order to distinguish gray matter lesions. Cortical demyelinating lesions were classified according to Peterson et al. (2001): type I lesions (leukocortical lesions); type II lesions (intracortical lesions); and type III lesions (subpial lesions). The morphological features and extent of the lesions were scored and shown as follows: normal white matter; large lesions in white matter; small lesions in gray matter with moderate MBP and intense Kluver–Barrera staining; lesions in gray matter with scarce MBP and pale Kluver–Barrera staining; and large lesions in gray matter with neither MBP nor Kluver–Barrera staining (Fig. 2).

Immunohistochemistry

After quenching endogenous peroxidase by a 10-min incubation with 5% H2O2 in 5% methanol in phosphate buffered saline (PBS), sections (30–40 μm thick) were incubated for 24–48 h in PBS–0.3% Triton X-100 and 2% normal donkey serum at 4 °C, with primary antisera/antibodies as specified in Table 2. Sections were then incubated either with biotinylated donkey antimouse, biotinylated donkey antirabbit, or biotinylated donkey antigoat secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA), followed by avidin–biotin–peroxidase reactions (Vectastain, ABC kit, Vector, Burlingame, CA), using 3,3′-diaminobenzidine (Sigma) as a chromogen. Sections were mounted on poly-lysine slides and air dried for at least 24 h. In order to assess the extent of demyelination and the expression of additional markers, the sections were counterstained with Luxol fast blue. The histological preparations were examined using an Axioskop 2 light microscope (Zeiss, Iena, Germany). Images were taken with a digital camera (ProgRes C10 plus, Zeiss) interfaced to a computer with IAS 2000 software (Delta Sistemi, Rome, Italy).

Table 2

Primary antibodies/antisera used for the study

Antigen Clone Target Dilution Source 
MBP Mature oligodendrocytes/myelin 1:100 Chemicon 
MOG NYRMOG Oligodendrocytes/myelin 1:100 Santa Cruz 
NFL Polyclonal NFL 1:100 Santa Cruz 
Nonphosphorylated neurofilament protein (SMI32) smi32 Nonphosphorylated epitope of neurofilament heavy polypeptide 1:1000 Sternberger Monoclonals Inc. 
HLA- DP, DQ, DR (MHC II) CR3/43 Microglia cells 1:100 Dako 
CD68 EBM11 Microglia/macrophages 1:100 Dako 
GFAP G-A-5 Astrocytes 1:400 Sigma 
P2Y12 receptor Polyclonal P2Y12 receptor 1:100–300 Alomone 
P2X1,2,3,4,6,7–P2Y1,2,6,11,14 receptors Polyclonal P2X1,2,3,4,6,7–P2Y1,2,6,11,14 receptors 1:100–500 Alomone 
Antigen Clone Target Dilution Source 
MBP Mature oligodendrocytes/myelin 1:100 Chemicon 
MOG NYRMOG Oligodendrocytes/myelin 1:100 Santa Cruz 
NFL Polyclonal NFL 1:100 Santa Cruz 
Nonphosphorylated neurofilament protein (SMI32) smi32 Nonphosphorylated epitope of neurofilament heavy polypeptide 1:1000 Sternberger Monoclonals Inc. 
HLA- DP, DQ, DR (MHC II) CR3/43 Microglia cells 1:100 Dako 
CD68 EBM11 Microglia/macrophages 1:100 Dako 
GFAP G-A-5 Astrocytes 1:400 Sigma 
P2Y12 receptor Polyclonal P2Y12 receptor 1:100–300 Alomone 
P2X1,2,3,4,6,7–P2Y1,2,6,11,14 receptors Polyclonal P2X1,2,3,4,6,7–P2Y1,2,6,11,14 receptors 1:100–500 Alomone 

Note: HLA, human leukocyte antigen; CD68, transmembrane glycoprotein.

Double and Triple Immunofluorescence

Sections (30–40 μm thick) were processed for double and triple immunofluorescence studies. Nonspecific binding was blocked with 10% normal donkey serum in 0.3% Triton X-100 in PBS, for 1 h at room temperature. The sections were incubated with a mixture of primary antisera/antibodies (as specified above) in 0.3% Triton X-100 and 2% normal donkey serum in PBS, for 24–48 h at 4 °C, (see also Table 2). The secondary antibodies used for double labeling were Cy3-conjugated donkey antirabbit immunoglobulin G (IgG) (1:100, Jackson Immunoresearch, red immunofluorescence), Cy2-conjugated donkey antimouse IgG (1:100, Jackson Immunoresearch, green immunofluorescence), or Cy2-conjugated donkey antigoat IgG (1:100, Jackson Immunoresearch, red immunofluorescence). For the third color labeling, Cy5-conjugated donkey antigoat IgG (1:100, Jackson Immunoresearch, blue immunofluorescence) was used. The sections were washed in PBS 3 times for 5 min each and then incubated in a solution containing a mixture of the secondary antibodies in 0.3% Triton X-100 and 2% normal donkey serum in PBS, for 3 h at room temperature. After rinsing, the sections were mounted on slide glasses, allowed to air dry, and coverslipped with gel/mount antifading medium (Biomeda, Foster City, CA).

Triple Immunofluorescence with Zenon Technology

After double immunofluorescence, the sections were mounted on slide glasses, and allowed to air dry. A rectangle was then drawn around the sections with a PAP pen. To allow the use of a second mouse antibody in the same immunolabeling protocol, the unlabeled monoclonal antiMBP (mouse IgG1 isotype) was labeled with Zenon technology (Molecular Probes, OR). Briefly, mouse anti-MBP (1:100, Chemicon International) was incubated with Zenon Alexa Fluor 647 mouse IgG1 labeling reagent (molar ratio 6:1), which contains fluorophore-labeled (Ex/Em 650/668) antimouse Fab fragments. The labeled Fab fragments bind to the Fc portion of the monoclonal antibodies and excess Fab fragments are neutralized by the addition of a nonspecific IgG (Zenon blocking reagent mouse IgG). The addition of nonspecific IgG prevents cross-labeling of the Fab fragment, in experiments where multiple primary antibodies of the same type are present. After rehydration in PBS, the sections were incubated in a humidified chamber with the staining solution in PBS containing 0.5% Triton X-100 (PBT), for 2 h at room temperature. The sections were washed twice in PBT and for 5 min in PBS at room temperature. They were then fixed in 4% paraformaldehyde in PB for 15 min at room temperature, to avoid the dissociation of the Zenon-Fab fragment from the primary antibody. Finally, the sections were washed 3 times with PBS, allowed to air dry, and coverslipped with gel/mount antifading medium.

Confocal Microscopy

Double or triple label immunofluorescence was analyzed by means of a confocal laser scanning microscope (LSM 510, Zeiss, Arese Mi-Italy) equipped with argon laser emitting at 488 nm, helium–neon laser emitting at 543 nm, and helium–neon laser emitting at 633 nm. Signal specificity was positively proved by performing confocal analysis in the absence of the primary antibodies/antisera but in the presence of either antirabbit, antimouse, or antigoat secondary antibodies. Specificity was further confirmed for the P2Y12 receptor antiserum by performing immunoreactions in the simultaneous presence of the P2Y12 receptor neutralizing immunogenic peptide. The polyclonal P2Y12 receptor antiserum used in this study was raised against a highly purified peptide (identity confirmed by mass spectrography and amino acid analysis, as indicated in the certificate of analysis provided by the manufacturer), corresponding to an epitope not present in any other known protein.

Protein Extraction and Western Blotting

Snap-frozen blocks from cases MS114, MS125, and MS163 were homogenized in Ripa buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] in PBS, containing protease inhibitors). After a short sonication, the homogenates were incubated on ice for 1 h and centrifuged at 14,000 rpm for 10 min at 4 °C. Protein quantification was performed from the supernatants by Bradford colorimetric assay (Biorad, Milan, Italy). Proteins (80 μg) were separated by electrophoresis on 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose Hybond-C-extra membranes (Amersham Biosciences, Cologno Monzese, Italy). The filter was prewetted in 2% blocking agent in TBS-T (10 mM Tris pH 8, 150 mM NaCl, and 0.1% Tween 20) and hybridized overnight with P2X and P2Y antisera used at the following dilutions: 1:200 (P2X3,6–P2Y11,12,14); 1:300 (P2Y6); 1:400 (P2Y1,2); and 1:500 (P2X1,2,4,7). Incubations of all P2X and P2Y receptor antisera were performed either in the absence or in the presence of the neutralizing immunogenic peptides used in a 1:1 protein ratio. The antisera were immunodetected with an antirabbit horse radish peroxidase–conjugated antibody (1:2500) and developed by enhanced chemiluminescence (Amersham Biosciences), using Kodak Image Station (KDS IS440CF).

Results

Classification of SPMS Cases, Morphological Appearance of Cortical Lesions, and Presence of P2Y12 Receptor

The first question addressed in this work was the presence of the P2Y12 receptor protein in MS frontal cortex (Fig. 1). We analyzed 15 different cases of SPMS patients, with age at death ranging from 34 to 80 years, variable causes of death, stable or progressive activities of disease, and disease durations spanning between 11 and 50 years. For each case, we examined 2–4 different tissue blocks (37 total blocks), and for each block, we inspected 8–48 different serial slices (777 total slices) (Table 1). As internal controls, all tissue slices were examined in areas completely devoid of visible damage, although independent analysis was performed also in brain sample from patients who died of nonneurological diseases (data not shown). As previously observed in rat in vivo (Amadio et al. 2006), the P2Y12 protein was found abundant and widespread in human frontal cortex (Fig. 1). The receptor was homogeneously distributed throughout the gray matter (Fig. 1, arrows) and enriched in differently sized fiber bundles of white matter (Fig. 1, asterisks). P2Y12 receptor immunoreactivity always colocalized with MBP protein (Fig. 1) but not with neuronal markers (see Fig. 4). Immunoreactivity for P2Y12 receptor was at all times abolished in the presence of the neutralizing P2Y12 receptor immunogenic peptide or in the absence of the primary antiserum (data not shown). The presence of P2Y12 protein in MS frontal cortex was confirmed by western blot analysis (Fig. 1, inset). The receptor was recognized as a major protein band of 47–49 kDa (lanes a–c), which was abolished in the presence of the neutralizing P2Y12 receptor immunogenic peptide (lane p) or in the absence of the primary antiserum (data not shown).

Figure 1.

P2Y12 receptor protein is present in SPMS frontal cortex and colocalizes with MBP. Double immunofluorescence and confocal microscopy analysis was performed on sections from SPMS frontal cortex (case MS088) using antibodies for MBP (green, Cy2 immunofluorescence) and P2Y12 receptor (red, Cy3 immunofluorescence). Arrows indicate gray matter and asterisks show white matter fiber bundles. Scale bar = 100 μm. Inset: P2Y12 receptor in human frontal cortex was detected by western blot analysis. Equal amount of total protein (80 μg/well) from snap-frozen blocks (case MS114 lane a, case MS125 lane b and case MS163 lane c) was separated by SDS-PAGE and transferred to nitrocellulose. Filters were stained with Ponceau-S and immunostained with rabbit anti-P2Y12 serum, in the absence or presence (p) of the neutralizing immunogenic peptide. Molecular masses of 64, 49, and 37 kDa are indicated.

Figure 1.

P2Y12 receptor protein is present in SPMS frontal cortex and colocalizes with MBP. Double immunofluorescence and confocal microscopy analysis was performed on sections from SPMS frontal cortex (case MS088) using antibodies for MBP (green, Cy2 immunofluorescence) and P2Y12 receptor (red, Cy3 immunofluorescence). Arrows indicate gray matter and asterisks show white matter fiber bundles. Scale bar = 100 μm. Inset: P2Y12 receptor in human frontal cortex was detected by western blot analysis. Equal amount of total protein (80 μg/well) from snap-frozen blocks (case MS114 lane a, case MS125 lane b and case MS163 lane c) was separated by SDS-PAGE and transferred to nitrocellulose. Filters were stained with Ponceau-S and immunostained with rabbit anti-P2Y12 serum, in the absence or presence (p) of the neutralizing immunogenic peptide. Molecular masses of 64, 49, and 37 kDa are indicated.

Figure 2.

Classification of representative SPMS cases and morphological appearance of cortical lesions. Cortical tissue was provided from UK Multiple Sclerosis Tissue Bank at Imperial College, in London. Schematic maps of lesions from frontal cortical sections stained with Kluver–Barrera staining and MBP were obtained. Lesion intensities were scored and shown: normal white matter (dark blue); large lesions in white matter (blue); small lesion in gray matter with moderate MBP and intense Kluver–Barrera staining (green); lesions in gray matter with scarce MBP and pale Kluver–Barrera staining (azure); extensive lesions in gray matter with neither MBP nor Kluver–Barrera staining (pink). Abbreviations: GM (gray matter), WM (white matter), and NAWM (normal appearing white matter).

Figure 2.

Classification of representative SPMS cases and morphological appearance of cortical lesions. Cortical tissue was provided from UK Multiple Sclerosis Tissue Bank at Imperial College, in London. Schematic maps of lesions from frontal cortical sections stained with Kluver–Barrera staining and MBP were obtained. Lesion intensities were scored and shown: normal white matter (dark blue); large lesions in white matter (blue); small lesion in gray matter with moderate MBP and intense Kluver–Barrera staining (green); lesions in gray matter with scarce MBP and pale Kluver–Barrera staining (azure); extensive lesions in gray matter with neither MBP nor Kluver–Barrera staining (pink). Abbreviations: GM (gray matter), WM (white matter), and NAWM (normal appearing white matter).

We next characterized the lesions of our SPMS tissue, with the aim of correlating the P2Y12 receptor to the extent of demyelination (Fig. 2). In white matter, the lesions were characterized as active (with abundant amoeboid, round microglia) or inactive (with dense astrocytic scarring and ramified microglia), according to the morphological appearance of both major histocompatibility complex (MHC) II or glial fibrillary acidic protein (GFAP)–immunopositive cells. Gray matter lesions were classified as types I–III (Fig. 2), according to Peterson et al. (2001). Kluver–Barrera staining and MBP immunohistochemistry on all SPMS lesions are shown in Figure 2 by representative digital images. We observed the typical features of cortical demyelination (Magliozzi et al. 2007; Moll et al. 2008; reviewed in Peterson and Trapp 2005) in all SPMS cases. Severe myelin loss was mostly observed in subpial lesions (type III lesions), very close to the subarachnoid space, involving either a part of a cortical gyrus or often encompassing adjacent gyri. The remaining lesions were either intracortical (type II lesions) or deeper leukocortical lesions (type I). The cortical lesions contained very little inflammatory activity, with a modest T-cell infiltration and microglia activation (data not shown).

P2Y12 Receptor Protein Is Present in Myelin and Interlaminar Astrocytes

A further aim was to investigate the phenotypic distribution of the P2Y12 receptor in MS frontal cortex (Figs. 3 and 4). In all sections with small gray matter lesions and intense Kluver–Barrera staining, the P2Y12 protein was found in myelin sheaths, on long, thick, and thin parallel myelinated nerve fibers forming a large- and a close-mesh network in the superficial and deep layers of the cortex (Fig. 1 and 3A). A strong colocalization between P2Y12 receptor and MBP identified both longitudinal (arrows) and transverse myelinated fibers (arrowheads) (Fig. 3A). P2Y12 receptor immunoreactivity was also found in the processes of astrocytes classified as interlaminar (their somata were primarily present in cortical layer I, and their fibers extended into the deeper cortical layers, Oberheim et al. 2006) (Fig. 3B arrows; Fig. 3C). Conversely, P2Y12 protein was absent from the most abundant protoplasmic astrocytes residing in the deeper cortical layers (Fig. 3D), absent from MHC II–immunoreactive microglia (Fig. 3E), or CD68-positive macrophages, and NeuN-labeled neuronal cell bodies (data not shown). As previously reported in rat brain (Amadio et al. 2006), P2Y12 receptor immunofluorescence only apparently colocalized with some neurofilament light polypeptide (NFL)–positive longitudinal fragments (Fig. 4A, arrows) and transversally oriented neuronal fibers and bundles (Fig. 4B–C, arrowheads), due to close vicinity and tight association of axonal and myelin structures. We never observed the presence of P2Y12 receptor immunoreactivity on either demyelinated neuronal fibers (Fig. 4D) or somata (Fig. 4E), which were positive for the nonphosphorylated epitope of the neurofilament heavy polypeptide SMI32 (Trapp et al. 1998).

Figure 3.

P2Y12 receptor protein is present in myelin and interlaminar astrocytes. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for different immunoreactive markers. (A) Case MS114: P2Y12 (red, Cy3 immunofluorescence), MBP (green, Cy2 immunofluorescence), and merged (yellow). The arrow shows a longitudinal fiber, whereas the arrowhead indicates a transverse fiber. (B) Case MS125, and (C) Case MS092: P2Y12 (red, Cy3 immunofluorescence) and astroglial marker GFAP (green, Cy2 immunofluorescence). The arrows indicate identical fiber immunolabeled by both P2Y12 and GFAP antisera. (D) Case MS154: merged field of P2Y12 (red, Cy3 immunofluorescence) and GFAP (green, Cy2 immunofluorescence), showing lack of colocalization. (E) Case MS092: merged field of P2Y12 (red, Cy3 immunofluorescence) and microglia marker MHC II (green, Cy2 immunofluorescence), showing lack of colocalization. Minor nonspecific red neuronal lipofuscin autofluorescence is visible in the background (panel B). Scale bar = 20 μm in A and B, 10 μm in C; 50 μm in D; and 20 μm in E.

Figure 3.

P2Y12 receptor protein is present in myelin and interlaminar astrocytes. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for different immunoreactive markers. (A) Case MS114: P2Y12 (red, Cy3 immunofluorescence), MBP (green, Cy2 immunofluorescence), and merged (yellow). The arrow shows a longitudinal fiber, whereas the arrowhead indicates a transverse fiber. (B) Case MS125, and (C) Case MS092: P2Y12 (red, Cy3 immunofluorescence) and astroglial marker GFAP (green, Cy2 immunofluorescence). The arrows indicate identical fiber immunolabeled by both P2Y12 and GFAP antisera. (D) Case MS154: merged field of P2Y12 (red, Cy3 immunofluorescence) and GFAP (green, Cy2 immunofluorescence), showing lack of colocalization. (E) Case MS092: merged field of P2Y12 (red, Cy3 immunofluorescence) and microglia marker MHC II (green, Cy2 immunofluorescence), showing lack of colocalization. Minor nonspecific red neuronal lipofuscin autofluorescence is visible in the background (panel B). Scale bar = 20 μm in A and B, 10 μm in C; 50 μm in D; and 20 μm in E.

Figure 4.

Absence of P2Y12 receptor protein from demyelinated axons in gray matter. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for different immunoreactive markers. (A–C) Case MS143: merged field of P2Y12 (red Cy3, immunofluorescence) and NFL (green, Cy2 immunofluorescence), indicating only proximity of signals. In A, longitudinal fibers are indicated by arrows, whereas in B–C, transverse fiber bundles are marked by arrowheads. In C, partial loss of P2Y12 receptor immunoreactivity is visible within a single bundle of fibers, in which the red P2Y12 signal surrounding yellow dots is in close proximity to residual black holes within the green-NFL field. (D) Case MS143, and (E) case MS143: merged fields of P2Y12 (red Cy3, immunofluorescence) and SMI32 (green, Cy2 immunofluorescence) showing lack of colocalization. Nonspecific red/yellow neuronal lipofuscin autofluorescent signal is visible in the background of panel D. Scale bar = 10 μm in A; 5 μm in B and C; 50 μm in D; and 20 μm in E.

Figure 4.

Absence of P2Y12 receptor protein from demyelinated axons in gray matter. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for different immunoreactive markers. (A–C) Case MS143: merged field of P2Y12 (red Cy3, immunofluorescence) and NFL (green, Cy2 immunofluorescence), indicating only proximity of signals. In A, longitudinal fibers are indicated by arrows, whereas in B–C, transverse fiber bundles are marked by arrowheads. In C, partial loss of P2Y12 receptor immunoreactivity is visible within a single bundle of fibers, in which the red P2Y12 signal surrounding yellow dots is in close proximity to residual black holes within the green-NFL field. (D) Case MS143, and (E) case MS143: merged fields of P2Y12 (red Cy3, immunofluorescence) and SMI32 (green, Cy2 immunofluorescence) showing lack of colocalization. Nonspecific red/yellow neuronal lipofuscin autofluorescent signal is visible in the background of panel D. Scale bar = 10 μm in A; 5 μm in B and C; 50 μm in D; and 20 μm in E.

P2Y12 Receptor Signal Disappears from Gray Matter Lesions Prior to MBP but Later Than Myelin Oligodendrocyte Glycoprotein (MOG)

We next asked if the P2Y12 receptor was correlated with the extent of demyelination in lesioned gray matter (Fig. 5). In areas with pale Kluver–Barrera staining (confront Fig. 2, azure lesions), we first performed triple immunofluorescence confocal analysis with antibodies against MBP, P2Y12 receptor, and NFL (Fig. 5). By comparing an axon with strong and continuous NFL immunoreactivity with a transected axon with weaker, thinner, and interrupted NFL signal, we found that although MBP persisted in both cases (green panel), P2Y12 receptor was decreased in the intact axons but nearly lost in the transected fibers (Fig. 5A). Nevertheless, P2Y12 receptor immunoreactivity weakly persisted on myelin sheaths even in the absence of axonal content (absent NFL signal but persistent MBP labeling, Bitsch et al. 2000; Bjartmar et al. 2001) (Fig. 5A, ellipse). In all the different 15 SPMS cases studied, P2Y12 receptor immunoreactivity was much weaker and thinner than MBP immunostaining in gray matter areas with pale or absent Kluver–Barrera staining. On the contrary, the MBP and P2Y12 receptor signals were always found more similar in intensity and continuity in gray matter areas with no apparent lesion and intense Kluver–Barrera staining (confront Fig. 3A).

Figure 5.

P2Y12 receptor signal in gray matter lesions disappears prior to MBP but later than MOG. Triple immunofluorescence visualized by confocal analysis with different immunoreactive markers was performed on sections from SPMS frontal cortex. (A) Case MS143: MBP (green, Cy2 immunofluorescence), P2Y12 receptor (red, Cy3 immunofluorescence), and NFL (blue, Cy5 immunofluorescence). A continuous axon (arrow), a transected fiber (arrowheads), and a myelin sheath deprived of axonal content (ellipse) were compared for expression of the different markers. In the merged field, triple immunofluorescent white signal was found in segments simultaneously positive for MBP, P2Y12 receptor, and NFL. In segments lacking P2Y12 receptor, the immunoreactive signal was light blue. In segments lacking NFL, but maintaining MBP and P2Y12 receptor, the immunoreactive signal was finally yellow. We never observed pink axonal segments, eventually derived from lack of MBP and persistence of P2Y12 and NFL. (B) Case MS154: MOG (green, Cy2 immunofluorescence), P2Y12 receptor (red, Cy3 immunofluorescence), and NFL (blue, Cy5 immunofluorescence). In the merged field, triple immunofluorescent white signal was found in intact axonal segments simultaneously positive for MOG, P2Y12 receptor, and NFL. In pink are shown P2Y12 NFL–positive axonal segments and fibers (arrowheads), whereas the asterisks show rare fibers positive only for P2Y12 receptor (red). Several NFL positive nude axons are also seen (blue). (C) Case MS143: MOG (green, Cy2 immunofluorescence), P2Y12 receptor (red, Cy3 immunofluorescence), and MBP (blue, Cy5 immunofluorescence). In this fiber, the signal for MOG is lower than for the P2Y12 receptor, which in turn is lower than MBP. Scale bar = 5 μm in A; 20 μm in B, and 10 μm in C.

Figure 5.

P2Y12 receptor signal in gray matter lesions disappears prior to MBP but later than MOG. Triple immunofluorescence visualized by confocal analysis with different immunoreactive markers was performed on sections from SPMS frontal cortex. (A) Case MS143: MBP (green, Cy2 immunofluorescence), P2Y12 receptor (red, Cy3 immunofluorescence), and NFL (blue, Cy5 immunofluorescence). A continuous axon (arrow), a transected fiber (arrowheads), and a myelin sheath deprived of axonal content (ellipse) were compared for expression of the different markers. In the merged field, triple immunofluorescent white signal was found in segments simultaneously positive for MBP, P2Y12 receptor, and NFL. In segments lacking P2Y12 receptor, the immunoreactive signal was light blue. In segments lacking NFL, but maintaining MBP and P2Y12 receptor, the immunoreactive signal was finally yellow. We never observed pink axonal segments, eventually derived from lack of MBP and persistence of P2Y12 and NFL. (B) Case MS154: MOG (green, Cy2 immunofluorescence), P2Y12 receptor (red, Cy3 immunofluorescence), and NFL (blue, Cy5 immunofluorescence). In the merged field, triple immunofluorescent white signal was found in intact axonal segments simultaneously positive for MOG, P2Y12 receptor, and NFL. In pink are shown P2Y12 NFL–positive axonal segments and fibers (arrowheads), whereas the asterisks show rare fibers positive only for P2Y12 receptor (red). Several NFL positive nude axons are also seen (blue). (C) Case MS143: MOG (green, Cy2 immunofluorescence), P2Y12 receptor (red, Cy3 immunofluorescence), and MBP (blue, Cy5 immunofluorescence). In this fiber, the signal for MOG is lower than for the P2Y12 receptor, which in turn is lower than MBP. Scale bar = 5 μm in A; 20 μm in B, and 10 μm in C.

We then performed immunofluorescence for MOG, an important constituent of myelin sheaths (Quarles 2002; Zhou et al. 2006), which was found only on sporadic intact myelin segments showing a robust signal also for P2Y12 receptor (Fig. 5B). By triple immunofluorescence, we demonstrated that several fibers were immunoreactive for NFL, a few for P2Y12 receptor, but just one for MOG. Remarkably, we never observed MOG NFL–positive axons deprived of P2Y12 receptor. In general, the intensity and continuity of MOG immunoreactivity was lower than P2Y12 receptor and in turn lower than MBP (Fig. 5C).

The Expression of P2Y12 Protein in Inactive Gray Matter SPMS Cortex Varies According to the Distance from the Lesion

We then asked if the level of expression of P2Y12 receptor might depend on the distance from the gray matter lesion (Fig. 6). Using Kluver–Barrera staining and MBP immunohistochemistry, we identified 3 areas progressively distant from a severe type III subpial lesion (Fig. 6A–C), which was characterized by a consistent GFAP-positive glia scar (Fig. 6D) and abundant ramified/reactive MHC II microglia (Fig. 6E). We found that closer to the glial scar at the edge of each SPMS section, both MBP immunohistochemistry (Fig. 6C) and immunofluorescence decreased but to lesser extent than P2Y12 receptor immunoreactivity. This result was extended to cortical gray matter lesion types I and II (data not shown).

Figure 6.

The expression of P2Y12 receptor protein varies according to the distance from the lesion. Sections from SPMS frontal cortex (case MS154) were subjected to Kluver–Barrera staining (panels A,B) to immunohistochemistry for MBP, GFAP, and MHC II (panels CE) and to Luxol fast blue/MHC II staining (panel F). We performed double immunofluorescence and confocal microscopy with sera against P2Y12 receptor (red, Cy3 immunofluorescence) and MBP (green, Cy2 immunofluorescence) (fields 1–3, panels green, red and merged). The 3 chosen aligned fields (circled 1–3) are increasingly distant from the subpial glial scar at the edge of the section (panels BD). In parallel, they presented an increasing MBP immunohistochemistry signal (panel C) and MBP immunofluorescence (in green), going from fields 1 to 3. We noticed a few, disordered but well visible myelinated fibers in field 1, which became more abundant and oriented in field 2 and thick and bundled in field 3. The expression of P2Y12 receptor protein (in red) from fields 1 to 3 showed a comparable trend, although the immunoreactivity appeared always less intense than MBP. Although rare P2Y12 receptor–positive fibers (in red) were visible in fields 1 and 2, they remained highly fragmented, discontinuous, and disordered in field 2 but became abundant and oriented to an extent almost equivalent to MBP only in field 3. Nonspecific green/red/yellow neuronal lipofuscin autofluorescent signal is visible in the background of fields 1 and 2. Scale bar = 300 μm in B, C, and E; 400 μm in D; 10 μm in F; and 40 μm in fields 1–3.

Figure 6.

The expression of P2Y12 receptor protein varies according to the distance from the lesion. Sections from SPMS frontal cortex (case MS154) were subjected to Kluver–Barrera staining (panels A,B) to immunohistochemistry for MBP, GFAP, and MHC II (panels CE) and to Luxol fast blue/MHC II staining (panel F). We performed double immunofluorescence and confocal microscopy with sera against P2Y12 receptor (red, Cy3 immunofluorescence) and MBP (green, Cy2 immunofluorescence) (fields 1–3, panels green, red and merged). The 3 chosen aligned fields (circled 1–3) are increasingly distant from the subpial glial scar at the edge of the section (panels BD). In parallel, they presented an increasing MBP immunohistochemistry signal (panel C) and MBP immunofluorescence (in green), going from fields 1 to 3. We noticed a few, disordered but well visible myelinated fibers in field 1, which became more abundant and oriented in field 2 and thick and bundled in field 3. The expression of P2Y12 receptor protein (in red) from fields 1 to 3 showed a comparable trend, although the immunoreactivity appeared always less intense than MBP. Although rare P2Y12 receptor–positive fibers (in red) were visible in fields 1 and 2, they remained highly fragmented, discontinuous, and disordered in field 2 but became abundant and oriented to an extent almost equivalent to MBP only in field 3. Nonspecific green/red/yellow neuronal lipofuscin autofluorescent signal is visible in the background of fields 1 and 2. Scale bar = 300 μm in B, C, and E; 400 μm in D; 10 μm in F; and 40 μm in fields 1–3.

P2Y12 Receptor Protein is Phagocytosed by Microglia in SPMS White Matter

A further question addressed in this work was the presence of the P2Y12 receptor in MS white matter (Fig. 7). We confirmed the presence of the P2Y12 protein on MBP-positive myelinated fibers and its decline in proximity to the injured tissue (Fig. 7A). Although the immunoreactive signal for MBP was decreased only at the edge of the lesion (arrow), the area lacking the P2Y12 signal was more extended. At higher magnification (Fig. 7B,C), several fibers showed the typical features of axonal swelling (arrowheads), with the presence of terminal spheroids (arrows) (reviewed in Peterson and Trapp 2005). This was demonstrated by both immunofluorescence for MBP–P2Y12 protein (Fig. 7B) and immunohistochemistry for P2Y12 receptor counterstained with Luxol fast blue (Fig. 7C). Moreover, by examining a white matter plaque surrounding a blood vessel (Fig. 7D,E), we noticed that, although P2Y12 protein decreased inside the lesion (red), reactivity for MHC II increased (green). In close proximity to the blood vessel walls, the microglia/phagocytic macrophages contained P2Y12-positive material (Fig. 7E), as confirmed by double immunofluorescence with CD68 (Fig. 7E, inset). The same was previously seen for myelin proteins that are phagocytosed by macrophages during the early stages of MS demyelination (Gobin et al. 2001).

Figure 7.

In SPMS white matter, the P2Y12 receptor protein is found in phagocytic microglia. Double immunofluorescence was performed on sections from SPMS frontal cortex (Fig. 7A,B, case MS154) with antibody against MBP (green, Cy2 immunofluorescence) and antiserum for P2Y12 receptor (red, Cy3 immunofluorescence). The asterisk indicates the white matter lesion, and the arrow shows proximity to the lesion rim. Especially in the merged field, the green MBP signal is much wider than the yellow MBP–P2Y12 one. Panel B shows a higher magnification of the merged MBP–P2Y12 receptor field, with arrowheads illustrating axonal swelling and arrows showing terminal spheroids. This is also depicted in panel C, where sections from SPMS frontal cortex (case MS154) were subjected to Luxol fast blue/P2Y12 staining. In panels DE (case MS154), double immunofluorescence was performed with antibody against MHC II (green, Cy2 immunofluorescence) and antiserum for P2Y12 receptor (red, Cy3 immunofluorescence). The inset in panel E (case MS143) shows higher magnification of the merged CD68–P2Y12 signals (black arrowhead). Scale bar = 100 μm in A and D; 20 μm in B and E; 10 μm in C; and 5 μm in inset in E.

Figure 7.

In SPMS white matter, the P2Y12 receptor protein is found in phagocytic microglia. Double immunofluorescence was performed on sections from SPMS frontal cortex (Fig. 7A,B, case MS154) with antibody against MBP (green, Cy2 immunofluorescence) and antiserum for P2Y12 receptor (red, Cy3 immunofluorescence). The asterisk indicates the white matter lesion, and the arrow shows proximity to the lesion rim. Especially in the merged field, the green MBP signal is much wider than the yellow MBP–P2Y12 one. Panel B shows a higher magnification of the merged MBP–P2Y12 receptor field, with arrowheads illustrating axonal swelling and arrows showing terminal spheroids. This is also depicted in panel C, where sections from SPMS frontal cortex (case MS154) were subjected to Luxol fast blue/P2Y12 staining. In panels DE (case MS154), double immunofluorescence was performed with antibody against MHC II (green, Cy2 immunofluorescence) and antiserum for P2Y12 receptor (red, Cy3 immunofluorescence). The inset in panel E (case MS143) shows higher magnification of the merged CD68–P2Y12 signals (black arrowhead). Scale bar = 100 μm in A and D; 20 μm in B and E; 10 μm in C; and 5 μm in inset in E.

Additional P2X and P2Y Receptor Proteins Are Present in SPMS Cortex

The last issue addressed in this work was to map the presence of all additional P2X and P2Y receptor proteins in SPMS frontal cortex, by both western blotting and immunohistochemistry. Major single bands were detected for P2X1,2,4,7 and P2Y2,6,11 subtypes, whereas P2X3 and P2Y1,14 receptors provided 2 major bands each. No specific signal was seen for P2X6 protein (Table 3). Specificity was confirmed for all receptor subtypes by the use of corresponding neutralizing immunogenic peptides. Similar results were obtained for all the different cases analyzed. When evaluated by immunohistochemistry, strong signals were observed in the entire frontal cortex for P2X3,4,7 and P2Y2,11 receptors, whereas P2X1,2 and P2Y6,14 immunoreactivities were weaker but more localized to small areas. The P2X6 and P2Y1 receptors were not detected in SPMS frontal cortex (Table 3).

Table 3

Synoptic view of P2 receptors in SPMS frontal cortex

P2 receptors WB (kDa) IHC 
P2X1 60 
P2X2 35 
P2X3 30 and 35 
P2X4 ∼60 
P2X5 
P2X6 Ns — 
P2X7 45 
P2Y1 68 and 82 — 
P2Y2 70 
P2Y4 
P2Y6 70 
P2Y11 41 
P2Y13 
P2Y14 37 and 55 
P2 receptors WB (kDa) IHC 
P2X1 60 
P2X2 35 
P2X3 30 and 35 
P2X4 ∼60 
P2X5 
P2X6 Ns — 
P2X7 45 
P2Y1 68 and 82 — 
P2Y2 70 
P2Y4 
P2Y6 70 
P2Y11 41 
P2Y13 
P2Y14 37 and 55 

Note: IHC: immunohistochemistry; *: reactivity not confirmed in human; ns: not specific signal; and WB: western blotting.

Discussion

The most important pathological events underlying the progression of neurological disability in MS are axonal damage and demyelination (Irvine and Blakemore 2008; Stadelmann et al. 2008; reviewed in Lassmann 1998) caused by cytotoxic factors released from immune cells, excitotoxicity, and loss of trophic support (Bitsch et al. 2000; reviewed in Stadelmann and Brück 2008). Extracellular purine/pyrimidine nucleotides can be released by immune cells, can cause excitotoxicity, and also act as trophic factors (reviewed in Burnstock 2008; Gonçalves and Queiroz 2008; Volonté, D’Ambrosi, and Amadio 2008; Burnstock et al. 2009; Volonté and D'Ambrosi 2009). Their potential role in MS is thus very plausible (Yiangou et al. 2006; Matute et al. 2007; reviewed in Agresti, Meomartini, Amadio, Ambrosini, Volonté, et al. 2005), and it represents the central aim of this work.

Indeed, electrical activity in neurons causes them to release ATP (reviewed in Burnstock 2006), which in turn serves as a stimulus for myelin formation. ATP does not act directly on oligodendrocytes, instead induces astrocytes to secrete the cytokine leukemia inhibitory factor, a regulatory protein that promotes the myelinating activity of oligodendrocytes (Ishibashi et al. 2006). Because we previously demonstrated the purinergic P2Y12 receptor in rat myelin sheaths (Amadio et al. 2006), here we investigated the expression of this same subtype in demyelinating SPMS frontal cortex and all P2X and P2Y receptors. The established localization of P2Y12 immunoreactivity to myelin and interlaminar astrocytes, but absence from protoplasmic astrocytes, neurons, and microglia, would suggest a role in signaling between the axon and the oligodendrocyte/myelin unit, and in a number of astrocyte functions, for instance maintenance of the blood-brain barrier, transmitter and potassium reuptake and release (reviewed in Kettenmann and Verkhratsky 2008). ATP activating P2Y12 receptors on oligodendrocytes and astrocytes might also likely perform a direct and/or indirect role in the promotion of myelination. All this could be mediated by P2Y12 receptor–dependent signal transduction mechanisms (He and McCarthy 1994) and cytoplasmic Ca2+ fluxes from intracellular stores, which are indeed known to be induced by ATP/ADP in oligodendrocytes in vitro (Kirischuk et al. 1995) and in vivo, in mouse and rat “corpus callosum” and optic nerve (Bernstein et al. 1996; James and Butt 2001). P2Y12 protein on oligodendrocytes and astrocytes at the axon–glial interface might even contribute to the extension and adhesion of the oligodendroglial processes to the axons to be myelinated. This would be sustained by the well-established role that P2Y12 receptor plays in both human platelets, as a mediator of cell contact, adhesion and thrombus stability (reviewed in Cattaneo 2007; Michelson 2008), and in rat microglia, as a mediator of chemotaxis (Nasu-Tada et al. 2005).

The further aim of our work was to correlate the level of P2Y12 receptor expression with axonal damage and gray matter demyelination occurring in frontal cortex during the secondary progressive phase of MS. The reduction in P2Y12 protein expression indeed well correlated with increasing demyelination and overall reduction of MBP in myelin sheaths and oligodendrocytes. However, the reduced P2Y12 receptor expression might also occur on interlaminar astrocytes operating as a nonsynaptic pathway for long-distance signaling and integration of activity within cortical columns. Because this particular type of glia is known to be markedly altered or even absent in neurodegenerative conditions (for instance Alzheimer's disease, Colombo et al. 2002), the reduced P2Y12 protein expression in MS might also be a detrimental astroglial consequence of the neurodegenerative process. Nevertheless, we cannot exclude that a compensatory replacement of interlaminar astrocytes with other astrocytic phenotypes, and a general mitogenic activity, hypertrophy of astrocytes, and elongation of processes might instead occur and involve the P2Y12 receptor function. Such events are actually known to be promoted in vivo by direct activation of the P2Y12 subunit (Franke et al. 2001). This would then suggest a contribution from the P2Y12 receptor to both destructive and restorative phases of MS, in agreement with the dual role that glial cells exert in MS disease progression.

In conclusion, our analysis of frontal cortex has determined the simultaneous presence of several purinergic P2X and P2Y receptors, as well as the altered expression of the P2Y12 subtype at the axon–myelin interface in white and gray matter of patients with SPMS. The extent of P2Y12 protein was found to be inversely proportional to demyelination and lesion formation. We speculate that a reduction in P2Y12 receptor might become an additional marker of the development of the lesions in the disease. Because the therapeutic choice at present in MS is limited and relies on mildly to moderately effective immunomodulatory treatments, a combined restorative strategy could now likely include also the modulation of the ATP signaling pathways.

Funding

Cofinanziamento Ministero della Salute RF05.105V and RC09.C.

All postmortem MS samples were supplied by the UK MS Tissue Bank (www.ukmstissuebank.imperial.ac.uk), funded by the Multiple Sclerosis Society of Great Britain and Northern Ireland (registered charity 207495). We thank Dr F. Florenzano (CNR/Santa Lucia Foundation) for technical suggestions. Conflict of Interest: None declared.

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