Synaptopathy Connects Inflammation and Neurodegeneration in Multiple

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Synaptopathy connects inflammation and neurodegeneration in multiple sclerosis Georgia Mandolesi, Antonietta Gentile, Alessandra Musella, Diego Fresegna, Francesca De Vito, Silvia Bullitta, Helena Sepman, Girolama A. Marfia and Diego Centonze

Abstract | Multiple sclerosis (MS) has long been regarded as a chronic inflammatory disease of the white matter that leads to demyelination and eventually to neurodegeneration. In the past decade, several aspects of MS pathogenesis have been challenged, and degenerative changes of the grey matter, which are independent of demyelination, have become a topic of interest. CNS inflammation in MS and experimental autoimmune encephalomyelitis (EAE; a disease model used to study MS in rodents) causes a marked imbalance between GABAergic and glutamatergic transmission, and a loss of synapses, all of which leads to a diffuse ‘synaptopathy’. Altered synaptic transmission can occur early in MS and EAE, independently of demyelination and axonal loss, and subsequently causes excitotoxic damage. Inflammation-driven synaptic abnormalities are emerging as a prominent pathogenic mechanism in MS—importantly, they are potentially reversible and, therefore, represent attractive therapeutic targets. In this Review, we focus on the connection between inflammation and synaptopathy in MS and EAE, which sheds light not only on the pathophysiology of MS but also on that of primary neurodegenerative disorders in which inflammatory processes contribute to disease progression.

Mandolesi, G. et al. Nat. Rev. Neurol. advance online publication 20 November 2015; doi:10.1038/nrneurol.2015.222 Introduction The pathogenesis of multiple sclerosis (MS) begins with an conducted in animals with experimental autoimmune inflammatory cascade in the CNS, which is mainly caused encephalomyelitis (EAE; considered by many researchers by inappropriately activated T cells that trigger an immune as a validated rodent model of human MS18) have provided response against myelin and myelin-forming cells (oligo- solid evidence in support of these distinct processes that dendrocytes). Autoreactive T cells from the bloodstream affect grey matter.19,20 Many researchers have focused on penetrate into the CNS by crossing the blood–brain barrier axonal damage because it is a crucial determinant of dis- (BBB) and produce inflammatory cytokines, causing addi- ability, and have identified several effector molecules and tional damage to the myelin and surrounding tissue. The downstream mechanisms through which the immune resulting formation of large demyelinating plaques in the system triggers the neurodegenerative process that results white matter leads to neurodegeneration.1,2 in axonal damage.19,20 Over the past decade, MRI studies and analysis of Accumulating evidence from proteomic, transcrip- autopsy tissue from patients with MS have revolution- tomic, neurophysiological and histological studies of MS ized our understanding of MS pathogenesis.3,4 Grey indicates that diffuse synaptic dysfunction and loss, col- matter damage is now known to occur early in the disease lectively known as synaptopathy (Box 1), is a hallmark of IRCCS Fondazione 5–12 5–9 Santa Lucia/Centro course, to be partly independent of demyelination, MS pathophysiology. Most of the studies assessing synaptic 10–15 Europeo per la Ricerca and to be associated with clinical disability and cog- dysfunction and loss in MS report perturbations of both sul Cervello (CERC), nitive dysfunction.10,12–15 Grey matter atrophy in patients excitatory (mediated by glutamate) and inhibitory (medi- Via del Fosso di Fiorano 64, 00143 Rome, Italy with MS seems to result from a combination of demyelinated by γ‑aminobutyric acid [GABA]) neurotransmission, (G.M., A.M., D.F., S.B., ation, neuronal loss and/or atrophy, neurite transaction7 which are critically involved in the correct functioning of H.S.). Dipartimento di 8,16 Medicina dei Sistemi, and reduced numbers of synapses and glia, suggest- the CNS. Of note, a long-lasting perturbation of synaptic Università Tor Vergata, ing that inflammatory and neurodegenerative events are homeostasis can become detrimental, leading to excito- Via Montpellier 1, intermingled rather than sequential.17 Several studies toxic damage and neuronal death. Here, we review studies 00133 Rome, Italy (A.G., F.D.V., H.S., relating to synaptic aspects of grey matter pathology in G.A.M.). IRCCS Istituto MS and EAE, including loss of synapses (Box 1), synaptic Neurologico Competing interests Mediterraneo (INM) D.C. is an Advisory Board member for Bayer Schering, Merck- dysregulation as a result of perturbations of the molecular Neuromed, Via Serono and Teva, and has received honoraria for speaking or machinery responsible for neurotransmitter homeostasis, Atinense 18, consultation fees from Almirall, Bayer Schering, Biogen Idec, 86077 Pozzilli, Italy and synaptic dysfunction, including functional altera- Genzyme, GW Pharmaceuticals, Merck Serono, Novartis, Sanofi- (D.C.). tion of neurotransmission. Particular emphasis is given to Aventis and Teva. He is also the principal investigator in clinical inflammatory-dependent synaptopathy, which is poten- Correspondence to: trials for Bayer Schering, Biogen Idec, Novartis, Merck Serono, D.C. Sanofi-Aventis and Teva. The other authors declare no tially reversible and might represent a novel therapeutic [email protected] competing interests. target in MS.

Key points (synaptophysin, synaptotagmin, postsynaptic density protein‑95 [PSD‑95] and Ca2+-calmodulin-dependent ■■ Progressive synaptic loss and dysfunction—also known as synaptopathy —occur early in multiple sclerosis (MS), and in experimental autoimmune protein kinase II [CASK; a peripheral plasma mem- encephalomyelitis (EAE), which is used to study MS in rodent models brane protein that is involved in synaptic vesicle release ■■ Along with demyelination and axonal damage or transection, synaptopathy and interacts with neurexin]), even though hippocampal is a pathophysiological hallmark observed in MS and EAE; moreover, it is neuronal loss was minimal.16 Moreover, the integrity independent of axonal transection and demyelination of the cholinergic neurotransmitter system has been ■■ Synaptopathy has long-lasting effects that can be detrimental for motor and reported to be compromised in both demyelinated and cognitive functions nondemyelinated hippocampi.9 Recently, components ■ In MS and EAE, neuroinflammation alters the balance between the GABAergic ■ of the complement system—C1q and C3—have been and glutamatergic systems in the brain and spinal cord identified as mediators of synapse elimination in the ■■ Proinflammatory cytokines released during acute MS attacks increase 21 glutamate-mediated synaptic transmission and reduce γ-aminobutyric acid- hippocampus in patients with MS, suggesting a direct mediated synaptic signalling, resulting in unbalanced synaptic hyperexcitation link between inflammation and synaptopathy in MS. and possibly also to neurodegeneration In cortical demyelinating lesions of patients with ■■ Targeting of mechanisms that stabilize, protect, repair or help regenerate relapsing–remitting MS (RRMS) or secondary pro- synapses would enable clinical intervention at both early and late stages of gressive MS (SPMS), the loss of both synaptophysin the disease —a presynaptic protein involved in synaptic vesicle release—and glial excitatory amino acid transporters (EAATs; mostly involved in glutamate transport) cor- Box 1 | Glossary relates with infiltration of the CNS by activated microg- Synaptopathy lia.22 A substantial reduction in synaptophysin was also Synaptopathy refers to pathological alterations of synaptic structure and function, found independent of local demyelination in the neo- and has been implicated in many CNS disorders, including Alzheimer disease, cortex.8 Together, these studies provide new insights Huntington disease, schizophrenia, autism and—recently—multiple sclerosis. into synaptic pathology in MS (Figure 1), suggesting Synaptic loss that the inflammatory microenvironment in the CNS is Loss of synapses—the structural connections between neurons—can happen involved in reducing the number of synapses, which can in both healthy and pathological conditions. During development or learning take place independently of grey matter demyelination and memory consolidation, surplus synapses are removed (synaptic pruning), but synapses can also be lost as a result of neuronal injury (synaptic stripping) and neuronal loss. caused by, for example, misfolded proteins and excitotoxic insults. Glutamate excitotoxicity Studies in EAE animal models Dysregulation of glutamate signalling, including sustained activation of EAE models of MS can be induced in several rodent ionotropic glutamate receptors or reduced glutamate uptake, impairs cellulal species and strains with immunization protocols involv- calcium homeostasis and activates nitric oxide synthesis, leading to free radical ing injections of antigenic material against myelin pro- generation and cell death. teins. EAE can reproduce several pathological features of Excitatory postsynaptic currents (EPSCs) MS, and despite some limitations (the whole spectrum Binding of glutamate to the postsynaptic glutamate-gated ion channels triggers of MS symptoms and pathology cannot be covered in an EPSC—a temporary influx of positive ions into the postsynaptic neuron—which a single EAE model, or even in several different EAE results in an excitatory postsynaptic potential (EPSP). Spatial and temporal models), it is an established model to study the immune accumulation of EPSPs at the postsynaptic neuron increases the likelihood of 18 the neuron firing an action potential. alterations that are typical of human MS, and also some of the neuropathological characteristics of this Inhibitory postsynaptic currents (IPSCs) disease.23,24 Assessment of synaptic changes in different Release of γ‑aminobutyric acid (GABA) into the synaptic cleft induces an IPSC —a flux of negative ions—either through the GABA-gated channels localized at the stages of the disease is easier and more controllable in postsynaptic membrane or through a shunt of cell input resistance. IPSCs result rodent models of EAE than in patients. in an inhibitory postsynaptic potential (IPSP) that prevents the membrane from The development of motor symptoms in EAE is reaching the threshold to fire an action potential. dependent on the infiltration of activated mononuclear cells into the CNS from the periphery,18 an event that is accompanied by microglial activation, astrogliosis, Loss of synapses in MS and EAE and abundant proinflammatory cytokine production, Histochemical and biochemical analyses, as well as demyelination and macroscopic plaque formation. genetic profiling of postmortem brains, have demon- In EAE models, structural synaptic alterations have strated synaptic loss (Box 1) in patients with MS and in been detected in several areas of the CNS, including the rodents with EAE. The inflammatory microenvironment spinal cord, hippocampus, cerebellum, striatum and seems to directly influence the structural modifications cortex. In spinal cord tissue derived from relapsing– and loss of the presynatic and postsynaptic elements. remitting EAE rats25,26 or mice,27 immunohistochemical and ultrastructural analysis reveal a pronounced plastic Studies in patients with MS retraction of both dendrites and synaptic terminals of In autopsy study of patients with progressive MS, the spinal motor neurons during disease exacerbation, and demyelinated hippocampi showed decreased levels this loss of synapses correlates with astrogliosis.25–27 of proteins that are crucial to synaptic maintenance Similarly to motor deficits, most of these synaptic (neurexin–neuroligin complex) and synaptic function alterations have the potential for rapid and spontaneous

a Healthy synapse Synaptophysin Synaptotagmin Glutamatergic Neurexin terminal Neuroligin

Na+ Microglia CASK Astroglia PSD-95

Ca2+ Glutamate GABA α TNF β IL-1β

GABAergic NMDA Na+ terminal Ca2+ AMPA mGluR Postsynaptic site Glutamine synthetase sEPSC Glutamine dehydrogenase Glutaminase – Cl EAAT sIPSC GABAR

b Synaptic dysfunction α Activated c Synaptic loss β T lymphocytes α Glutamatergic β terminal Activated Glutamatergic Activated + astroglia Na microglia terminal β β β Ca2+ β Ca2+ β α α α α α β β β α α α α β α β α α α β α β α β α β α β GABAergic Na+ terminal GABAergic Ca2+ terminal

α Postsynaptic Postsynaptic site β site sEPSC

Cl– sIPSC Figure 1 | Inflammatory synaptopathy in MS and EAE. Perturbations of the presynaptic and postsynaptic cellular and molecular machineries can profoundly affect synaptic transmission; moreover, altered neurotransmissionNature Reviews can induce | Neurology a maladaptive response in the CNS microenvironment. a | In the healthy synapse, the concentrations of excitatory (glutamate) and inhibitory (GABA) neurotransmitters at the synaptic cleft are modulated by strict control of transmitter release, degradation or reuptake. b | Proinflammatory cytokines released by autoreactive lymphocytes and activated microglia induce synaptic dysfunction in both glutamatergic and GABAergic systems in MS and EAE. c | Furthermore, inflammation can induce structural alterations, comprising synaptic loss characterized by degeneration of the presynaptic and/or postsynaptic site and alterations of the glial compartment. Representative postsynaptic electrophysiological traces (sEPSC and sIPSC) are reported in each panel. Abbreviations: AMPA, α‑amino‑3-hydroxy‑5-methyl‑4-isoxazolepropionic acid; CASK, Ca2+-calmodulin-dependent protein kinase II; EAAT, excitatory amino acid transporter; EAE, experimental autoimmune encephalomyelitis; GABA, γ‑aminobutyric acid; GABAR, GABA receptor; MS, multiple sclerosis; mGluR, metabotropic glutamate receptor; NMDAR, N‑methyl‑d-aspartate receptor; PSD‑95, postsynaptic density protein 95; sEPSC, spontaneous excitatory postsynaptic current; sIPSC, spontaneous inhibitory synaptic current; TNF, tumour necrosis factor.

recovery that, interestingly, is independent of the processes,27 and probably presenting an impediment for much slower remyelination process.26 Of note, during the re-establishment of neural connections.28 the remission phases, the ratio between excitatory In the striatum of EAE mice, neuroinflammation was and inhibitory inputs increases, indicating the poten- found to result in a marked reduction in the spine density tial for development of excitotoxic neurodegenerative on striatal medium spiny neurons (MSNs), whereas the

Table 1 | Potential pharmaceutical MS treatments that target the glutamatergic system MS type Drugs Trial type and number of Dose/ Effects patients administration Relapsing– Amantadine (weak Double-blind multicentre 10 mg twice daily Reduced relapse rate remitting NMDAR antagonist) study; 53 patients77 (oral) All types Memantine (NMDAR Double-blind, randomized, 10 mg twice daily No improvement in cognitive blocker) placebo-controlled trial; (oral) performance 114 patients146 Primary Riluzole (sodium channel Pilot study; 16 patients80 50 mg twice daily Beneficial effect on lesion evolution progressive blocker, kainate and (oral) and axonal loss, but no clear effect NMDAR antagonist) on formation of new lesions

Abbreviations: MS, multiple sclerosis; NMDAR, N‑methyl‑d-aspartate receptor.

numbers of primary dendrites and cell somata were at the levels of synthesis, release, degradation and reup- unaffected.29 Of note, the loss of spines could be prevented take. In both MS and EAE, the molecular machinery by in vivo treatment with a selective inhibitor for iono- involved in these processes is perturbed, and the expres- tropic α‑amino‑3-hydroxy‑5-methyl‑4-isoxazolepropionic sion of receptors involved in neurotransmission is altered glutamate receptors (AMPARs; discussed below).29 (Figure 1 and Supplementary Tables 1–3 online). Besides spine loss, apoptotic-like events at the synaptic level were detected in striatal synaptosomal preparations, The glutamatergic system in MS and EAE as indicated by increased labelling of annexin A5 (a mol- Abnormal accumulation of glutamate in the synaptic ecule that has a high specificity and affinity for phospho- cleft, as a result of increased release and/or deficient reup- lipid phosphatidylserine, which is exposed on the cell take of glutamate into astrocytes, causes excessive stimu- surface during the early stages of apoptosis).29 The fact lation of glutamate receptors (GluRs) and, consequently, that synapses begin to show this sign of apoptosis in the excitotoxic damage of neurons and oligodendrocytes. acute phase of EAE, even in the absence of overt neuronal In MS and EAE, glutamate excitotoxicity (Box 1) is an loss,29 supports the idea that synapses are early targets of important link between neuroinflammation and neuro apoptotic events in this disease, because they are highly degeneration.41–46 Accumulating evidence points to sensitive to the subtle inflammation-induced changes in increased availability of glutamate in both MS and EAE cellular homeostasis. Moreover, in the sensorimotor cortex (Figure 1 and Supplementary Tables 1 and 2 online). of EAE rats, reductions in spine density and dendritic Despite a few controversial results,47–51 probably reflect- length were observed to correlate with the inflammatory ing differences in technique sensitivity, sampling, and burden.24 In the hippocampi of EAE mice, expression MS or EAE phenotypes, high levels of glutamate have of certain synaptic proteins (synapsin 1, a modulator of been detected in the brains of EAE model animals,52–54 neurotransmitter release, and PSD‑95, a scaffold protein and in the cerebrospinal fluid (CSF), white matter that binds to N‑methyl‑d-aspartate receptors [NMDARs] and grey matter of patients with MS (Supplementary and neuroligin) and the number of presynaptic terminals Table 1 online).55–59 in the CA1 region were substantially reduced.30–32 Several processes that depend on the interplay between Finally, a decrease in the density of inhibitory presyn- immune and nervous systems are involved in the aptic terminals has been repeatedly described in different observed increase in glutamate concentration. Besides brain areas of EAE models. For example, in the cerebellum, neurons, T cells, macrophages, astroglia, and activated the density of basket and stellate inputs that impinge on microglia all have the potential to synthesize and release Purkinje cells is markedly reduced,33 and in the striatum34 glutamate,60 providing a continuous local supply of this and the primary motor cortex,35 the numbers of synap- neurotransmitter. The metabolizing enzymes glutamate tic terminals detected by the vesicular GABA transporter dehydrogenase and glutamine synthetase are down- (VGAT) marker are reduced. These events occur in the regulated in the EAE brain53,61 and in oligodendrocytes presence of chronic microglial activation and infiltrating of patients with MS,54 whereas the glutaminase enzyme, blood-borne immune cells,30,32,34,36,37 and in parallel with which is responsible for glutamate synthesis, is upregu- selective degeneration of a subpopulation of GABAergic lated in EAE and active MS lesions (Supplementary interneurons that are detected mainly by the presence Tables 1 and 2 online).54 Impaired glutamate uptake of the calcium-binding protein parvalbumin.30,32,34–37 Of from the synaptic cleft also contributes to impaired glu- note, parvalbumin-positive interneurons seem to be par- tamate homeostasis. Activated microglia generate reac- ticularly susceptible to degeneration not only in EAE, but tive oxygen and nitrogen species and proinflammatory also in several brain areas (including the cortex, thalamus, cytokines, all of which suppress the expression of amygdala and hippocampus) in patients with MS.38–40 glutamate transporters (GluTs) that are involved in glutamate uptake.45 Indeed, the levels of neuronal and glial Dysregulation of neurotransmitters glutamate transporters (EAAT1, EAAT2 and EAAT3) The concentrations of glutamate, GABA and other are downregulated in both EAE36,62–64,52 and MS.22,41,54 neurotransmitters at the synaptic cleft are modulated Seemingly contradictory results—enhanced glutamate

Table 2 | EAE studies evaluating pharmaceutical treatments that target the glutamatergic system Drug EAE type and Administration Effects model animal NBQX (AMPAR blocker) MBP SJL/J mice Prophylactic (SC) Ameliorates disease Increases oligodendrocyte survival Neuroprotective in the spinal cord41 NBQX, fanapanel, talampanel, MBP Lewis rats Prophylactic and Ameliorates disease GYKI52466 (AMPAR antagonists) therapeutic (IP) Neuroprotective in the spinal cord75,147 Tag‑G-Gpep (peptide against MOG C57 mice Therapeutic Ameliorates disease GluR2–GAPDH complex) (daily IP) Neuroprotective in the spinal cord72 Dizocilpine, memantine (NMDAR Sch Lewis rats Prophylactic and Ameliorates disease blockers), therapeutic (IP) Reduced lesion development (only observed with prophylactic administration)148 Reduced blood–brain barrier breakdown149 Memantine MBP Lewis rats Therapeutic (IP) Ameliorates disease No effect on inflammation150 Amantadine (weak NMDAR blocker Sch Lewis rats Prophylactic (IP) Ameliorates disease and glutamatergic and dopamine agonist),memantine defects74,151,152 Dizocilpine MOG C57 mice Prophylactic (ICV Ameliorates disease osmotic minipump) Restores synaptic function101 NBQX–GPE (combined MOG C57 mice Therapeutic (50% IP Ameliorates disease AMPAR + NMDAR antagonist) and 50% IV) Promotes CNS repair when combined with anti-inflammatory treatment153,154 LY367385 (mGluR1 antagonist), Sch Lewis rats Prophylactic (IP) No effect on disease74,151,152 MPEP (mGluR5 antagonist) Riluzole (Na+ channel blocker, MOG C57 mice Prophylactic and Ameliorates disease kainate and NMDAR antagonist) therapeutic (IP) Reduced inflammation, demyelination and axonal damage78 Carbenoxolone (gap junction blocker) MOG C57 mice Prophylactic (IP) Attenuates symptoms79 6-diazo‑5-oxo‑l-norleucine (glutaminase inhibitor) Aniracetam (AMPAR agonist) PLP Biozzi mice Prophylactic (IP) Exacerbates disease75

l-AP4 (mGluR4 agonist) MBP Lewis rat Prophylactic SC Increases recovery rate155 osmotic minipump

Abbreviations: AMPAR, α‑amino‑3-hydroxy‑5-methyl‑4-isoxazolepropionic acid receptor; EAE, experimental autoimmune encephalomyelitis, GAPDH, glyceraldehyde‑3-phosphate dehydrogenase; GPE, glycine–proline–glutamic acid; ICV, intracerebroventricular; IP, intraperitoneal; IV, intravenous; MBP, myelin basic protein; mGluR, metabotropic glutamate receptor; MOG, myelin oligodendrocyte glycoprotein; NMDAR, N‑methyl‑d-aspartate receptor; PLP, proteolipid protein; PPMS, primary progressive MS; RRMS, relapsing–remitting MS; SC, subcutaneous; Sch, spinal cord homogenate.

uptake activity and increased expression of GluT immune cells (T cells, B cells, antigen-presenting cells, mRNA—have been reported in studies assessing the macrophages, and activated microglia).76 Glutamate optic nerves and spinal cords of patients with MS65–67 and might also have an essential role in MS, not only through of EAE rodents.63,68 These glutamatergic alterations have excitotoxic mechanisms but also by enhancing the pro- been interpreted as an adaptive response to enhanced liferation of autoreactive T cells in response to myelin release of the neurotransmitter from neurons and proteins, as reported in one study involving 14 patients astrocytes in an attempt to limit excitotoxicity.65–67 with MS.76 In both MS67,69–72 and EAE,29,43,52,62,70,73,74 a perma- It should be noted that pharmacological blockade of nent increase in proinflammatory cytokine levels and GluRs has limited clinical use, because GluRs have a vital increased availability of glutamate can upregulate role in maintaining normal synaptic transmission, and neuronal GluR expression and exacerbate synaptic total blockade of ionotropic GluRs (iGluRs) could lead dysfunction, thereby reinforcing the local glutamate to numerous adverse effects. Investigators are, therefore, excitotoxicity (Figure 1 and Supplementary Tables 1 and currently focusing on preventing excessive activation of 2 online). Because this mechanism has been proposed iGluRs or metabotropic GluRs (mGluRs; Tables 1 and 2). as a major determinant of the neurodegeneration in MS Only a few positive human studies have been completed and EAE, drugs that regulate the function and expression so far, but treatment of MS patients with amantadine of GluRs might have a protective effect against excito- (a weak NMDAR antagonist) was shown to reduce toxic cell death (Tables 1 and 2). Indeed, pharmacologi- the relapse rate60 and improve pendular nystagmus cal treatment with GluR antagonists has been shown to (Table 2).77 ameliorate motor deficits and neuropathology in murine Agents that inhibit glutamate release from neural EAE models.41,45,75 The beneficial effect of GluR antago- and immune cells have been reported to have beneficial nist treatment might also be mediated by the GluRs in effects in EAE (Table 2),78,79 and suppression of glutamate

Table 3 | EAE studies evaluating pharmaceutical treatments that target the GABAergic system Drugs EAE model Administration Effects

Exogenous GABA MOG35–55 C57BL/6 mice IP prophylactic Increases disease severity Enhances MOG-dependent proliferation Increases TNF and IL-6 in spinal cord94

94 Vigabatrin (GABA-transaminase MOG35–55 C57BL/6 mice IP prophylactic Protects from disease development inhibitor, enhances endogenous GABA)

94 Vigabatrin MOG35–55 C57BL/6 mice IP therapeutic Ameliorates disease severity Vigabatrin PLP SJL/J mice Oral prophylactic Protection or amelioration of disease Topiramate development via inhibition of inflammation92 Vigabatrin PLP SJL/J mice Oral therapeutic Ameliorates disease severity92

95 Phenobarbitone sodium (GABAA Immunization with spinal cord IP prophylactic Ameliorates disease severity receptor allosteric modulator) homogenate from Wistar rats

and sodium valproate (GABAA level enhancer)

95 Diazepam (GABAA receptor Immunization with spinal cord IP prophylactic No effect agonist) homogenate from Wistar rats Diazepam Intradermal inoculation of IP prophylactic/ Ameliorates disease severity96 bovine MBP in Wistar rats therapeutic

Abbreviations: GABA, γ‑aminobutyric acid; IP, intraperitoneal; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; TNF, tumour necrosis factor.

release from immune cells has also shown promising ameliorate the motor symptoms in EAE rats,95 suggest- results in patients with MS. (Table 1)80 Thus, modula- ing that neuronal and glial susceptibility to injury can be tion of extracellular glutamate levels seems to be one reduced by local modulation of GABA levels in the CNS. of the most promising therapeutic strategies to prevent Mixed results have been obtained in EAE studies evalu

neurodegeneration in MS. ating the effects of treatments with GABAA receptor agonists, such as diazepam and phenobarbitone sodium,95,96 The GABAergic system in MS and in EAE indicating that further studies are required. In patients Accumulating evidence points to dysregulation of the with MS, GABA agonists can improve MS symptoms, GABAergic system in both MS and EAE (Supplementary such as spasticity and nystagmus (Table 4).97–100 Table 3 online). Reduced GABA levels have been detected in the CSF of patients with MS,49,81 as well as in Inflammatory synaptic dysfunction the spinal cord and cerebral cortex of EAE rodents,50,52,82 The mechanisms through which the myelin-targeting though a few contrasting results have been published.83,84 autoimmune reaction results in abnormal synaptic The studies reporting reduced GABA in the CSF and transmission are not fully understood. However, brain- CNS are in line with earlier findings of decreased GABA infiltrating autoreactive T cells and resident CNS immune levels in the blood of patients with MS,81,85 and with cells—such as microglia and astroglia, which show more-recent transcriptomic and proteomic analyses pronounced activation in MS and EAE29,32,33,36,37,101,102 showing that the molecular machinery that controls —are known to extensively modulate synaptic transmis- GABA levels is dysregulated in patients with MS71,82,86–90 sion. Like infiltrating T cells, microglia and astroglia are and in EAE.71,82,90,91 crucial sensors of the local microenvironment, and can Along with its neurotransmitter function, GABA is secrete substantial quantities of cytokines, growth factors an important modulator of immunity,92,93 affecting a and/or neurotransmitters. Therefore, these cells are con- variety of immune cell functions, including cytokine sidered to be potent and far-reaching regulators of the release, cell proliferation, and phagocytic activity.92 The extended neuron–glia network.103 Activated microglia can role of GABA in the regulation of immune system func- also influence synaptic structure and function by shed- tion has hampered our understanding of its action as a ding microvesicles,104 or by initiating synaptic stripping neuroprotective and antiexcitotoxic molecule, and could after an inflammatory insult (Box 1 and Supplementary perhaps partly explain why GABAergic pharmacological Table 4 online).105 treatment in EAE models has had seemingly contradic- Recent studies have shown that immune cells can tory effects: exogenous GABA administration exacerbates trigger synaptic alterations similar to those seen in EAE. disease severity, probably because it increases immune Incubation of brain slices derived from healthy mice with cell activity.92,94 By sharp contrast, enhancement of endo CD3+ T cells isolated from the spleens of EAE mice was genous GABA activity by systemic treatment with the found to alter glutamatergic and GABAergic synaptic GABA transaminase inhibitor vigabatrin has beneficial transmission.29,36,37 Remarkably, the synaptic alterations effects on EAE symptoms (Table 3).93,94 Sodium valpro- observed in striatal, hippocampal and cerebellar slices ate, an enhancer of GABA levels, has also been shown to incubated with EAE CD3+ T cells were reminiscent of the

Table 4 | Potential pharmaceutical MS treatments that target the GABAergic system slices to replicate EAE-like alterations (Supplementary Table 4 online). MS type Drug Trial type and Dose/ Effects number of patients administration In MS, two approaches are typically applied to reveal inflammation-dependent modulation of synaptic func- PPMS Gabapentin Prospective blind 1.2 g daily (oral) Improves and SPMS crossover trial; acquired tion: correlation of cortical excitability and plasticity—as 11 patients nystagmus98 measured by transcranial magnetic stimulation (TMS)— 32 RRMS Gabapentin Single-blind 1.2 g daily (oral) Improves with levels of cytokines in the CSF, or exploration of the crossover trial; acquired effects of cytokines in patient-derived CSF on synaptic eight patients nystagmus99 transmission in brain slices from healthy rodents.110 CPMS Gabapentin Prospective, double- 0.3–0.9 g three Improves blinded, placebo- times daily (oral) spasticity100 Glutamatergic and GABAergic transmission in EAE controlled crossover Glutamatergic transmission in the striatum trial; 21 patients An early electrophysiological study conducted in a RRMS Gabapentin Open-label trial; 0.6 g daily (oral) Reduces painful myelin oligodendrocyte glycoprotein peptide 33–55 and SPMS 24 patients spasms156 (MOG33–35) EAE mouse model (Supplementary Table 4 RRMS Vigabatrin Single-blind 2 g daily (oral) No effect on online) demonstrated dramatic changes in glutamate- crossover trial; acquired 29 eight patients nystagmus99 mediated transmission in MSNs. Of note, these alterations started in the presymptomatic phase of the disease RRMS, Baclofen Doubleblind, 25–50 μg injected No effect on gait SPMS placebo-controlled into the lumbar and postural (7–9 days post injection [dpi]) before the appearance of and PPMS crossover trial; subarachnoid stability157 motor deficits, and were still present at the peak of the 14 patients space acute phase of the disease (20–25 dpi),29 and even in Not Baclofen Nine patients 20–60 mg daily Increases the the late, chronic stages of EAE (50 dpi).111 Furthermore, specified with spasticity (oral) soleus stretch these synaptic abnormalities were seen in regions devoid 158 reflex of overt inflammatory foci, and persisted when micro Not Baclofen 10 patients with 15–60 mg daily Reduces ankle glial activation declined in the late phase of EAE.29 These specified spasticity in the (oral) joint stiffness159 lower extremities findings suggest that the synaptic abnormalities represent virtually irreversible alterations, triggered by inflam- Abbreviations: CPMS, chronic progressive MS; GABA, γ‑aminobutyric acid; MS, multiple sclerosis; PPMS, primary progressive multiple sclerosis; RRMS, relapsing–remitting MS; SPMS, secondary progressive MS. mation but persisting after its resolution, resulting in a chronic synaptic insult that ultimately leads to neuronal excitotoxic damage. defects seen in the brains of the EAE mice.29,36,37 Similar Both the presynaptic and postsynaptic compartments synaptic defects characteristic of EAE have been seen in of excitatory synapses are compromised in EAE, result- experiments where brain slices were incubated with acti- ing in increased frequency and enhanced duration of the vated microglia.29,37 Notably, the effects of CD3+ T cells and spontaneous glutamate current (Supplementary Table 4 activated microglia on synaptic function can be almost online).29,101,111 These glutamatergic alterations are in completely reversed by specific cytokine antagonists,29,37 part caused by increased presynaptic NMDAR activa- as described below (Supplementary Table 4 online). tion and increased postsynaptic AMPA receptor expres- Proinflammatory and anti-inflammatory cytokines sion and sensitivity (Supplementary Table 4 online),29,101 have an essential role in the regulation of bidirectional which contribute to degeneration of dendritic spines and communication between glia and neurons, and in the development of motor deficits. Pharmacological block- modulation of synaptic transmission.106–108 Therefore, in ade of AMPA receptors29 and preventive intracerebroven- pathological conditions, the final downstream effects of tricular (ICV) NMDAR blockade101 have been associated cytokines on synaptic transmission and neuronal sur- with reversal of these synaptic deficits and reduced vival depend on the synaptic cytokine concentrations, the EAE severity (Table 2). In line with these findings, EAE balance between proinflammatory and anti-inflammatory mice with genetically enhanced NMDAR signalling cytokines, and the subcellular expression of their receptors showed exacerbated synaptic defects (Supplementary in specific neuronal compartments (Figure 1).109 Table 4 online).101 Neuroinflammation can affect synaptic transmission A concurrent mechanism that is responsible for at different levels, with a tremendous impact on synap- enhanced synaptic release of glutamate in the EAE stri tic excitability and, therefore, neuronal function.106–108 atum involves a reversal in the direction of the axonal Several characteristics of the spontaneous excitatory and Na+/Ca2+ cotransporter.111 This reversal alters the intra- inhibitory postsynaptic currents (sEPSCs and sIPSCs; axonal Na+/Ca2+ balance so that Ca2+ levels exceed those Box 1) recorded from neurons are affected by inflamma- of Na+, and is likely to be secondary to focal axonal tion. In particular, inflammation alters the frequency of demyelination and the associated abnormal functioning presynaptic neurotransmitter release, and the amplitude of voltage-dependent Na+ channels, rather than to diffuse and duration of the postsynaptic currents.29,33,34,36 The inflammation (Supplementary Table 4 online).111 effects of cytokines can be explored via administration Unlike the synaptic alterations triggered by inflamma- of cytokine antagonists to prevent or reverse the synaptic tory cytokines, which affect the postsynaptic compart- deficits in EAE animals or brain slices, or by administra- ment of neurons and are detectable in the early as well tion of a specific cytokine to a healthy animal or brain as in the late phase of EAE,29,111 the alterations caused

by axonal demyelination affect presynaptic transmitter studies reported impairments in basal excitatory synaptic release, and disappear in the chronic phases of EAE, transmission that involved AMPAR-mediated changes when a certain degree of remyelination presumably takes in synaptic currents and faster decay rates of NMDAR- place.111 EAE-related synaptic abnormalities that occur mediated currents.31 Furthermore, altered paired-pulse in other brain structures and affect other neurotrans- facilitation (an indicator of a presynaptic alterations) mitter systems could, therefore, be caused by a com- has been observed.114 Crossmodality correlation analy- bination of diffuse and focal inflammation and axonal sis revealed that deficits in excitatory synaptic trans- demyelination (Supplementary Table 4 online). mission correlated with reductions in trans-synaptic Application of either activated microglia or tumour protein binding partners that are known to modu- necrosis factor (TNF) to control striatal slices increases late excitatory synaptic transmission.114 Contrasting sEPSC duration, thereby mimicking the effects of EAE, results were obtained in another EAE model (one

and making TNF a candidate for the cytokine respon- boost of MOG35–55 immunization in female mice): no sible for the induction of the synaptic deficits seen in the difference was found in the efficacy of glutamatergic striatum of EAE animal models (Supplementary Table 4 transmission in the CA1 area, or in sEPSC amplitudes online).29 Similar effects have been reported following or frequency.37 The effects of proinflammatory cyto- in vivo treatment: ICV administration of the TNF sig- kines on hippocampal glutamatergic transmission are nalling inhibitor etanercept prevented sEPSC altera- still unknown. tions in EAE mice, whereas ICV treatment with TNF in healthy mice altered sEPSC duration in a way that was GABAergic transmission reminiscent of abnormal glutamate transmission in EAE The most established and common GABAergic alteration (Supplementary Table 4 online).112 IL‑1β has also been observed in the cerebellum, hippocampus and striatum implicated in synaptic modulation: it can increase sEPSC in EAE mice with a chronic disease course is a reduction frequency via modulation of transient receptor poten- in the frequency of GABAergic spontaneous synaptic tial vanilloid 1 (TRPV1) channels110 in striatal MSNs, currents (Figure 1 and Supplementary Table 4 online). corroborating a role for IL‑1β in synaptic alterations in The reduced sIPSC frequency typically manifests during EAE and MS. the acute phase of the disease, and strongly correlates with selective loss of GABAergic interneurons in these Glutamatergic transmission in the cerebellum brain areas. Moreover, the amplitude of GABAergic Electrophysiological recordings from Purkinje cells sIPSCs is reduced in the striatum34 and hippocampus,37 33 of MOG33‑35 EAE mice revealed enhanced glutamate but remains unaffected in the cerebellum. transmission during the symptomatic phase of EAE Although TNF does not seems to interfere with (21–25 dpi; Supplementary Table 4 online).36 The GABAergic transmission (at least in the striatum), IL‑1β increased duration of sEPSCs is caused by reduced signalling impairs GABAergic transmission in all three expression and functioning of EAAT1 (also known as studied brain structures, partly resembling the synaptic GLAST), the most abundant GluT expressed by the alterations in EAE.32–34,37,115 In support of these observa- Bergmann astroglia. A direct link between inflammation tions, ICV delivery of IL‑1ra rescues GABAergic function and such synaptic dysregulation has been demonstrated in EAE mice.32,36 in ex vivo and in vivo studies: although TNF did not have any effect on cerebellar synaptic transmission, IL‑1β— Inflammation-dependent neurotransmission in MS released by a subset of activated microglia and macro- Cortical excitability in patients with MS phages and infiltrating T cells—enhanced glutamatergic Cortical excitability and plasticity in MS can be assessed transmission.36 Brief incubation of normal cerebellar by measuring the response to different TMS protocols slices with IL‑1β replicated EAE modifications through that target the motor cortex.116 TMS techniques, such as rapid EAAT1 downregulation, whereas treatment with paired-pulse and twin-coil TMS, enable measurement of the IL‑1β receptor antagonist (IL-1ra) blocked sEPSC cortical neuronal populations that are functionally con- alterations, and normalized EAAT1 expression.36 nected to pyramidal cells. With these techniques, disability in MS has been shown to correlate with reduced Glutamatergic transmission in the hippocampus measures of short-interval intracortical inhibition,117,118 Neuroimaging studies suggest that hippocampal pathol- and with reduced transcallosal inhibitory and facilitatory ogy is involved in MS‑associated memory defects,48 and connectivity.119–121 Furthermore, intracortical facilita- a correlation with CNS glutamate levels and memory tion, a TMS measure of glutamate transmission, corre- has recently been observed in patients with the disease.48 lates with a high IL‑1β–IL-1ra ratio in the CSF of MS Hippocampal neurodegeneration and resulting spatial patients with active lesions,110 suggesting a link between learning impairment has been described to occur in inflammation and excitotoxicity in MS. During a relapse, 30 MOG33–35 EAE mice; moreover, synaptic dysfunc- patients with MS show decreased short-interval intra tion was reported in the CA1, which is the main output cortical inhibition,122 which is known to be regulated 123 124 of the hippocampus to the cortex (Supplementary by both NMDAR and GABAA receptors. Ex vivo Table 4 online).31,37,113,114 experiments have demonstrated that the reduced

In support of a role for alterations to the hippocampal GABAA-mediated neuronal inhibition depends on IL‑1β glutamatergic system in EAE, earlier electrophysiological (see below).125

In ammation

Reparative process

In ammation

Reparative process

In ammation

Reparative process

Lymphocyte Macrophage Microglia

Figure 2 | Demyelination, axonal loss and synaptopathy characterize multiple sclerosis and experimentalNature Reviews autoimmune | Neurology encephalomyelitis pathophysiology. Inflammation driven by lymphocytes triggers a chain (red arrows) of inflammatory events (microglial and macrophage activation), leading to demyelination (1), axon transection (2) and synaptopathy (3) in the CNS. Axons that remain demyelinated for long periods of time (1) undergo maladaptive processes, such as redistribution of ion channels, which promote chronic neurodegeneration. Similarly, axonal loss (2) is usually not reversible and leads to neuronal death. Long-lasting but potentially reversible synaptic dysfunction (3) in the presence of chronic microglial activation can lead to excitotoxic damage and subsequent neurodegeneration.

Effects of MS CSF on synaptic transmission progressive forms of MS. Moreoever, CSF from patients Ex vivo studies in murine striatal slices incubated in CSF with MS who had acute brain lesions can impair derived from MS patients who had active brain lesions on GABAergic transmission in murine striatal slices.125 IL‑1β MRI support a role for cytokines in unbalanced synaptic is likely to be responsible for this effect, because it could hyperexcitation and, possibly, excitotoxic neurodegenera- be blocked by IL‑1ra incubation, and application of exo tion (Supplementary Table 4 online).110,125,126 As in EAE genous IL‑1β mimicked the effect of MS CSF on synapses studies, inflammatory cytokines released during acute MS (Supplementary Table 4 online).125 attacks can modulate both glutamatergic and GABAergic synaptic transmission. In particular, murine striatal slices The synapse as an MS drug target incubated with MS CSF exhibit a substantial increase in Synaptic dysfunction could be reversible sEPSC frequency and glutamate-mediated neuronal On the basis of the findings discussed above, which point swelling, both of which are markers of excitotoxicity.110 to diffuse synaptopathy (Box 1) as a pathophysiological These effects are dependent on increased IL‑1β signalling hallmark of MS, we suggest that synaptic damage should and AMPAR stimulation, as suggested by rescue experi- also be considered as a target for disease-modifying ments conducted in the presence of specific inhibitors.110 treatments. Such treatments could aim for synaptic In another ex vivo MS‑CSF incubation study of striatal repair and regeneration. This strategy is particularly slices, TNF—which is expressed at higher levels in the appealing, because—unlike loss of neurons—synaptic CSF of patients with progressive MS than in patients with dysfunction and loss of synapses are reversible. Synapses RRMS—was shown to promote glutamatergic transmis- are, in fact, highly dynamic and plastic, such that dys- sion, but by increasing the duration rather than the fre- functional synapses can be repaired and new synapses quency of sEPSCs, leading to neuronal swelling.126 These can be formed, as occurs during synaptic plasticity or data point to TNF as a primary neurotoxic molecule in rehabilitative processes.127

Spine degeneration Synaptic loss Presynaptic terminal Autoreactive degeneration CNS T-lymphocyte Cytokine in ammation inltration release Reduced into the CNS Excitotoxic GABA release damage Impaired Astroglial glutamate uptake and microglial activation Increased Synaptic postsynaptic dysfunction glutamate receptor expression and sensitivity

Enhanced glutamate release Figure 3 | Cascades leading to inflammation-induced excitotoxicity. Inflammatory events (blue) in the CNS trigger structural Nature Reviews | Neurology (orange) and functional (green) alterations to the synaptic compartment. These parallel maladaptive changes lead to synaptic dysfunction and loss of synapses and, consequently, to excitotoxic damage (red). Abbreviation: GABA, γ‑aminobutyric acid.

Potential neuroprotection by current MS drugs Consistent with this hypothesis, laquinimod can regu- Currently, all disease-modifying therapies (DMTs) in MS late synaptic transmission in vitro: direct application of act by restoration of a dysregulated immune response laquinimod to striatal slices results in increased sIPSCs and/or by preventing the migration of autoreactive and reduced sEPSCs.136 T cells into the CNS in an immunomodulatory or immunosuppressive fashion. However, MS DMTs have Effects on microglial activation adverse effects associated with their immunomodula- Laquinimod,140 dimethyl fumarate137,141 and fingoli- tory function, and are all ineffective in patients with mod135 can inhibit microglial activation and thereby progressive MS.128–130 reduce axonal injury in EAE models. Dimethyl fumarate In patients with RRMS, effective treatment with is metabolized to monomethyl fumarate, which can cross immunomodulatory or immunosuppressive DMTs the BBB and has the potential for direct neuroprotective reduces brain atrophy. In fact, treatment with some of effects. A recent study reported that monomethyl fuma- these DMTs is associated with more-pronounced brain rate was able to directly modulate glutamate release from tissue preservation than would be expected from their presynaptic nerve endings, and to affect the synapto- activity on the immune system, raising the hypothesis that toxicity of activated microglia.137 In vitro studies suggest these drugs could have direct neuroprotective effects.131–133 that the modulatory effect of monomethyl fumarate on Fingolimod (or its analogue siponimod), laquinimod, and microglial function and, thereby, its indirect protective dimethyl fumarate are examples of small molecules that effect on glutamatergic transmission is independent of are potentially capable of crossing the BBB and exert- its activity on peripheral immune cells.137 In EAE mice, ing direct neuroprotective effects on the CNS. Clinical oral fingolimod can restore presynaptic and postsynap- trials in progressive forms of MS have been designed to tic alterations of glutamatergic transmission and promote test the supposed neuroprotective effect of these drugs.134 the recovery of dendritic spines,135 probably owing to its Importantly, these drugs have all been demonstrated to capacity to suppress activation of microglia.142 Fingolimod mitigate the effects of inflammation of synaptic transmis- can also protect against excitotoxic insult in cultured cor- sion in rodents135–137 and, at least in the case of laquinimod tical neurons143 and, more interestingly, in patients with and dimethyl fumarate, these protective effects on synaptic RRMS, by reducing glutamate-mediated intracortical transmission were independent of their activity on periph- excitability as measured by paired-pulse TMS.144 eral immune cells.136,137 Both therapeutic and preventive treatment with laquinimod has been shown to ameliorate Conclusions motor disability, reduce CNS inflammation, and increase In the context of grey matter pathology in MS, much the number of myelinated axons in EAE.136,138 attention has been focused on axonal damage and transection. Insights from both clinical and experi- Effects on GABAergic and glutamatergic pathways mental studies, however, are establishing a role for syn- In EAE, laquinimod can notably ameliorate synaptic dys- aptic dysfunction in the development of disability in MS function: the alterations in striatal GABAergic synaptic (Figure 2). First, alterations of the synaptic compart- transmission can be fully reversed by both preventive ment are not only a consequence of axonal or neuro and therapeutic laquinimod treatment, and the kinetic nal damage, but are also part of an ongoing process that alterations of striatal glutamatergic transmission are does not depend on axonal transection (because synaptic improved by preventive treatment.136 Given that laqui- dysfunction can begin early in the MS and EAE disease nimod is a small molecule that freely diffuses across course, and continues throughout the progression of the BBB,139 a direct neuroprotective effect is plausible. the disease). Second, in both MS and EAE, the balance

between glutamatergic and GABAergic transmission is Review criteria perturbed in the brain and spinal cord, and this imbal- Literature for this Review were identified through ance between excitatory and inhibitory transmission searches of PubMed and ProteinQuest (BioDigitalValley; is detrimental for both motor and cognitive functions. a platform for biomedical literature retrieval and analysis, Third, the inflammatory microenvironment is a key which integrates data from scientific literature, biological determinant of diffuse synaptopathy in MS and EAE. images and data repositories from articles, clinical Inflammatory cytokines released during acute MS trials and patents). We searched for articles published in English up to August 2015. The keywords “multiple attacks can both increase glutamate-mediated synaptic sclerosis” and/or “EAE” were used in combination transmission and reduce GABA-mediated synaptic sig- with: “pathogenesis”, “grey matter”, “synaptic loss”, nalling, resulting in synaptic hyperexcitation and, possi- “synaptic dysfunction”, “inhibitory transmission”, “GABA”, bly, excitotoxic neurodegeneration. Last, on the basis that “glutamate”, “glutamate receptor”, “NMDA”, “AMPA”, synaptic dysfunction and loss are reversible, (Figure 3), “pro-inflammatory cytokines”, “IL‑1β”, “TNF”, “TMS” targeting of mechanisms that stabilize and protect, or (single or multiple items from the list). Additional filters were applied for brain regions (hippocampus, cortex, repair and regenerate synapses would enable clinical spinal cord, cerebellum, cortex, striatum) and for drugs interventions at both early and late stages of the disease. related to clinical and preclinical treatments (including A deeper understanding of the fundamental roles of glutamate and GABA antagonist and/or agonist). We also inflammatory and non-inflammatory mechanisms identified articles through searches of the reference lists of synaptic dysfunction and degeneration could have of the articles found with the above-cited search terms, clinical implications not only for MS, but also for other and of the authors’ own files. The final reference list was neurological disorders145 in which an interplay between generated on the basis of originality and relevance to the scope of this Review. synaptopathy and neuroinflammation is implicated.

1. Peterson, J. W., Bö, L., Mörk, S., Chang, A. & multiple sclerosis. Neurology 65, 280–285 autoimmune encephalomyelitis reveals Trapp, B. D. Transected neurites, apoptotic (2005). brain cortex remodeling. J. Neurosci. 35, neurons, and reduced inflammation in cortical 14. Sanfilipo, M. P., Benedict, R. H., Sharma, J., 10088–10100 (2015). multiple sclerosis lesions. Ann. Neurol. 50, Weinstock-Guttman, B. & Bakshi, R. The 25. Zhu, B., Luo, L., Moore, G. R., Paty, D. W. & 389–400 (2001). relationship between whole brain volume and Cynader, M. S. Dendritic and synaptic pathology 2. Compston, A. & Coles, A. Multiple sclerosis. disability in multiple sclerosis: a comparison in experimental autoimmune encephalomyelitis. Lancet 372, 1502–1517 (2008). of normalized gray vs. white matter with Am. J. Pathol. 162, 1639–1650 (2003). 3. Ciccarelli, O. et al. Pathogenesis of multiple misclassification correction. Neuroimage 26, 26. Marques, K. B., Santos, L. M. & Oliveira, A. L. sclerosis: insights from molecular and 1068–1077 (2005). Spinal motoneuron synaptic plasticity during metabolic imaging. Lancet Neurol. 13, 807–822 15. Roosendaal, S. D. et al. Grey matter volume in the course of an animal model of multiple (2014). a large cohort of MS patients: relation to MRI sclerosis. Eur. J. Neurosci. 24, 3053–3062 4. Geurts, J. J. G., Calabrese, M., Fisher, E. & parameters and disability. Mult. Scler. 17, (2006). Rudick, R. A. Measurement and clinical effect 1098–1106 (2011). 27. Freria, C. M., Zanon, R. G., Santos, L. M. & of grey matter pathology in multiple sclerosis. 16. Dutta, R. et al. Demyelination causes synaptic Oliveira, A. L. Major histocompatibility complex Lancet Neurol. 11, 1082–1092 (2012). alterations in hippocampi from multiple class I expression and glial reaction influence 5. Bø, L., Vedeler, C. A., Nyland, H. I., Trapp, B. D. sclerosis patients. Ann. Neurol. 69, 445–454 spinal motoneuron synaptic plasticity during & Mørk, S. J. Subpial demyelination in the (2011). the course of experimental autoimmune cerebral cortex of multiple sclerosis patients. 17. Calabrese, M. et al. Exploring the origins of grey encephalomyelitis. J. Comp. Neurol. 518, J. Neuropathol. Exp. Neurol. 62, 723–732 (2003). matter damage in multiple sclerosis. Nat. Rev. 990–1007 (2010). 6. Kutzelnigg, A. et al. Widespread demyelination Neurosci. 16, 147–158 (2015). 28. Kerschensteiner, M. et al. Remodeling of axonal in the cerebellar cortex in multiple sclerosis. 18. Mix, E., Meyer-Rienecker, H., Hartung, H. P. connections contributes to recovery in an animal Brain Pathol. 17, 38–44 (2007). & Zettl, U. K. Animal models of multiple model of multiple sclerosis. J. Exp. Med. 200, 7. Peterson, J. W., Bö, L., Mörk, S., Chang, A. & sclerosis—potentials and limitations. 1027–1038 (2004). Trapp, B. D. Transected neurites, apoptotic Prog. Neurobiol. 92, 386–404 (2010). 29. Centonze, D. et al. Inflammation triggers neurons, and reduced inflammation in cortical 19. Friese, M. A., Schattling, B. & Fugger, L. synaptic alteration and degeneration in multiple sclerosis lesions. Ann. Neurol. 50, Mechanisms of neurodegeneration and axonal experimental autoimmune encephalomyelitis. 389–400 (2001). dysfunction in multiple sclerosis. Nat. Rev. J. Neurosci. 29, 3442–3452 (2009). 8. Wegner, C., Esiri, M. M., Chance, S. A., Palace, J. Neurol. 10, 225–238 (2014). 30. Ziehn, M. O., Avedisian, A. A., Tiwari-Woodruff, S. & Matthews, P. M. Neocortical neuronal, 20. Ellwardt, E. & Zipp, F. Molecular mechanisms & Voskuhl, R. R. Hippocampal CA1 atrophy and synaptic, and glial loss in multiple sclerosis. linking neuroinflammation and neurodegeneration synaptic loss during experimental autoimmune Neurology 67, 960–967 (2006). in MS. Exp. Neurol. 262, 8–17 (2014). encephalomyelitis, EAE. Lab. Invest. 90, 9. Kooi, E.‑J. et al. Cholinergic imbalance in 21. Michailidou, I. et al. Complement 774–786 (2010). the multiple sclerosis hippocampus. Acta C1q‑C3‑associated synaptic changes in 31. Ziehn, M. O. et al. Therapeutic testosterone Neuropathol. 122, 313–322 (2011). multiple sclerosis hippocampus. Ann. Neurol. administration preserves excitatory synaptic 10. De Stefano, N. et al. Evidence of early cortical 77, 1007–1026 (2015). transmission in the hippocampus during atrophy in MS: relevance to white matter 22. Vercellino, M. et al. Altered glutamate reuptake autoimmune demyelinating disease. J. Neurosci. changes and disability. Neurology 60, in relapsing-remitting and secondary progressive 32, 12312–12324 (2012). 1157–1162 (2003). multiple sclerosis cortex: correlation with 32. Mori, F. et al. Interleukin‑1β promotes long-term 11. Audoin, B. et al. Atrophy mainly affects the limbic microglia infiltration, demyelination, and potentiation in patients with multiple sclerosis. system and the deep grey matter at the first neuronal and synaptic damage. J. Neuropathol. Neuromolecular Med. 16, 38–51 (2013). stage of multiple sclerosis. J. Neurol. Neurosurg. Exp. Neurol. 66, 723–729 (2007). 33. Mandolesi, G. et al. GABAergic signaling and Psychiatry 81, 690–695 (2010). 23. Kornek, B. et al. Multiple sclerosis and chronic connectivity on Purkinje cells are impaired in 12. Calabrese, M. et al. Cortical lesions and autoimmune encephalomyelitis: a comparative experimental autoimmune encephalomyelitis. atrophy associated with cognitive impairment quantitative study of axonal injury in active, Neurobiol. Dis. 46, 414–424 (2012). in relapsing-remitting multiple sclerosis. inactive, and remyelinated lesions. Am. J. Pathol. 34. Rossi, S. et al. Impaired striatal GABA Arch. Neurol. 66, 1144–1150 (2009). 157, 267–276 (2000). transmission in experimental autoimmune 13. Tedeschi, G. et al. Brain atrophy and lesion 24. Tambalo, S. et al. Functional magnetic encephalomyelitis. Brain Behav. Immun. 25, load in a large population of patients with resonance imaging of rats with experimental 947–956 (2011).

35. Falco, A., Pennucci, R., Brambilla, E. & excitotoxic factors in experimental autoimmune receptors in multiple sclerosis. Brain 126, De Curtis, I. Reduction in parvalbumin-positive encephalomyelitis. Neurosci. Lett. 560, 92–97 1755–1766 (2003). interneurons and inhibitory input in the cortex (2014). 70. Fazio, F. et al. Switch in the expression of mGlu1 of mice with experimental autoimmune 53. Castegna, A. et al. Oxidative stress and reduced and mGlu5 metabotropic glutamate receptors in encephalomyelitis. Exp. Brain Res. 232, glutamine synthetase activity in the absence of the cerebellum of mice developing experimental 2439–2449 (2014). inflammation in the cortex of mice with autoimmune encephalomyelitis and in autoptic 36. Mandolesi, G. et al. Interleukin‑1β alters experimental allergic encephalomyelitis. cerebellar samples from patients with multiple glutamate transmission at purkinje cell Neuroscience 185, 97–105 (2011). sclerosis. Neuropharmacology 55, 491–499 synapses in a mouse model of multiple 54. Werner, P., Pitt, D. & Raine, C. S. Multiple (2008). sclerosis. J. Neurosci. 33, 12105–12121 sclerosis: Altered glutamate homeostasis in 71. Han, M. H. et al. Proteomic analysis of active (2013). lesions correlates with oligodendrocyre and multiple sclerosis lesions reveals therapeutic 37. Nisticò, R. et al. Inflammation subverts axonal damage. Ann. Neurol. 50, 169–180 targets. Nature 451, 1076–1081 (2008). hippocampal synaptic plasticity in experimental (2001). 72. Zhai, D. et al. Blocking GluR2-GAPDH multiple sclerosis. PLoS ONE 8, e54666 55. Stover, J. F. et al. Neurotransmitters in ameliorates experimental autoimmune (2013). cerebrospinal fluid reflect pathological encephalomyelitis. Ann. Clin. Transl. Neurol. 2, 38. Clements, R. J., McDonough, J. & Freeman, E. J. activity. Eur. J. Clin. Invest. 27, 1038–1043 388–400 (2015). Distribution of parvalbumin and calretinin (1997). 73. Wheeler, E., Bolton, C., Mullins, J. & Paul, C. immunoreactive interneurons in motor cortex 56. Sarchielli, P., Greco, L., Floridi, A., Floridi, A. & Identification of the NMDA receptor subtype from multiple sclerosis post-mortem tissue. Gallai, V. Excitatory amino acids and multiple involved in the development of disease during Exp. Brain Res. 187, 459–465 (2008). sclerosis: evidence from cerebrospinal fluid. experimental autoimmune encephalomyelitis.

39. Freeman, L. et al. The neuronal component of Arch. Neurol. 60, 1082–1088 (2003). pA2 [online], http://www.pa2online.org/ gray matter damage in multiple sclerosis: a [11C] 57. Srinivasan, R., Sailasuta, N., Hurd, R., Nelson, S. Vol1Issue1abst018P.html (2003). flumazenil positron emission tomography study. & Pelletier, D. Evidence of elevated glutamate in 74. Sulkowski, G., Da˛browska-Bouta, B., Ann. Neurol. 78, 554–567 (2015). multiple sclerosis using magnetic resonance Chalimoniuk, M. & Struz˙yn´ska, L. Effects of 40. Nicholas, R., Magliozzi, R., Campbell, G., spectroscopy at 3 T. Brain 128, 1016–1025 antagonists of glutamate receptors on pro- Mahad, D. & Reynolds, R. Temporal lobe cortical (2005). inflammatory cytokines in the brain cortex of pathology and inhibitory GABA interneuron cell 58. Vrenken, H. et al. MR spectroscopic evidence for rats subjected to experimental autoimmune loss are associated with seizures in multiple glial increase but not for neuro-axonal damage in encephalomyelitis. J. Neuroimmunol. 261, sclerosis. Mult. Scler. http://dx.doi.org/ MS normal-appearing white matter. Magn. Reson. 67–76 (2013). 10.1177/1352458515579445. Med. 53, 256–266 (2005). 75. Groom, A. J., Smith, T. & Turski, L. Multiple 41. Pitt, D., Werner, P. & Raine, C. S. Glutamate 59. Baranzini, S. E. et al. Genetic variation sclerosis and glutamate. Ann. N. Y. Acad. Sci. excitotoxicity in a model of multiple sclerosis. influences glutamate concentrations in brains 993, 229–275 (2003). Nat. Med. 6, 67–70 (2000). of patients with multiple sclerosis. Brain 133, 76. Starck, M., Albrecht, H., Pöllmann, W., 42. Stojanovic, I. R., Kostic, M. & Ljubisavljevic, S. 2603–2611 (2010). Straube, A. & Dieterich, M. Drug therapy for The role of glutamate and its receptors in 60. Ganor, Y. & Levite, M. The neurotransmitter acquired pendular nystagmus in multiple multiple sclerosis. J. Neural Transm. 121, glutamate and human T cells: glutamate sclerosis. J. Neurol. 244, 9–16 (1997). 945–955 (2014). receptors and glutamate-induced direct and 77. Plaut, G. S. Effectiveness of amantadine in 43. Centonze, D. et al. The link between potent effects on normal human T cells, reducing relapses in multiple sclerosis. inflammation, synaptic transmission and cancerous human leukemia and lymphoma J. R. Soc. Med. 80, 91–93 (1987). neurodegeneration in multiple sclerosis. T cells, and autoimmune human T cells. J. Neural 78. Gilgun-Sherki, Y., Panet, H., Melamed, E. & Cell Death Differ. 17, 1083–1091 (2010). Transm. 121, 983–1006 (2014). Offen, D. Riluzole suppresses experimental 44. Azevedo, C. J. et al. In vivo evidence of glutamate 61. Hardin-Pouzet, H. et al. Glutamate metabolism autoimmune encephalomyelitis: Implications for toxicity in multiple sclerosis. Ann. Neurol. 76, is down-regulated in astrocytes during the treatment of multiple sclerosis. Brain Res. 269–278 (2014). experimental allergic encephalomyelitis. Glia 20, 989, 196–204 (2003). 45. Bolton, C. & Paul, C. Glutamate receptors in 79–85 (1997). 79. Shijie, J. et al. Blockade of glutamate release neuroinflammatory demyelinating disease. 62. Olechowski, C. J. et al. A diminished response from microglia attenuates experimental Mediators Inflamm. 2006, 93684 (2006). to formalin stimulation reveals a role for the autoimmune encephalomyelitis in mice. 46. Mehta, A., Prabhakar, M., Kumar, P., glutamate transporters in the altered pain Tohoku J. Exp. Med. 217, 87–92 (2009). Deshmukh, R. & Sharma, P. L. Excitotoxicity: sensitivity of mice with experimental 80. Killestein, J., Kalkers, N. F. & Polman, C. H. bridge to various triggers in neurodegenerative autoimmune encephalomyelitis (EAE). Pain 149, Glutamate inhibition in MS: the neuroprotective disorders. Eur. J. Pharmacol. 698, 6–18 (2013). 565–572 (2010). properties of riluzole. J. Neurol. Sci. 233, 47. MacMillan, E. L. et al. Progressive multiple 63. Ohgoh, M. Altered expression of glutamate 113–115 (2005). sclerosis exhibits decreasing glutamate and transporters in experimental autoimmune 81. Manyam, N. V, Katz, L., Hare, T. A., Gerber, J. C. & glutamine over two years. Mult. Scler. http:// encephalomyelitis. J. Neuroimmunol. 125, Grossman, M. H. Levels of gamma-aminobutyric dx.doi.org/10.1177/1352458515586086. 170–178 (2002). acid in cerebrospinal fluid in various neurologic 48. Muhlert, N. et al. Memory in multiple sclerosis is 64. Mitosek-Szewczyk, K., Sulkowski, G., disorders. Arch. Neurol. 37, 352–355 (1980). linked to glutamate concentration in grey matter Stelmasiak, Z. & Struz˙yn´ska, L. Expression of 82. Gottesfeld, Z., Teitelbaum, D., Webb, C. & regions. J. Neurol. Neurosurg. Psychiatry 85, glutamate transporters GLT‑1 and GLAST in Arnon, R. Changes in the GABA system in 834–840 (2014). different regions of rat brain during the course experimental allergic encephalomyelitis-induced 49. Qureshi, G. A. & Baig, M. S. Quantitation of free of experimental autoimmune encephalomyelitis. paralysis. J. Neurochem. 27, 695–699 (1976). amino acids in biological samples by high- Neuroscience 155, 45–52 (2008). 83. Achar, V. S., Welch, K. M., Chabi, E., Bartosh, K. performance liquid chromatography. Application 65. Vallejo-Illarramendi, A., Domercq, M., Pérez- & Meyer, J. S. Cerebrospinal fluid gamma- of the method in evaluating amino acid levels in Cerdá, F., Ravid, R. & Matute, C. Increased aminobutyric acid in neurologic disease. cerebrospinal fluid and plasma of patients with expression and function of glutamate Neurology 26, 777–780 (1976). multiple sclerosis. J. Chromatogr. 459, 237–244 transporters in multiple sclerosis. Neurobiol. Dis. 84. Zepeda, A. T., Ortiz Nesme, F. J., Méndez- (1988). 21, 154–164 (2006). Franco, J., Otero-Siliceo, E. & 50. Honegger, C. G., Krenger, W. & Langemann, H. 66. Lieury, A. et al. Tissue remodeling in periplaque Pérez de la Mora, M. Free-GABA levels in the Changes in amino acid contents in the spinal regions of multiple sclerosis spinal cord lesions. cerebrospinal fluid of patients suffering from cord and brainstem of rats with experimental Glia 62, 1645–1658 (2014). several neurological diseases Its potential use autoimmune encephalomyelitis. J. Neurochem. 67. Newcombe, J. et al. Glutamate receptor for the diagnosis of diseases which course with 53, 423–427 (1989). expression in multiple sclerosis lesions. Brain inflammation and tissular necrosis. Amino Acids 51. Musgrave, T. et al. The MAO inhibitor phenelzine Pathol. 18, 52–61 (2008). 9, 207–216 (1995). improves functional outcomes in mice with 68. Marte, A. et al. Alterations of glutamate release 85. Demakova, E. V, Korobov, V. P. & Lemkina, L. M. experimental autoimmune encephalomyelitis in the spinal cord of mice with experimental Determination of gamma-aminobutyric acid (EAE). Brain Behav. Immun. 25, 1677–1688 autoimmune encephalomyelitis. J. Neurochem. concentration and activity of glutamate (2011). 115, 343–352 (2010). decarboxylase in blood serum of patients with 52. Kan, Q.‑C., Zhang, S., Xu, Y.‑M., Zhang, G.‑X. & 69. Geurts, J. J. G. et al. Altered expression patterns multiple sclerosis [Russian]. Klin. Lab. Diagn. 4, Zhu, L. Matrine regulates glutamate-related of group I and II metabotropic glutamate 15–17 (2003).

86. Lock, C. et al. Gene-microarray analysis of grey matter pathology in multiple sclerosis. stimulation in patients with central motor multiple sclerosis lesions yields new targets Prog. Neurobiol. 127–128, 1–22 (2015). disorders. Electroencephalogr. Clin. Neurophysiol. validated in autoimmune encephalomyelitis. 103. Wrona, D. Neural-immune interactions: an 81, 243–250 (1991). Nat. Med. 8, 500–508 (2002). integrative view of the bidirectional relationship 123. Schwenkreis, P. et al. Influence of the N‑methyl‑d- 87. Massella, A. et al. Gender effect on between the brain and immune systems. aspartate antagonist memantine on human neurodegeneration and myelin markers in J. Neuroimmunol. 172, 38–58 (2006). motor cortex excitability. Neurosci. Lett. 270, an animal model for multiple sclerosis. BMC 104. Verderio, C. et al. Myeloid microvesicles are 137–140 (1999). Neuroscience 13, 12 (2012). a marker and therapeutic target for 124. Ziemann, U., Lönnecker, S., Steinhoff, B. J. 88. Vanheel, A. et al. Identification of protein neuroinflammation. Ann. Neurol. 72, 610–624 & Paulus, W. Effects of antiepileptic drugs networks involved in the disease course of (2012). on motor cortex excitability in humans: experimental autoimmune encephalomyelitis, an 105. Kettenmann, H., Kirchhoff, F. & Verkhratsky, A. a transcranial magnetic stimulation study. animal model of multiple sclerosis. PLoS ONE 7, Microglia: new roles for the synaptic stripper. Ann. Neurol. 40, 367–378 (1996). e35544 (2012). Neuron 77, 10–18 (2013). 125. Rossi, S. et al. Inflammation inhibits GABA 89. Wang, Y. et al. γ-Aminobutyric acid transporter 1 106. Vitkovic, L., Bockaert, J. & Jacque, C. transmission in multiple sclerosis. Mult. Scler. negatively regulates T cell-mediated immune “Inflammatory” cytokines: neuromodulators 18, 1633–1635 (2012). responses and ameliorates autoimmune in normal brain? J. Neurochem. 74, 457–471 126. Rossi, S. et al. Tumor necrosis factor is elevated inflammation in the CNS. J. Immunol. 181, (2000). in progressive multiple sclerosis and causes 8226–8236 (2008). 107. Bauer, S., Kerr, B. J. & Patterson, P. H. The excitotoxic neurodegeneration. Mult. Scler. 20, 90. Paul, A. M. et al. GABA transport and neuropoietic cytokine family in development, 304–312 (2014). neuroinflammation are coupled in multiple plasticity, disease and injury. Nat. Rev. Neurosci. 127. Morgen, K. et al. Training-dependent plasticity sclerosis: Regulation of the GABA 8, 221–232 (2007). in patients with multiple sclerosis. Brain 127, transporter‑2 by ganaxolone. Neuroscience 273, 108. Schäfers, M. & Sorkin, L. Effect of cytokines 2506–2517 (2004). 24–38 (2014). on neuronal excitability. Neurosci. Lett. 437, 128. Vosoughi, R. & Freedman, M. S. Therapy of MS. 91. Tajouri, L. et al. Quantitative and qualitative 188–193 (2008). Clin. Neurol. Neurosurg. 112, 365–385 (2010). changes in gene expression patterns characterize 109. Steinman, L. Inflammatory cytokines at the 129. Ransohoff, R. M., Hafler, D. A. & Lucchinetti, C. F. the activity of plaques in multiple sclerosis. Brain summits of pathological signal cascades in Multiple sclerosis—a quiet revolution. Nat. Rev. Res. Mol. Brain Res. 119, 170–183 (2003). brain diseases. Sci. Signal. 6, pe3 (2013). Neurol. 11, 134–142 (2015). 92. Bhat, R. et al. Inhibitory role for GABA in 110. Rossi, S. et al. Interleukin‑1β causes synaptic 130. Franklin, R. J., ffrench-Constant, C., Edgar, J. M. & autoimmune inflammation. Proc. Natl Acad. Sci. hyperexcitability in multiple sclerosis. Ann. Smith, K. J. Neuroprotection and repair in multiple USA 107, 2580–2585 (2010). Neurol. 71, 76–83 (2012). sclerosis. Nat. Rev. Neurol. 8, 624–634 (2012). 93. Jin, Z., Mendu, S. K. & Birnir, B. GABA is an 111. Rossi, S. et al. Abnormal activity of the Na/Ca 131. Arnold, D. L. et al. Effects of delayed-release effective immunomodulatory molecule. Amino exchanger enhances glutamate transmission in dimethyl fumarate on MRI measures in the Acids 45, 87–94 (2013). experimental autoimmune encephalomyelitis. Phase 3 DEFINE study. J. Neurol. 261, 94. Carmans, S. et al. Systemic treatment with the Brain Behav. Immun. 24, 1379–1385 (2010). 1794–1802 (2014). inhibitory neurotransmitter gamma-aminobutyric 112. Haji, N. et al. TNF‑α-mediated anxiety in a mouse 132. Filippi, M. et al. Placebo-controlled trial of oral acid aggravates experimental autoimmune model of multiple sclerosis. Exp. Neurol. 237, laquinimod in multiple sclerosis: MRI evidence encephalomyelitis by affecting proinflammatory 296–303 (2012). of an effect on brain tissue damage. J. Neurol. immune responses. J. Neuroimmunol. 255, 113. Weiss, S., Mori, F., Rossi, S. & Centonze, D. Neurosurg. Psychiatry 85, 851–858 (2014). 45–53 (2013). Disability in multiple sclerosis: when synaptic 133. Cohen, J. a. et al. Long-term (up to 4.5 years) 95. Gilani, A. A., Dash, R. P., Jivrajani, M. N., long-term potentiation fails. Neurosci. Biobehav. treatment with fingolimod in multiple sclerosis: Thakur, S. K. & Nivsarkar, M. Evaluation of Rev. 43, 88–99 (2014). results from the extension of the randomised GABAergic transmission modulation as a novel 114. Ziehn, M. O., Avedisian, A. A., Dervin, S. M., TRANSFORMS study. J. Neurol. Neurosurg. functional target for management of multiple O’Dell, T. J. & Voskuhl, R. R. Estriol preserves Psychiatry http://dx.doi.org/10.1136/ sclerosis: Exploring inhibitory effect of GABA synaptic transmission in the hippocampus jnnp-2015-310597. on glutamate-mediated excitotoxicity. during autoimmune demyelinating disease. 134. Ontaneda, D., Fox, R. J. & Chataway, J. Clinical Adv. Pharmacol. Sci. 2014, 632376 (2014). Lab. Invest. 92, 1234–1245 (2012). trials in progressive multiple sclerosis: lessons 96. Bibolini, M. J. et al. Inhibitory role of diazepam 115. Musumeci, G. et al. Transient receptor potential learned and future perspectives. Lancet Neurol. on autoimmune inflammation in rats with vanilloid 1 channels modulate the synaptic 14, 208–223 (2015). experimental autoimmune encephalomyelitis. effects of TNF‑α and of IL‑1β in experimental 135. Rossi, S. et al. Oral fingolimod rescues the Neuroscience 199, 421–428 (2011). autoimmune encephalomyelitis. Neurobiol. Dis. functional deficits of synapses in experimental 97. Rode, G., Maupas, E., Luaute, J., Courtois- 43, 669–677 (2011). autoimmune encephalomyelitis. Br. J. Pharmacol. Jacquin, S. & Boisson, D. Medical treatment 116. Rossini, P. M. et al. Non-invasive electrical and 165, 861–869 (2012). of spasticity [French]. Neurochirurgie 49, magnetic stimulation of the brain, spinal cord 136. Ruffini, F. et al. Laquinimod prevents inflammation- 247–255 (2003). and roots: basic principles and procedures induced synaptic alterations occurring in 98. Starck, M., Albrecht, H., Pöllmann, W., for routine clinical application. Report of an experimental autoimmune encephalomyelitis. Dieterich, M. & Straube, A. Acquired pendular IFCN committee. Electroencephalogr. Clin. Mult. Scler. 19, 1084–1094 (2013). nystagmus in multiple sclerosis: an examiner- Neurophysiol. 91, 79–92 (1994). 137. Parodi, B. et al. Fumarates modulate microglia blind cross-over treatment study of memantine 117. Conte, A. et al. Intracortical excitability in activation through a novel HCAR2 signaling and gabapentin. J. Neurol. 257, 322–327 patients with relapsing-remitting and secondary pathway and rescue synaptic dysregulation in (2010). progressive multiple sclerosis. J. Neurol. 256, inflamed CNS. Acta Neuropathol. 130, 279–295 99. Bandini, F., Castello, E., Mazzella, L., 933–938 (2009). (2015). Mancardi, G. L. & Solaro, C. Gabapentin but 118. Vucic, S. et al. Cortical dysfunction underlies 138. Moore, S. et al. Therapeutic laquinimod not vigabatrin is effective in the treatment of disability in multiple sclerosis. Mult. Scler. 18, treatment decreases inflammation, initiates acquired nystagmus in multiple sclerosis: how 425–432 (2012). axon remyelination, and improves motor deficit valid is the GABAergic hypothesis? J. Neurol. 119. Schmierer, K., Irlbacher, K., Grosse, P., Röricht, S. in a mouse model of multiple sclerosis. Brain Neurosurg. Psychiatry 71, 107–110 (2001). & Meyer, B.‑U. Correlates of disability in multiple Behav. 3, 664–682 (2013). 100. Cutter, N. C., Scott, D. D., Johnson, J. C. & sclerosis detected by transcranial magnetic 139. Brück, W. & Wegner, C. Insight into the Whiteneck, G. Gabapentin effect on spasticity stimulation. Neurology 59, 1218–1224 (2002). mechanism of laquinimod action. J. Neurol. Sci. in multiple sclerosis: a placebo-controlled, 120. Codecà, C. et al. Differential patterns of 306, 173–179 (2011). randomized trial. Arch. Phys. Med. Rehabil. 81, interhemispheric functional disconnection 140. Mishra, M. K. et al. Laquinimod reduces 164–169 (2000). in mild and advanced multiple sclerosis. neuroaxonal injury through inhibiting microglial 101. Grasselli, G. et al. Abnormal NMDA receptor Mult. Scler. 16, 1308–1316 (2010). activation. Ann. Clin. Transl. Neurol. 1, 409–422 function exacerbates experimental autoimmune 121. Mori, F. et al. Short interval intracortical (2014). encephalomyelitis. Br. J. Pharmacol. 168, facilitation correlates with the degree of 141. Wilms, H. et al. Dimethylfumarate inhibits 502–517 (2013). disability in multiple sclerosis. Brain Stimul. 6, microglial and astrocytic inflammation by 102. Mallucci, G., Peruzzotti-Jametti, L., 67–71 (2013). suppressing the synthesis of nitric oxide, IL‑1β, Bernstock, J. D. & Pluchino, S. The role of 122. Caramia, M. D. et al. ’Excitability changes TNF-α and IL‑6 in an in-vitro model of brain immune cells, glia and neurons in white and of muscular responses to magnetic brain inflammation. J. Neuroinflammation 7, 30 (2010).

142. Noda, H., Takeuchi, H., Mizuno, T. & Suzumura, A. 150. Wallström, E. et al. Memantine abrogates treating nocturnal painful spasms in multiple Fingolimod phosphate promotes the neurological deficits, but not CNS inflammation, sclerosis. Mult. Scler. 6, 192–193 (2000). neuroprotective effects of microglia. in Lewis rat experimental autoimmune 157. Ørsnes, G., Crone, C., Krarup, C., Petersen, N. J. Neuroimmunol. 256, 13–18 (2013). encephalomyelitis. J. Neurol. Sci. 137, 89–96 & Nielsen, J. The effect of baclofen on the 143. Di Menna, L. et al. Fingolimod protects cultured (1996). transmission in spinal pathways in spastic cortical neurons against excitotoxic death. 151. Sulkowski, G., Da˛browska-Bouta, B., Salin´ska, E. multiple sclerosis patients. Clin. Neurophysiol. Pharmacol. Res. 67, 1–9 (2013). & Struz˙yn´ska, L. Modulation of glutamate 111, 1372–1379 (2000). 144. Landi, D. et al. Oral fingolimod reduces transport and receptor binding by glutamate 158. Nielsen, J. F., Anderson, J. B. & Sinkjaer, T. glutamate-mediated intracortical excitability receptor antagonists in EAE rat brain. PLoS ONE Baclofen increases the soleus stretch reflex in relapsing-remitting multiple sclerosis. 9, e113954 (2014). threshold in the early swing phase during Clin. Neurophysiol. 126, 165–169 (2015). 152. Sulkowski, G., Da˛browska-Bouta, B., & walking in spastic multiple sclerosis patients. 145. Glass, C. K., Saijo, K., Winner, B., Struz˙yn´ska, L. Modulation of neurological Mult. Scler. 6, 105–114 (2000). Marchetto, M. C. & Gage, H. Mechanisms deficits and expression of glutamate 159. Nielsen, J. F. & Sinkjær, T. Peripheral and central underlying inflammation in neurodegeneration. receptors during experimental autoimmune effect of baclofen on ankle joint stiffness in Cell 140, 918–934 (2010). encephalomyelitis after treatment with multiple sclerosis. Muscle Nerve 23, 98–105 146. Lovera, J. F. et al. Memantine for cognitive selected antagonists of glutamate (2000). impairment in multiple sclerosis: a randomized receptors. Biomed. Res. Int. 2013, 186068 placebo-controlled trial. Mult. Scler. 16, 715–723 (2013). Acknowledgements (2010). 153. Kanwar, J. R., Kanwar, R. K. & Krissansen, G. W. This work was supported by the Italian National 147. Smith, T., Groom, A., Zhu, B. & Turski, L. Simultaneous neuroprotection and blockade of Ministero della Salute (Progetto Giovani Ricercatori) Autoimmune encephalomyelitis ameliorated inflammation reverses autoimmune to G.M. (No. GR‑2011‑02347036) and A.M. (No. by AMPA antagonists. Nat. Med. 6, 62–66 encephalomyelitis. Brain 127, 1313–1331 GR‑2011‑02351422) and by Fondazione Italiana (2000). (2004). Sclerosi Multipla (No. FISM 2010/S/2) to D.C. 148. Bolton, C. & Paul, C. MK‑801 limits neurovascular 154. Kanwar, J. R. Anti-inflammatory immunotherapy Author contributions dysfunction during experimental allergic for multiple sclerosis/experimental autoimmune D.C., G.M., A.G., A.M., D.F., F.D.V., S.B. and H.S. encephalomyelitis. J. Pharmacol. Exp. Ther. 282, encephalomyelitis (EAE) disease. Curr. Med. researched data for the article. D.C. and G.M. wrote 397–402 (1997). Chem. 12, 2947–2962 (2005). the article. D.C., G.M., A.G., A.M. and G.A.M. provided 149. Paul, C. & Bolton, C. Modulation of blood-brain 155. Besong, G. et al. Activation of group III substantial contributions to discussion of content. All barrier dysfunction and neurological deficits metabotropic glutamate receptors inhibits the authors participated in reviewing and/or editing of the during acute experimental allergic production of RANTES in glial cell cultures. manuscript before submission. encephalomyelitis by the N‑methyl‑d-aspartate J. Neurosci. 22, 5403–5411 (2002). receptor antagonist memantine. J. Pharmacol. 156. Solaro, C., Uccelli, M. M., Guglieri, P., Uccelli, A. Supplementary information is linked to the online Exp. Ther. 302, 50–57 (2002). & Mancardi, G. L. Gabapentin is effective in version of the paper at www.nature.com/nrneurol.