Molecular mechanisms linking neuroinflammation and neurodegeneration in MS.

YEXNR-11654; No. of pages: 10; 4C: 2 Experimental Neurology xxx (2014) xxx–xxx

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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

Review

Molecular mechanisms linking neuroinflammation and neurodegeneration in MS Erik Ellwardt, Frauke Zipp ⁎ Focus Program Translational Neurosciences (FTN), Rhine Main Neuroscience Network (rmn2), Department of Neurology, University Medical Center of the Johannes-Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany

a r t i c l e

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Article history: Received 30 October 2013 Revised 31 January 2014 Accepted 7 February 2014 Available online xxxx Keywords: Multiple sclerosis Neuroinflammation Neurodegeneration Mitochondrial dysfunction Channelopathies

a b s t r a c t Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disorder of the central nervous system (CNS) and one of the leading causes of neurological deficits and disability in young adults in western countries. Current medical treatment mainly influences disease progression via immunomodulatory or immunosuppressive actions. Indeed, MS research has been foremost focused on inflammation in the CNS, but more recent evidence suggests that chronic disability in MS is caused by neurodegeneration. Imaging studies show an early involvement of neurodegeneration as brain atrophy and gray matter lesions can be observed at disease onset. Thus, neuroprotective treatment strategies and the elucidation of the molecular mechanisms underlying neurodegeneration in MS have attracted the attention of the scientific community. Experimental autoimmune encephalomyelitis (EAE; the most commonly used animal model for MS), novel in-vivo imaging techniques such as twophoton microscopy and recently discovered molecular changes have offered new insights into the pathogenesis of neuroinflammation as well as neurodegeneration in MS. This review focuses on the interaction between components of the immune system and the neuronal compartment, as well as describing the most important molecular mechanisms that lead to axonal and neuronal degeneration in MS and EAE. © 2014 Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal models for studying pathogenesis of MS: EAE . . . . . . . . . . Cellular mechanisms combining neuroinflammation and neurodegeneration Immune effector mechanisms . . . . . . . . . . . . . . . . . . . CD8 cells . . . . . . . . . . . . . . . . . . . . . . . . . CD4 cells . . . . . . . . . . . . . . . . . . . . . . . . . Macrophages/microglia . . . . . . . . . . . . . . . . . . B-cells and autoantibodies . . . . . . . . . . . . . . . . . Natural killer cells . . . . . . . . . . . . . . . . . . . . . Proposed mechanisms provoking injury of neuronal structures in MS . . . Mitochondrial dysfunction . . . . . . . . . . . . . . . . . . . . Redistribution of sodium channels . . . . . . . . . . . . . . . . . TASK1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of ASIC-1 and TRPM4 . . . . . . . . . . . . . . . . . . . Brain-derived neurotrophic factor (BDNF) . . . . . . . . . . . . . Glutamate excitotoxicity . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Neurology, Focus Program Translational Neuroscience (FTN), Rhine Main Neuroscience Network (rmn2), University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany. Fax: +49 6131 17 5697. E-mail address: [email protected] (F. Zipp).

http://dx.doi.org/10.1016/j.expneurol.2014.02.006 0014-4886/© 2014 Published by Elsevier Inc.

Please cite this article as: Ellwardt, E., Zipp, F., Molecular mechanisms linking neuroinflammation and neurodegeneration in MS, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.02.006

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E. Ellwardt, F. Zipp / Experimental Neurology xxx (2014) xxx–xxx

Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) affecting mainly young people aged between 20 and 40 at disease onset. Initial symptoms are diverse but the most frequent ones are visual disturbances, paresthesias, ataxia and muscle weakness. The typical disease course, occurring in about 85% of patients, is relapsing–remitting (RR)-MS, in which there is full recovery between initial relapses. After a time, patients do not completely recover between relapses and develop secondary progressive (SP)-MS. 15% of patients do not have classical relapses but a primary progressive form of MS (PPMS) (Lublin et al., 1996). MS is the major cause of disability resulting from CNS inflammation in young adults in western countries. Research over the last decades has focused on inflammatory mechanisms leading to demyelination, mainly in the white matter of the brain and the spinal cord. New imaging techniques allow lesions associated with acute and chronic inflammation to be monitored. Magnetic resonance imaging (MRI) is one of the most important tools for the diagnosis and monitoring of the disease course of MS. MRI and also MR spectroscopy have revealed that atrophy of gray matter begins at an early stage of the disease course and can even precede white matter lesions (Chard and Miller, 2009). It is now well accepted that MS is not only an inflammatory disease but also a neurodegenerative pathology that involves axonal transection and neuronal damage (Trapp et al., 1998; Vogt et al., 2009). Neuroinflammation and neurodegeneration have been shown to be coupled not only in MS but in many pathologies affecting the CNS, such as Parkinson's or Alzheimer's diseases (Fuller et al., 2010; Mcgeer et al., 2003; Tansey et al., 2007). The question arises whether inflammatory processes precede neurodegeneration or vice versa. This is

important in terms of prevention or early intervention as there are currently few neurorestorative approaches in the treatment of MS. The etiology of MS still remains poorly understood, although it is known that environmental factors and susceptible genes are involved in disease pathogenesis. It is commonly accepted that MS is an autoimmune disease and that misdirection of the immune system is one of the main effector mechanisms (Herz et al., 2010). Pathological hallmarks include crossing of the blood–brain barrier (BBB) by T cells, activation of autoimmune T cells, demyelination, remyelination, gliosis, and axonal/ neuronal degeneration (Siffrin et al., 2007). Demyelination and degeneration of neurons can be monitored via electrophysiology (e.g., visual evoked potentials) and MRI. Oligoclonal bands, found in the cerebrospinal fluid (CSF) in MS patients, are a correlate for the autochthone production of antibodies. The CNS possesses the capacity for remyelination, which initially contributes to full clinical remission (Chari, 2007; Franklin and FfrenchConstant, 2008), and neuronal injury may even be repaired up to a certain point. However, once an axon is completely transected or the integrity of the neuronal cell body is severely disturbed, repair mechanisms are insufficient (Bellmann-Strobl et al., 2009; Nikic et al., 2011; Siffrin et al., 2010). As the disease progresses, the “lesion load” in the CNS increases and ultimately leads to persistent neurological disability (Frohman et al., 2006). The proposed cause of this neurological damage is an increased loss of axons and neurons following both demyelination and demyelinationindependent mechanisms (Deluca et al., 2006; Minagar et al., 2004). Knowing that neurodegeneration as well as neuroinflammation play a key role in the disease course of MS, attention has shifted towards the development of neuroprotective strategies to ameliorate long-term neurological disability. In this review, we will give an overview of the cells, cytokines and pathways that are thought to be involved in the

Fig. 1. Channels/receptors accounting for intracellular calcium and sodium increase in MS. Demyelination of axons leads to a redistribution of sodium channels to restore electrical nerve conduction. The following intracellular sodium accumulation challenges the Na+/K+ ATPase, which is not able to fully compensate for increasing sodium levels due to an energy failure caused by dysfunctional mitochondria. The Na+/Ca++ exchanger is upregulated and partially stabilizes intracellular sodium concentrations. However, in place of sodium, intracellular calcium accumulates. Furthermore, sensitized NMDA/AMPA receptors, TRPM4 and ASIC1 all contribute to sustained elevated sodium and calcium levels, which precede cell death in demyelinated axons.



crosstalk between neuroinflammation and neurodegeneration and of promising targets for potential therapies. Animal models for studying pathogenesis of MS: EAE Animal models for human diseases provide an excellent opportunity to gain insights into underlying pathomechanisms and disease courses. The most commonly used animal model for MS is experimental autoimmune encephalomyelitis (EAE) (Croxford et al., 2011). Currently, there are two broad methods available to induce EAE (Massacesi et al., 1995). Briefly, in the first (active EAE), mice are immunized subcutaneously with peptides that are normally found in the CNS (e.g., in the myelin sheath). In order to boost the immune system reaction (a prerequisite for developing clinically visible EAE), adjuvants such as Complete Freund's Adjuvant (CFA) and/or pertussis toxin are used. The BBB, which normally impedes migration of autoreactive lymphocytes into the brain tissue, becomes more permeable as a consequence of the immunization. Commercially manufactured kits are available to perform this easy-to-apply method. In the other approach (adoptive transfer, passive EAE), T cells from the spleen and lymph nodes are isolated from mice that have already been sensitized against peptides in the CNS. Those encephalitogenic T cells are differentiated to CD4 + cells by applying specific cytokines and co-culturing with antigen-presenting cells loaded with myelin peptides to stimulate the CD4+ immune cells via major histocompatibility complex (MHC) class II. Once cells are differentiated, they can be transferred to other mouse lines ('T Hart et al., 2011) and may induce EAE. This passive EAE method has the advantage of allowing the application of fluorescence-marked encephalitogenic T cells and induction of EAE in many different transgenic mouse lines, e.g., for imaging purposes. After a period of several days, depending on the mouse strain and method used, mice exhibit typical symptoms such as tail and hind limb paralysis and weight loss. One of the more recently developed techniques to analyze mice is two-photon microscopy. With multiphoton microscopy, cellular interactions and subcellular changes (Cahalan et al., 2002; Niesner et al., 2013) can be monitored in the CNS of living animals, which gives unique information about the pathological processes involved in the disease. FACS analyses of

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encephalitogenic T cells and cytokines, on the other hand, can be performed after processing of the brain tissue. Animal models, especially in MS, are valuable tools for the preliminary screening of potential immunosuppressive and neuroprotective drugs. Further improvement and adjustment of current disease models and new transgenic mouse strains will facilitate the development of new treatment options. Cellular mechanisms combining neuroinflammation and neurodegeneration Immune effector mechanisms CD8 cells In recent work, it has been shown that lymphocytes from the peripheries are able to cross the BBB and invade the CNS, which under normal circumstances is considered to be an “immune-privileged” organ (Alun Brown, 2001; Hickey, 1991; Ransohoff et al., 2003). However, in infectious CNS diseases as well as in autoimmune disorders, lymphocytes migrate via the BBB and combat viral or bacterial antigens. The mechanisms responsible for BBB dysfunction include chemokine and cytokine induced endothelial disruption and upregulation of cell adhesion molecules (Minagar and Alexander, 2003). The activation of T cells occurs normally with the help of antigen presenting cells (APCs). Professional APCs (e.g., dendritic cells, B cells, macrophages) present antigens via MHC class II and activate CD4+ T cells if the appropriate T-cell receptor (TCR) for the exposed antigen is present (Grakoui et al., 1999). CD8+ cells, on the other hand, interact only with MHC class I, which is expressed in almost all nucleated cells (non-professional APCs), including neurons (Neumann et al., 1995). The expression of MHC I, and therefore the presentation of possible antigens to CD8+ cytotoxic T lymphocytes (CTL), can be upregulated by proinflammatory cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α or by suppressing electrical activity, e.g., through sodium channel blockers (Medana et al., 2000; Neumann et al., 1997; Shatz, 2009). Interestingly, MHC class I upregulation was found in all cell types of the CNS in MS lesions (Höftberger et al., 2004), including neurons. Thus, CTL can recognize antigens presented by oligodendrocytes and neurons. Once activated, they may be partly responsible for the

Table 1 Mechanisms leading to neurodegeneration in MS. Different molecular mechanisms that cause neuronal/axonal damage due to an imbalance of energy supply and demand in axons following demyelination during inflammation with T cells and macrophages/microglia. Mitochondrial dysfunction, redistribution of sodium channels, channelopathies, TRAIL and others all contribute to an energy breakdown and an intracellular calcium increase. A sustained calcium increase precedes neuronal death. Involved pathway/key players

Mechanism leading to neurodegeneration

References

TRAIL

Ligand which induces apoptosis, secreted by CD4+ and CD8+ cells

Perforin, granzymes A + B

Secreted by CD8+ (and to a lower degree by CD4+ cells) after antigen recognition via MHC; induces membrane instability and apoptosis Induces apoptosis by binding to Fas ligand; expressed mainly on CD8+ cells and NKTs Different cytokines secreted by CD4+ and CD8+ cells sensitize glutamate (excitotoxic) receptors and increase glutamate excitotoxicity ROS/RNS (and possibly proinflammatory molecules) produced by macrophages/ microglia damage mitochondria To restore nerve conduction, sodium channels are upregulated in formerly myelinated sections; this leads to less effective nerve conduction and increased energy demand; Na+/ K+ ATPase is overstrained and Na+/Ca++ exchanger accounts for calcium influx in response to a sodium efflux Potassium channel on T cells and neurons; blocking of TASK1 leads to less T-cell proliferation and reduced proinflammatory cytokines in EAE; also, axonal and neuronal degeneration was attenuated in MOG35–55 induced EAE BDNF produced by T and B cells in active MS lesions is neuroprotective. Glatiramer acetate increases BDNF production by lymphocytes and therefore contributes to neuroprotection Acid-sensing ion channel is upregulated in active MS/EAE lesions; allows the influx of sodium and calcium; amiloride antagonizes ASIC-1 and ameliorates disease in EAE Transient receptor potential melastatin 4 opens via increased intracellular calcium and decreased ATP concentrations; leads to sodium (and calcium) accumulation; glibenclamide blocks TRPM4 and acts neuroprotectively in EAE Elevated glutamate concentrations have been found in MS lesions; an excess of glutamate leads to axonal calcium increase and can be antagonized via AMPA or NMDA receptor blocking

Aktas et al. (2005), Nitsch et al. (2000) and Vogt et al. (2009) Huse et al. (2008) and Meuth et al. (2009)

Fas TNF-α, IFN-γ, IL-17 and other cytokines Mitochondrial dysfunction Redistribution of sodium channels on axons

TASK1

BDNF ASIC-1 TRPM4

Glutamate excitotoxicity

Giuliani et al. (2003) and Medana et al. (2000) Mizuno et al. (2008) and Neumann et al. (2002) Mahad et al. (2008) and Nikic et al. (2011) Craner et al. (2004), Moll et al. (1991) and Waxman (2006)

Bittner et al. (2009, 2012) and Meuth et al. (2008)

Hohlfeld et al. (2000), Kerschensteiner et al. (1999) and Linker et al. (2010) Friese et al. (2007) and Vergo et al. (2011) Schattling et al. (2012)

Nitsch et al. (2004), Pitt et al. (2000), Siffrin et al. (2010) and Srinivasan et al. (2005)


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demyelination and axonal/neuronal damage (Bitsch et al., 2000; Medana et al., 2000; Meuth et al., 2009) that occur in multiple sclerosis. CD8+ cells are found in the CNS in MS and expand clonally in MS lesions (Babbe et al., 2000; Neumann et al., 2002), a characteristic that has not been shown for CD4+ cells. There is still controversy about the molecular mechanisms by which CTLs harm axons and neurons in autoimmune disorders such as MS. After recognition of a presented antigen by MHC I, CTLs release the proinflammatory cytokines TNF-α and IFN-γ, as well as perforin, granzymes A and B, among others (Huse et al., 2008; Meuth et al., 2009; Mizuno et al., 2008) into the “immunological synapse”. TNF-α, for instance, can trigger cell death via the p55 receptor on neurons and silencing of survival signals (Venters et al., 2000). IFN-γ, on the other hand, increases glutamate neurotoxicity and calcium influx into neurons through binding to the IFN-γ receptor and modulating the IFN-γ/AMPA GluR1 receptor complex, which leads to dendritic-bead formation and consequently to cell death (Mizuno et al., 2008). Perforin and granzymes directly damage the membrane and cause a sodium and calcium influx that ultimately leads to energy breakdown in the cell. The interaction of the Fas-antigen on CD8+ cells with the Fas-ligand (FasL) on neurons is a further mechanism that activates the intracellular caspase cascade and causes axonal/neuronal damage (Giuliani et al., 2003; Medana et al., 2000). CD8+ cells do not only exert disease promoting effects with respect to CNS autoimmune disorders; they also have regulatory functions, as shown by Hu et al. (2004). They showed that the mouse protein Qa-1 (homologous to HLA-E in humans) has important immunoregulatory functions. CD8 + cells seem to restrain highly active CD4 + cells through a Qa-1-dependent pathway, as Qa-1 deficient mice are more susceptible to EAE owing to enhanced CD4+ cell activity. Those regulatory CD8+ T-cells or CD8αα+ Tregs could have an important role in the suppression of autoimmunity and this cell population may be impaired in MS patients (Beeston et al., 2010; Pannemans et al., 2013; Smith and Kumar, 2008). CD4 cells Whether CD8+ or CD4+ cells infiltrating the CNS during MS are the main pathogenic immune T-cell subtype is still under debate. Although CD4 + cells cannot bind to MHC class I molecules, they have a major detrimental impact on neurons under pathological conditions. In perfused ex-vivo brain slices, for instance, CD4 + cells induce antigenindependent calcium oscillations in neurons, which ultimately lead to cell death (Nitsch et al., 2004). In the same experiment, the involvement of glutamate-mediated NMDA/AMPA receptor excitotoxicity and a perforin pathway was suggested, as blockade with Mk-801/NBQX and concanamycin-A ameliorated calcium oscillations and cell death. For a sustained interaction between neurons and immune cells that is not mediated through MHC class molecules, such as CD4–neuron interactions, other molecular interactions move into focus. The lymphocyte function-associated antigen LFA-1 (CD18) and its counter receptor, the intercellular adhesion molecule (ICAM), on axons/neurons are responsible for the generation of an immunological synapse (Anikeeva et al., 2005; Miklossy et al., 2006) in antigen-independent T-cell–neuron contacts. LFA-1–ICAM interactions also play an important role in other neurodegenerative diseases such as Parkinson's disease (Miklossy et al., 2006). The recently published observation that mice without functional CD4 + cells do not develop EAE, but mice without functional CD8 + cells (and thus, normal CD4 + cells) do when actively immunized, supports the assumption that CD4+ cells are the key T-cell subset in the disease pathogenesis (Leuenberger et al., 2013). Another key pathway involved in CD4-mediated neuronal impairment is the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Aktas et al., 2005; Nitsch et al., 2000). The TRAIL/TRAIL receptor system is normally involved in the control of immune-cell activation and proliferation as well as induction of apoptosis in tumor cells. In the EAE model, the severity of symptoms was increased when TRAIL was

delivered directly intracerebrally as a result of apoptosis-induced neuronal cell death. Conversely, TRAIL-deficient lymphocytes transferred to mice were found to be less encephalitogenic but myelin specific (Aktas et al., 2005). CD4+ TRAIL-producing lymphocytes seem also to be involved in lower motor neuron loss in EAE as well as in MS patients, whereas TRAIL-deficient lymphocytes do not cause motor neuron loss in the spinal cord (Vogt et al., 2009). Various research groups, including our own, have recently begun to focus their attention on a particular subset of CD4 + cells, namely, CD4 +-secreting Il-17A and Il-17F cells, or Th17 cells, which are involved in combating extracellular bacterial and fungal infections (Ouyang et al., 2008). An excess of Th17 cells, however, is thought to be involved in the pathogenesis of autoimmune disorders such as MS (Harrington et al., 2005; Steinman, 2007). Indeed, sustained contact between Th17 cells and neurons, more precisely with axons in the brainstem, has been shown in the EAE model in mice using twophoton imaging (Siffrin et al., 2010). This TCR-independent contact leads to intracellular calcium oscillations that precede neuronal damage. These observed effects were reversible by blocking NMDA receptors with Mk-801 and partly reversible by blocking sodium channels with phenytoin. LFA-1 deficient Th17 cells induced less severe disease symptoms and cell death, emphasizing again the importance of the LFA-1–ICAM interaction in MHC-independent immunological synapses. CD4+ cells are undoubtedly a major contributor to pathogenesis in MS, leading to inflammation and subsequently to neurodegeneration, which causes persistent disability in humans. Several approved therapeutic options lessen the T cell- and especially the CD4+-mediated attack of neurons. Natalizumab, an antibody against the surface protein very late antigen 4 (VLA-4) on lymphocytes, is a very effective drug for MS as it blocks the migration of circulating lymphocytes through the BBB. Natalizumab lowers the CD4+/CD8+/CD19+/CD139+ cell numbers and the CD4 +/CD8 + ratio in the CSF, as well as the total number of CD4 + cells in the brain (Martin et al., 2008; Stüve et al., 2006a,b), emphasizing the strong suppressive effect on lymphocytes, especially on the CD4 + subpopulation. Very recently, secukinumab (AIN457) was administered to MS patients (Deiß et al., 2013), and preliminary data from the ongoing phase IIa clinical study of this IL-17A blocking antibody indicate that targeting Th17 cells might be a viable strategy in treating MS. The drug, which neutralizes Il-17A, significantly lowered MS-type brain lesions and showed a trend towards reduced relapse rates in a 6-month, placebo-controlled trial in 73 patients. However, not all is bad with CD4 + cells. Unlike CD4+ Th17 cells, which may be considered the “bad guys” in autoimmune disorders of the CNS, CD4+ CD25+ FoxP3+ cells (regulatory T cells) are supposed to be the “good guys” among T cells during inflammation of the CNS. Generally, they possess the capacity to control the immune response after a successfully defeated infection or to prevent autoimmune diseases (Marson et al., 2007). Also, in neuroinflammatory diseases such as MS, regulatory T cells proliferate mainly in peripheral lymphoid tissue (Korn et al., 2007) and interact with immune effector cells. A possible strategy to circumvent severe side effects in MS treatment, such as progressive multifocal leukoencephalopathy (PML) in the case of natalizumab, might be to promote the influence of CD4 + CD25 + FoxP3 + cells in the immunoneurological network (Carbone et al., 2014; Mcgeachy et al., 2005; Stephens et al., 2009). In animal studies, the transfer of FoxP3+ T-cells into EAE mice was found to substantially contribute to remission during disease as well as protection from neuroinflammation (Mcgeachy et al., 2005). Also, regulatory T-cell stimulants such as the CD28 superagonist (JJ316) attenuated the disease course in EAE (Beyersdorf et al., 2005; Tischner et al., 2006). In humans, evidence is emerging that clinical symptoms in MS and a decline in regulatory Tcell expansion are correlated (Carbone et al., 2014). Additionally, it has been shown that regulatory CD4+ CD25+ FoxP3+ T-cells ameliorate inflammation in in-vitro and ex-vivo studies by suppressing the cytotoxicity of CD8+ cells (Gobel et al., 2012).



In addition to the transcription factor FoxP3, interleukin (IL)-10, which is produced by a different subset of CD4+ regulatory T cells, is another molecule involved in stabilizing the immune response and preventing the development of autoimmune diseases (Fujio et al., 2010). For example, during active inflammation in a monkey model of MS, IL-10 was significantly lower than in healthy controls (Ma et al., 2009). However, the molecular pathways involving FoxP3 or IL-10 for regulatory T-cell functioning are still under debate. Pathomechanisms that lead to impairment of those cells need to be elucidated as they provide a promising target in fighting autoimmune disorders. Macrophages/microglia Macrophages in the brain, which are derived from peripheral monocytes, and microglia, which are resident macrophages, are part of the innate immune system and play a central role in protection against pathogens in the CNS (Ginhoux et al., 2013; Hanisch, 2002). Microglia are derived from the yolk-sac blood islands and migrate to the neuroepithelium during early development (Ginhoux et al., 2013; Gomez Perdiguero et al., 2013), whereas monocytes only enter the CNS during brain injury/inflammation where they differentiate into macrophages. They both produce a variety of cytokines and are at the same time an important target of cytokines. They also contribute to the activation of the adaptive immune system in infectious diseases, for instance by recruiting T cells from the periphery. Under normal circumstances, inactivated macrophages/microglia contribute to the homeostasis of the neuronal compartment. However, they also play a central role in autoimmune disorders, as they maintain ongoing inflammation once they are activated. After eliminating the pathogens/ autoantigens in MS, they are involved in the remission of the inflammation process (Raivich and Banati, 2004; Rawji and Yong, 2013). Once activated, CD4 + cells and macrophages/microglia could be considered to be the central players in orchestrating the immunological attack towards the myelin sheath and axon (Carson, 2002). Briefly, we can distinguish between the two activation states of macrophages/ microglia M1 and M2. Whereas M1 is considered to be a proinflammatory phenotype involved in pathogen clearance, M2 has rather an immunomodulatory function and promotes CNS repair (Mosser and Edwards, 2008). An imbalance between the M1/M2 ratio towards the M1 phenotype leads to a more severe disease course in EAE (Mikita et al., 2011), thus suggesting an involvement in disease pathogenesis. Interestingly, myelin degradation products themselves seem to stimulate pro-inflammatory microglia (Clarner et al., 2012) although phagocytosis in the CNS normally promotes anti-inflammatory conditions (Sierra et al., 2013). Nurr1, an intracellular receptor that is involved in the maintenance of dopaminergic neurons, seems to have important anti-inflammatory effects. Reduced Nurr1 expression leads to an increased inflammatory response, mainly due to an increased release of neurotoxic mediators in microglia (Saijo et al., 2009). However, activated microglia are responsible for remyelination of axons after demyelination through activation and proliferation of oligodendrocyte precursor cells (OPC) (Arnett et al., 2001; Kotter et al., 2001; Ludwin, 1984). Thus, simply blocking (pro-)inflammatory cytokines or their receptors might impair remyelination of axons. Microglia have been shown to become activated through toll-likereceptor 4 (TLR4), and once activated, they produce proinflammatory cytokines such as interleukins 1 and 6, IFN-γ and TNF-α, among others (Jack et al., 2005). In MS, macrophages/microglia indirectly damage oligodendrocytes and neurons through those proinflammatory cytokines via sensitization of axons to glutamate excitotoxicity (Pitt et al., 2000), as well as their phagocytic properties and presentation of antigens via MHC class II to CD4 + cells. Direct damage to neurons occurs through reactive oxygen and nitrogen species (ROS/RNS) produced by microglia and has been shown to induce mitochondrial dysfunction. This, together with an increased energy demand in demyelinated axons, contributes to axonal degeneration and subsequent neuronal cell death (Nikic et al., 2011).

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B-cells and autoantibodies Neuroinflammation in MS is thought to be mainly driven by T cells. However, in EAE, there is evidence for the involvement of B cells in the pathogenesis. From active EAE experiments, we know that the contribution of B cells to disease development varies depending on the immunization peptide or animal strain used (Weber et al., 2010). B cells operate as antigen presenting cells, and more controversially, as a source of auto-antibodies against CNS targets (Molnarfi et al., 2013; Stefferl et al., 1999). In passive EAE, immunoglobulin (Ig) G-secreting B cells were found mainly in areas of demyelination (Mannara et al., 2012). Rituximab, a monoclonal antibody that targets CD20-positive B-cells, is used off label in some patients as it reduces inflammatory lesion load in the MRI as well as clinical relapse rate (Hauser et al., 2008; Kappos et al., 2011), although it has no influence on disease progression in patients with PPMS (Hawker et al., 2009). Additionally, the oligoclonal bands (OCB) in the CSF, which are one of the important immunological diagnostic markers for MS, are provoked by an autochthone antibody production in the CNS compartment. The abovementioned aspects and the capacity of antigen presentation by B cells and the subsequent activation of T cells suggest the involvement of B lymphocytes (Disanto et al., 2012; Krumbholz et al., 2012; Molnarfi et al., 2013) in disease pathogenesis. At the molecular level, antibodies against neurofascin, a neuronal membrane protein expressed at the nodes of Ranvier, have been detected and associated with axonal damage in EAE (Mathey et al., 2007). Further research needs to be conducted in this field to confirm these results and evaluate the pathological impact of autoantibodies and B cells in MS. Natural killer cells Natural killer (NK) cells, part of the innate immune system, are involved in the pathogenesis of autoimmune disorders (Kaur et al., 2013). In fact, NK cells act as regulatory cells in the context of autoimmune disorders, as activated NK cells suppress autoimmune disease development (Linsen et al., 2005). This regulatory role is contrasted by proinflammatory functions, which are, however, thought to be minor. It should be noted that there are differences between the immunoregulatory effects of CD16+ and CD56+ NK cells, two subsets of NK cells, with the latter being thought to be more effective in suppressing T-cell activity (Nielsen et al., 2012; Takahashi et al., 2004). NK cells expressing CD95 (Fas) are able to suppress T-cell activation via inhibition of IFN-γ secretion by T cells. Furthermore, a higher frequency of this subset is found during remission in patients with a relapsing–remitting disease course, pointing to an immunosuppressive effect of NK cells including the Fas ligand (Takahashi et al., 2004). Patients with lower levels of NK cells or more functionally inactive NK cells are at a higher risk of experiencing relapses in MS (Kastrukoff et al., 2003). Apart from CD95-mediated T-cell suppression, the roles of other signal pathways such as LFA-1 and TRAIL are being investigated in the suppression of CD4 + T-cells by CD56 + NK cells in humans (Nielsen et al., 2012). Stimulation of CD56 + NK cells, in particular, could be a future therapeutic approach for the treatment of MS. One promising candidate is daclizumab, a humanized monoclonal antibody against the interleukin-2 receptor α chain that promotes CD56 + NK cell expansion in MS patients and is correlated to the treatment response (Bielekova et al., 2006; Martin et al., 2010; Sheridan et al., 2011). Proposed mechanisms provoking injury of neuronal structures in MS The primary inflammatory feature of the disease, caused through dysfunction of the BBB and inappropriate activation of the immune system towards the neuronal compartment, provokes a wide variety of clinical symptoms in MS and EAE. Good clinical response to corticoids in humans and animals during the acute phase emphasizes the inflammatory component of this disease. However, the typical relapsing– remitting disease course at disease onset (in 85% of all patients) changes


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to a secondary progressive course after several years in the vast majority of patients under the natural course of the disease. The secondary progressive disease course does not respond to corticoids, suggesting that inflammation may not be the primary pathogenesis at this stage. Important hallmarks of the pathogenesis in MS are therefore inflammation, demyelination and injury/degeneration of axons and neurons. Increasing evidence indicates that the neurodegenerative process in MS already begins at disease onset. The formation of axonal ovoids for example, which is an early sign of transected or damaged axons, was found in patients with short disease duration (Trapp et al., 1998) in acute active as well as chronic active lesions. MRI has opened up new possibilities for monitoring white-matter lesions; however, graymatter lesions are difficult to detect and only recently developed imaging techniques/sequences suggest the involvement of gray-matter lesions in MS. Interestingly, gray-matter damage seems to occur at an early stage (Chard and Miller, 2009) and causes progressive atrophy of the brain (Chard et al., 2002; Dalton et al., 2002). Both gray-matter lesions and thus brain atrophy, as well as axonal damage occurring in white and gray matter, already appear during early stages of the disease course, which is supported by reduced N-acetyl aspartate/creatinine ratios in MS patients (De Stefano et al., 2001). Non-motor symptoms such as cognitive impairment and fatigue as well as motor symptoms are thought to be partly driven by gray-matter pathology (Geurts and Barkhof, 2008; Geurts et al., 2012); but what are the possible molecular mechanisms leading to axonal transection and neuronal cell death in MS that are induced by the immune-neuronal crosstalk (Table 1)? Mitochondrial dysfunction Mitochondria are essential energy suppliers of cells. Impaired functioning of oxidative phosphorylation of the respiratory chain complexes in mitochondria leads to pathological conditions that can end in cell death. Dysfunction of mitochondria is supposed to be one major contributor to several different neurodegenerative disorders including Parkinson's and Huntington's diseases (Hauser and Hastings, 2013; Quintanilla and Johnson, 2009). Inflammation and demyelination of axons in the CNS in MS increase the energy demand of a neuron due to ineffective nerve conductance and thus challenge the mitochondrial machinery (Trapp and Stys, 2009). Neurons may be unable to overcome this challenge for several reasons: different respiratory chain complexes in the mitochondria are impaired during inflammation and ion homeostasis in the axon collapses, which leads to the accumulation of sodium and calcium (Craner et al., 2004; Dutta et al., 2006). The involvement of dysfunctional complexes I and III in the mitochondria has been previously suggested in the motor cortex in MS (Dutta et al., 2006). In other work, it was shown that complex IV activity is reduced in acute lesions in axons and oligodendrocytes, which might lead to sustained hypoxia-like tissue injury (Mahad et al., 2008). This dysfunction could be partially caused by soluble factors of the innate immune system, such as reactive oxygen and nitrogen species, as the density of macrophages and microglia was found to be increased in acute lesions (Mahad et al., 2009). Oxidative damage to mitochondria and subsequent focal axonal degeneration can be ameliorated in animal models by reducing levels of oxidative molecules during inflammation (Nikic et al., 2011), putting oxidative stress in the focus of neurodegeneration. Presumably as a compensatory mechanism, the content of mitochondria as well as mitochondrial size in the axons increases in demyelinated neurons (Campbell et al., 2012; Kiryu-Seo et al., 2010). In remyelinated axons, the density of mitochondria decreases although it remains at a higher level than in healthy controls measured by complex IV activity (Zambonin et al., 2011). Another interesting observation is that in demyelinated axons the transport velocity is increased, whereas this measure as well as mitochondrial size returns to normal levels after remyelination (Kiryu-Seo et al., 2010). The abovementioned

aspects are likely to be compensatory mechanisms responding to enhanced energy demand caused by the altered and therefore ineffective nerve conduction and ion imbalance. Redistribution of sodium channels Demyelination of axons disturbs the salutatory conduction of nerves. Sodium channels that are primarily expressed at the nodes of Ranvier (and nerve terminals) are upregulated in formerly myelinated axonal sections to restore nerve conduction in MS patients (Moll et al., 1991; Waxman, 2006). This redistribution of sodium channels causes an increased energy (ATP) demand to reduce increasing sodium levels via the Na+/K+ ATPase (Craner et al., 2004; Dutta et al., 2006). As stated above, mitochondria, which supply energy to the cells, are impaired themselves and are not able to fully compensate for the enhanced energy demands. To compensate for increased sodium levels within the cell, the Na+/Ca++ exchanger progressively secretes sodium on the one hand, and accumulates calcium intracellularly on the other (Craner et al., 2004). This leads to increased intracellular calcium levels, which activate proteases, damage microtubuli and are generally thought to be a toxic cell mediator over time in pathological concentrations. Under pathological conditions such as inflammation, calcium is also released from intracellular stores (e.g., the endoplasmic reticulum) and NMDA receptors become sensitized (Dutta et al., 2006; Trapp and Stys, 2009). All of these mechanisms lead to a sustained calcium overload in the neuron in EAE (Nitsch et al., 2004; Siffrin et al., 2010) and can result in cell death (Fig. 1). The expression of sodium channels in activated macrophages/ microglia is also enhanced (Craner et al., 2005; Morsali et al., 2013). By blocking sodium channels with phenytoin, for example, the inflammatory cell infiltration in MS lesions decreases (Craner et al., 2005). Sodium channels in macrophages seem to play an important role in the pathogenesis of MS. With safinamide and flecainide, two other sodium channel blockers, macrophages/microglia are silenced and symptoms are ameliorated in animal models of MS (Morsali et al., 2013). Blocking sodium channels seems to be a promising target for the development of disease-modifying drugs in MS, although serious adverse effects have to be monitored carefully. Lamotrigine, another sodium channel blocker approved for the treatment of generalized epilepsy is a drug that has shown improvements in clinical scores in EAE and neuroprotection on a cellular level has been confirmed histopathologically (Bechtold et al., 2006). However, clinical trials of lamotrigine in MS patients did not show advantages over placebo with respect to neuroprotection (Hayton et al., 2012; Kapoor et al., 2010). Thus, the involvement of sodium channels, both in axons and macrophages/microglia, in the pathogenesis of MS is evident. Although simple blocking of these channels does not lead to a significant improvement of clinical symptoms in MS patients, the promising nature of these drugs has been shown in invitro experiments and EAE. The contribution of sodium channels to disease pathogenesis appears to be more complex than first assumed. Specifically targeting subsets of sodium channels or the selective administration into inflamed tissue could be strategies for the future (Al-Izki et al., 2014). TASK1 There is also evidence that potassium channels might play an important role in MS. TWIK-related acid-sensitive potassium channel 1 (TASK1) is a membrane protein expressed on T cells and neurons and acting as a potassium channel (Bittner et al., 2009; Meuth et al., 2008). It is especially important for the resting membrane potential. Under pharmacological inhibition with anandamide or in the knock-out mouse model, less severe T-cell activation and axonal degeneration in the MOG35–55 induced EAE model could be observed (Bittner et al., 2009). Both TASK1−/− mice and selective blockade of TASK1 by pharmacological treatment led to an ameliorated disease course in EAE



with less T-cell proliferation and INF-γ production by CD4+ cells plus a neuroprotective effect confirmed by MRI brain volume analyses (Bittner et al., 2009, 2012). Potassium channels, particularly TASK1, seem to play a role in autoimmune neuroinflammation such as in EAE; whether it has relevance in MS still needs to be elucidated.

The role of ASIC-1 and TRPM4 Acid-sensing ion channel 1 (ASIC-1) is a protein expressed in the membrane of neurons, which under acidotic conditions such as acute injury or inflammation opens and allows a sodium and calcium influx (Wemmie et al., 2002; Xiong et al., 2008). As discussed above, sodium and calcium accumulation can lead to axonal degeneration and neuronal cell death. In EAE, ASIC-1 seems to have a significant influence on disease development, as clinical symptoms were considerably reduced in ASIC-1 deficient mice with significantly less axonal damage (Friese et al., 2007). The same group demonstrated that blocking ASIC-1 with amiloride could be a new therapeutic approach as it provided neuroprotection in EAE and in vitro. Amiloride, which is approved for the treatment of hypertension, ameliorated disability in EAE and decreased neuronal and oligodendroglial damage. Furthermore, the expression of ASIC-1 is enhanced in neurons and oligodendrocytes in lesions both in patients as well as in the animal model (Vergo et al., 2011), indicating a major contribution to disease development. Transient receptor potential melastatin 4 (TRPM4) is another candidate cation channel presumably involved in disease pathogenesis. TRPM4 is expressed by axons during inflammation both in MS patients as well as in EAE (Schattling et al., 2012). This channel can be activated through either an intracellular calcium increase or ATP decrease; both mechanisms are observed during early disease development caused by the intracellular energy failure. The activation of this channel leads mainly to a sodium influx and can be antagonized by glibenclamide, a drug approved for the treatment of diabetes. In EAE, glibenclamide attenuates the disease course and leads to less severe axonal and neuronal degeneration in the mouse model of MS without influencing the inflammatory response in active lesions (Schattling et al., 2012). TRPM4−/− mice show a less severe disease course. Increased activity of the Na+/Ca++ exchanger, redistribution of sodium channels, breakdown of the Na+/K+ ATPase, ASIC-1 and TRPM4 all contribute to increased intracellular sodium and calcium levels and are to a certain degree responsible for the axonal degeneration observed in MS. The exact roles of all of these putative targets still remain elusive. Blocking some of the abovementioned channels might be promising targets for the treatment of MS.

Brain-derived neurotrophic factor (BDNF) As stated above, many different ion channels are presumably involved in MS/EAE disease pathogenesis. At the same time, a large variety of neurotransmitters and trophic factors are present in the CNS that could have implications in MS. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), for instance, regulate many processes such as axonal growth or synaptic plasticity in the CNS (Binder and Scharfman, 2004). Interestingly, not only neurons produce BDNF, but also T and B cells are capable of producing BDNF in active lesions in EAE or MS (Hohlfeld et al., 2000; Kerschensteiner et al., 1999). In the EAE, BDNF protects axons from degeneration, and glatiramer acetate, a well-established immunomodulating drug for MS, promotes BDNF production by lymphocytes and therefore promotes the neuroprotective effect of BDNF (Linker et al., 2010; Ziemssen et al., 2002). This is another good example of the complexity of the regulation of CNS homeostasis and that neuroprotective approaches for the treatment of MS are possible.

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Glutamate excitotoxicity Glutamate excitotoxicity is a further important link between neuroinflammation and neurodegeneration. Glutamate, the main excitatory neurotransmitter of the CNS, modulates intracellular ion homeostasis through different receptors such as NMDA or AMPA (Zarate et al., 2010). Excessive glutamate causes an imbalance in cell homeostasis and can, via calcium increase, contribute to cell damage or cell death. Elevated glutamate levels have been described in active MS lesions (Srinivasan et al., 2005) and are associated with axonal, oligodendroglial and myelin damage (Micu et al., 2006). Different cell types (e.g., neurons, astrocytes and immune cells) are possible sources of glutamate production and have the capacity to secrete large amounts of glutamate under pathological conditions (Li et al., 1999; Matute et al., 2001; Pitt et al., 2000; Ye et al., 2003). On the other hand, upregulation of glutamate receptors has been described for oligodendrocytes in active MS lesions (Newcombe et al., 2008). The blockade of AMPA/kainate or NMDA receptor with NBQX or MK801 led to decreased axonal and oligodendroglial damage in the experimental animal model of MS (Nitsch et al., 2004; Pitt et al., 2000; Siffrin et al., 2010) and ameliorated clinical symptoms. A decreased intracellular calcium concentration was also found, indicating a robust interaction between glutamate and disease progression. There are already two well-known drugs available that modulate glutamate excitotoxicity. Memantine, an uncompetitive antagonist of the NMDA receptor is an approved treatment for moderate to severe Alzheimer's disease as it improves cognition (Reisberg et al., 2003). As a low-affinity open-channel blocker it mainly blocks excessive NMDA activity and therefore does not block all receptors unspecifically, which could have unacceptable side effects (Lipton, 2005). Amantadine, another uncompetitive NMDA receptor antagonist, is approved for the treatment of Parkinson's disease and also ameliorates excessive glutamate-driven signaling (Kornhuber et al., 1991). Owing to their neuroprotective characteristics, memantine and amantadine, as well as other NMDA receptor modulators are under clinical investigation for MS (Esfahani et al., 2012; Villoslada et al., 2009). Conclusion MS is a heterogeneous disease involving crosstalk between neuroinflammatory and neurodegenerative processes. Dysfunction of the BBB, migration of immune cells into the CNS and attack of antigens on neurons and oligodendrocytes account for the main inflammatory events. Axonal and neuronal degeneration is caused by several mechanisms directly and indirectly linked to the immune attack. Therapeutically, we are able to modulate the immune response and reduce acute symptoms and relapse rates. However, chronic disability caused by neurodegeneration remains a burden for affected patients. Cells, of both the innate and adaptive immune system, initialize the neurodegenerative process via different effector molecules such as cytokines, glutamate, Fas, TRAIL or reactive species. In recent years, several molecular mechanisms following those molecules and leading to axonal and neuronal degeneration have been discovered. Impaired mitochondrial functioning and dysregulation of channels seem to be the key players that finally lead to an energy and subsequent ion imbalance. Targeting specific channels or preventing mitochondrial dysfunction has already shown promising effects, mainly in animal models of MS (Bittner and Meuth, 2013). Neurodegeneration has been shown already to occur at disease onset. Eliminating neuronal cell death and therefore disease progression and chronic disability in MS might be achieved by inhibiting various targets at the same time. Based on current research, it is hopeful that, in the near future, new neuroprotective drugs such as specific sodium or potassium channel blockers or inducers of neurotrophins could supplement well-established immunosuppressive and immunomodulatory therapies. Finally, in later stages of the disease when


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neurodegeneration may also occur independent of inflammatory processes, regeneration may be the only possibility to influence the damage mechanisms (Deshmukh et al., 2013).

References 'T Hart, B.A., Gran, B., Weissert, R., 2011. EAE: imperfect but useful models of multiple sclerosis. Trends Mol. Med. 17, 119–125. Aktas, O., Smorodchenko, A., Brocke, S., Infante-Duarte, C., Topphoff, U.S., Vogt, J., Prozorovski, T., Meier, S., Osmanova, V., Pohl, E., Bechmann, I., Nitsch, R., Zipp, F., 2005. Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL. Neuron 46, 421–432. Al-Izki, S., Pryce, G., Hankey, D.J.R., Lidster, K., Von Kutzleben, S.M., Browne, L., Clutterbuck, L., Posada, C., Edith Chan, A.W., Amor, S., Perkins, V., Gerritsen, W.H., Ummenthum, K., Peferoen-Baert, R., Van Der Valk, P., Montoya, A., Joel, S.P., Garthwaite, J., Giovannoni, G., Selwood, D.L., Baker, D., 2014. Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis. Brain 137, 92–108. Alun Brown, K., 2001. Factors modifying the migration of lymphocytes across the blood– brain barrier. Int. Immunopharmacol. 1, 2043–2062. Anikeeva, N., Somersalo, K., Sims, T.N., Thomas, V.K., Dustin, M.L., Sykulev, Y., 2005. Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 102, 6437–6442. Arnett, H.A., Mason, J., Marino, M., Suzuki, K., Matsushima, G.K., Ting, J.P.Y., 2001. TNF [alpha] promotes proliferation of oligodendrocyte progenitors and remyelination. Nat. Neurosci. 4, 1116–1122. Babbe, H., Roers, A., Waisman, A., Lassmann, H., Goebels, N., Hohlfeld, R., Friese, M., Schröder, R., Deckert, M., Schmidt, S., Ravid, R., Rajewsky, K., 2000. Clonal expansions of Cd8+ T cells dominate the t cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction. J. Exp. Med. 192, 393–404. Bechtold, D., Miller, S., Dawson, A., Sun, Y., Kapoor, R., Berry, D., Smith, K., 2006. Axonal protection achieved in a model of multiple sclerosis using lamotrigine. J. Neurol. 253, 1542–1551. Beeston, T., Smith, T.R.F., Maricic, I., Tang, X., Kumar, V., 2010. Involvement of IFN-γ and perforin, but not Fas/FasL interactions in regulatory T cell-mediated suppression of experimental autoimmune encephalomyelitis. J. Neuroimmunol. 229, 91–97. Bellmann-Strobl, J., Stiepani, H., Wuerfel, J., Bohner, G., Paul, F., Warmuth, C., Aktas, O., Wandinger, K.P., Zipp, F., Klingebiel, R., 2009. MR spectroscopy (MRS) and magnetisation transfer imaging (MTI), lesion load and clinical scores in early relapsing remitting multiple sclerosis: a combined cross-sectional and longitudinal study. Eur. Radiol. 19, 2066–2074. Beyersdorf, N., Gaupp, S., Balbach, K., Schmidt, J., Toyka, K.V., Lin, C.-H., Hanke, T., Hünig, T., Kerkau, T., Gold, R., 2005. Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis. J. Exp. Med. 202, 445–455. Bielekova, B., Catalfamo, M., Reichert-Scrivner, S., Packer, A., Cerna, M., Waldmann, T.A., Mcfarland, H., Henkart, P.A., Martin, R., 2006. Regulatory CD56bright natural killer cells mediate immunomodulatory effects of IL-2Rα-targeted therapy (daclizumab) in multiple sclerosis. Proc. Natl. Acad. Sci. 103, 5941–5946. Binder, D.K., Scharfman, H.E., 2004. Brain-derived neurotrophic factor. Growth Factors 22, 123–131. Bitsch, A., Schuchardt, J., Bunkowski, S., Kuhlmann, T., Brück, W., 2000. Acute axonal injury in multiple sclerosis: correlation with demyelination and inflammation. Brain 123, 1174–1183. Bittner, S., Meuth, S.G., 2013. Targeting ion channels for the treatment of autoimmune neuroinflammation. Ther. Adv. Neurol. Disord. 6, 322–336. Bittner, S., Meuth, S.G., Göbel, K., Melzer, N., Herrmann, A.M., Simon, O.J., Weishaupt, A., Budde, T., Bayliss, D.A., Bendszus, M., Wiendl, H., 2009. TASK1 modulates inflammation and neurodegeneration in autoimmune inflammation of the central nervous system. Brain 132, 2501–2516. Bittner, S., Bauer, M.A., Ehling, P., Bobak, N., Breuer, J., Herrmann, A.M., Golfels, M., Wiendl, H., Budde, T., Meuth, S.G., 2012. The TASK1 channel inhibitor A293 shows efficacy in a mouse model of multiple sclerosis. Exp. Neurol. 238, 149–155. Cahalan, M.D., Parker, I., Wei, S.H., Miller, M.J., 2002. Two-photon tissue imaging: seeing the immune system in a fresh light. Nat. Rev. Immunol. 2, 872–880. Campbell, G.R., Ohno, N., Turnbull, D.M., Mahad, D.J., 2012. Mitochondrial changes within axons in multiple sclerosis: an update. Curr. Opin. Neurol. 25, 221–230. http:// dx.doi.org/10.1097/WCO.0b013e3283533a25. Carbone, F., De Rosa, V., Carrieri, P.B., Montella, S., Bruzzese, D., Porcellini, A., Procaccini, C., La Cava, A., Matarese, G., 2014. Regulatory T cell proliferative potential is impaired in human autoimmune disease. Nat. Med. 20, 69–74. Carson, M.J., 2002. Microglia as liaisons between the immune and central nervous systems: functional implications for multiple sclerosis. Glia 40, 218–231. Chard, D., Miller, D., 2009. Grey matter pathology in clinically early multiple sclerosis: evidence from magnetic resonance imaging. J. Neurol. Sci. 282, 5–11. Chard, D.T., Griffin, C.M., Parker, G.J.M., Kapoor, R., Thompson, A.J., Miller, D.H., 2002. Brain atrophy in clinically early relapsing–remitting multiple sclerosis. Brain 125, 327–337. Chari, D.M., 2007. Remyelination in multiple sclerosis. In: Alireza, M. (Ed.), International Review of Neurobiology. Academic Press. Clarner, T., Diederichs, F., Berger, K., Denecke, B., Gan, L., Van Der Valk, P., Beyer, C., Amor, S., Kipp, M., 2012. Myelin debris regulates inflammatory responses in an experimental demyelination animal model and multiple sclerosis lesions. Glia 60, 1468–1480.

Craner, M.J., Newcombe, J., Black, J.A., Hartle, C., Cuzner, M.L., Waxman, S.G., 2004. Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na+/Ca2+ exchanger. Proc. Natl. Acad. Sci. U. S. A. 101, 8168–8173. Craner, M.J., Damarjian, T.G., Liu, S., Hains, B.C., Lo, A.C., Black, J.A., Newcombe, J., Cuzner, M.L., Waxman, S.G., 2005. Sodium channels contribute to microglia/macrophage activation and function in EAE and MS. Glia 49, 220–229. Croxford, A.L., Kurschus, F.C., Waisman, A., 2011. Mouse models for multiple sclerosis: historical facts and future implications. Biochim. Biophys. Acta (BBA) — Mol. Basis Dis. 1812, 177–183. Dalton, C.M., Brex, P.A., Jenkins, R., Fox, N.C., Miszkiel, K.A., Crum, W.R., O'riordan, J.I., Plant, G.T., Thompson, A.J., Miller, D.H., 2002. Progressive ventricular enlargement in patients with clinically isolated syndromes is associated with the early development of multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 73, 141–147. De Stefano, N., Narayanan, S., Francis, G.S., et al., 2001. EVidence of axonal damage in the early stages of multiple sclerosis and its relevance to disability. Arch. Neurol. 58, 65–70. Deiß, A., Brecht, I., Haarmann, A., Buttmann, M., 2013. Treating multiple sclerosis with monoclonal antibodies: a 2013 update. Expert. Rev. Neurother. 13, 313–335. Deluca, G.C., Williams, K., Evangelou, N., Ebers, G.C., Esiri, M.M., 2006. The contribution of demyelination to axonal loss in multiple sclerosis. Brain 129, 1507–1516. Deshmukh, V.A., Tardif, V., Lyssiotis, C.A., Green, C.C., Kerman, B., Kim, H.J., Padmanabhan, K., Swoboda, J.G., Ahmad, I., Kondo, T., Gage, F.H., Theofilopoulos, A.N., Lawson, B.R., Schultz, P.G., Lairson, L.L., 2013. A regenerative approach to the treatment of multiple sclerosis. Nature 502, 327–332. Disanto, G., Morahan, J.M., Barnett, M.H., Giovannoni, G., Ramagopalan, S.V., 2012. The evidence for a role of B cells in multiple sclerosis. Neurology 78, 823–832. Dutta, R., Mcdonough, J., Yin, X., Peterson, J., Chang, A., Torres, T., Gudz, T., Macklin, W.B., Lewis, D.A., Fox, R.J., Rudick, R., Mirnics, K., Trapp, B.D., 2006. Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann. Neurol. 59, 478–489. Esfahani, M., Harandi, Z., Movasat, M., Nikdel, M., Adelpour, M., Momeni, A., Merat, H., Fard, M., 2012. Memantine for axonal loss of optic neuritis. Graefes Arch. Clin. Exp. Ophthalmol. 250, 863–869. Franklin, R.J.M., Ffrench-Constant, C., 2008. Remyelination in the CNS: from biology to therapy. Nat. Rev. Neurosci. 9, 839–855. Friese, M.A., Craner, M.J., Etzensperger, R., Vergo, S., Wemmie, J.A., Welsh, M.J., Vincent, A., Fugger, L., 2007. Acid-sensing ion channel-1 contributes to axonal degeneration in autoimmune inflammation of the central nervous system. Nat. Med. 13, 1483–1489. Frohman, E.M., Racke, M.K., Raine, C.S., 2006. Multiple sclerosis — the plaque and its pathogenesis. N. Engl. J. Med. 354, 942–955. Fujio, K., Okamura, T., Yamamoto, K., 2010. Chapter 4 — the family of IL-10-secreting CD4+ T cells. In: Frederick, W.A. (Ed.), Advances in Immunology. Academic Press. Fuller, S., Steele, M., Münch, G., 2010. Activated astroglia during chronic inflammation in Alzheimer's disease—do they neglect their neurosupportive roles? Mutat. Res. Fundam. Mol. Mech. Mutagen. 690, 40–49. Geurts, J.J.G., Barkhof, F., 2008. Grey matter pathology in multiple sclerosis. Lancet Neurol. 7, 841–851. Geurts, J.J.G., Calabrese, M., Fisher, E., Rudick, R.A., 2012. Measurement and clinical effect of grey matter pathology in multiple sclerosis. Lancet Neurol. 11, 1082–1092. Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T., 2013. Origin and differentiation of microglia. Front. Cell. Neurosci. 7. Giuliani, F., Goodyer, C.G., Antel, J.P., Yong, V.W., 2003. Vulnerability of human neurons to T cell-mediated cytotoxicity. J. Immunol. 171, 368–379. Gobel, K., Bittner, S., Melzer, N., Pankratz, S., Dreykluft, A., Schuhmann, M., Meuth, S., Wiendl, H., 2012. CD4+ CD25+ FoxP3+ regulatory T cells suppress cytotoxicity of CD8+ effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level. J. Neuroinflammation 9, 41. Gomez Perdiguero, E., Schulz, C., Geissmann, F., 2013. Development and homeostasis of “resident” myeloid cells: the case of the microglia. Glia 61, 112–120. Grakoui, A., Bromley, S.K., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M., Dustin, M.L., 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227. Hanisch, U.-K., 2002. Microglia as a source and target of cytokines. Glia 40, 140–155. Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M., Weaver, C.T., 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6, 1123–1132. Hauser, D.N., Hastings, T.G., 2013. Mitochondrial dysfunction and oxidative stress in Parkinson's disease and monogenic Parkinsonism. Neurobiol. Dis. 51, 35–42. Hauser, S.L., Waubant, E., Arnold, D.L., Vollmer, T., Antel, J., Fox, R.J., Bar-Or, A., Panzara, M., Sarkar, N., Agarwal, S., Langer-Gould, A., Smith, C.H., 2008. B-cell depletion with rituximab in relapsing–remitting multiple sclerosis. N. Engl. J. Med. 358, 676–688. Hawker, K., O'connor, P., Freedman, M.S., Calabresi, P.A., Antel, J., Simon, J., Hauser, S., Waubant, E., Vollmer, T., Panitch, H., Zhang, J., Chin, P., Smith, C.H., 2009. Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial. Ann. Neurol. 66, 460–471. Hayton, T., Furby, J., Smith, K.J., Altmann, D.R., Brenner, R., Chataway, J., Hunter, K., Tozer, D.J., Miller, D.H., Kapoor, R., 2012. Longitudinal changes in magnetisation transfer ratio in secondary progressive multiple sclerosis: data from a randomised placebo controlled trial of lamotrigine. J. Neurol. 259, 505–514. Herz, J., Zipp, F., Siffrin, V., 2010. Neurodegeneration in autoimmune CNS inflammation. Exp. Neurol. 225, 9–17. Hickey, W.F., 1991. Migration of hematogenous cells through the blood–brain barrier and the initiation of CNS inflammation. Brain Pathol. 1, 97–105. Höftberger, R., Aboul-Enein, F., Brueck, W., Lucchinetti, C., Rodriguez, M., Schmidbauer, M., Jellinger, K., Lassmann, H., 2004. Expression of major histocompatibility complex


E. Ellwardt, F. Zipp / Experimental Neurology xxx (2014) xxx–xxx class I molecules on the different cell types in multiple sclerosis lesions. Brain Pathol. 14, 43–50. Hohlfeld, R., Kerschensteiner, M., Stadelmann, C., Lassmann, H., Wekerle, H., 2000. The neuroprotective effect of inflammation: implications for the therapy of multiple sclerosis. J. Neuroimmunol. 107, 161–166. Hu, D., Ikizawa, K., Lu, L., Sanchirico, M.E., Shinohara, M.L., Cantor, H., 2004. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat. Immunol. 5, 516–523. Huse, M., Quann, E.J., Davis, M.M., 2008. Shouts, whispers and the kiss of death: directional secretion in T cells. Nat. Immunol. 9, 1105–1111. Jack, C., Ruffini, F., Bar-Or, A., Antel, J.P., 2005. Microglia and multiple sclerosis. J. Neurosci. Res. 81, 363–373. Kapoor, R., Furby, J., Hayton, T., Smith, K.J., Altmann, D.R., Brenner, R., Chataway, J., RaC, Hughes, Miller, D.H., 2010. Lamotrigine for neuroprotection in secondary progressive multiple sclerosis: a randomised, double-blind, placebo-controlled, parallel-group trial. Lancet Neurol. 9, 681–688. Kappos, L., Li, D., Calabresi, P.A., O'connor, P., Bar-Or, A., Barkhof, F., Yin, M., Leppert, D., Glanzman, R., Tinbergen, J., Hauser, S.L., 2011. Ocrelizumab in relapsing–remitting multiple sclerosis: a phase 2, randomised, placebo-controlled, multicentre trial. Lancet 378, 1779–1787. Kastrukoff, L.F., Lau, A., Wee, R., Zecchini, D., White, R., Paty, D.W., 2003. Clinical relapses of multiple sclerosis are associated with ‘novel’ valleys in natural killer cell functional activity. J. Neuroimmunol. 145, 103–114. Kaur, G., Trowsdale, J., Fugger, L., 2013. Natural killer cells and their receptors in multiple sclerosis. Brain 136, 2657–2676. Kerschensteiner, M., Gallmeier, E., Behrens, L., Leal, V.V., Misgeld, T., Klinkert, W.E.F., Kolbeck, R., Hoppe, E., Oropeza-Wekerle, R.-L., Bartke, I., Stadelmann, C., Lassmann, H., Wekerle, H., Hohlfeld, R., 1999. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J. Exp. Med. 189, 865–870. Kiryu-Seo, S., Ohno, N., Kidd, G.J., Komuro, H., Trapp, B.D., 2010. Demyelination increases axonal stationary mitochondrial size and the speed of axonal mitochondrial transport. J. Neurosci. 30, 6658–6666. Korn, T., Reddy, J., Gao, W., Bettelli, E., Awasthi, A., Petersen, T.R., Backstrom, B.T., Sobel, R.A., Wucherpfennig, K.W., Strom, T.B., Oukka, M., Kuchroo, V.K., 2007. Myelinspecific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat. Med. 13, 423–431. Kornhuber, J., Bormann, J., Hübers, M., Rusche, K., Riederer, P., 1991. Effects of the 1-aminoadamantanes at the MK-801-binding site of the NMDA-receptor-gated ion channel: a human postmortem brain study. Eur. J. Pharmacol. Mol. Pharmacol. 206, 297–300. Kotter, M.R., Setzu, A., Sim, F.J., Van Rooijen, N., Franklin, R.J.M., 2001. Macrophage depletion impairs oligodendrocyte remyelination following lysolecithin-induced demyelination. Glia 35, 204–212. Krumbholz, M., Derfuss, T., Hohlfeld, R., Meinl, E., 2012. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat. Rev. Neurol. 8, 613–623. Leuenberger, T., Paterka, M., Reuter, E., Herz, J., Niesner, R.A., Radbruch, H., Bopp, T., Zipp, F., Siffrin, V., 2013. The role of CD8+ T cells and their local interaction with CD4+ T cells in myelin oligodendrocyte glycoprotein35–55-induced experimental autoimmune encephalomyelitis. J. Immunol. 191, 4960–4968. Li, S., GaR, Mealing, Morley, P., Stys, P.K., 1999. Novel injury mechanism in anoxia and trauma of spinal cord white matter: glutamate release via reverse Na+-dependent glutamate transport. J. Neurosci. 19, RC16. Linker, R.A., Lee, D.-H., Demir, S., Wiese, S., Kruse, N., Siglienti, I., Gerhardt, E., Neumann, H., Sendtner, M., Lühder, F., Gold, R., 2010. Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis. Brain 133, 2248–2263. Linsen, L., Somers, V., Stinissen, P., 2005. Immunoregulation of autoimmunity by natural killer T cells. Hum. Immunol. 66, 1193–1202. Lipton, S.A., 2005. The molecular basis of memantine action in Alzheimer's disease and other neurologic disorders: low-affinity, uncompetitive antagonism. Curr. Alzheimer Res. 2, 155–165. Lublin, F.D., Reingold, S.C., Sclerosis* NMSSaCOCTONaIM, 1996. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 46, 907–911. Ludwin, S.K., 1984. Proliferation of mature oligodendrocytes after trauma to the central nervous system. Nature 308, 274–275. Ma, A., Xiong, Z., Hu, Y., Qi, S., Song, L., Dun, H., Zhang, L., Lou, D., Yang, P., Zhao, Z., Wang, X., Zhang, D., Daloze, P., Chen, H., 2009. Dysfunction of IL-10-producing type 1 regulatory T cells and CD4+ CD25 + regulatory T cells in a mimic model of human multiple sclerosis in Cynomolgus monkeys. Int. Immunopharmacol. 9, 599–608. Mahad, D., Ziabreva, I., Lassmann, H., Turnbull, D., 2008. Mitochondrial defects in acute multiple sclerosis lesions. Brain 131, 1722–1735. Mahad, D.J., Ziabreva, I., Campbell, G., Lax, N., White, K., Hanson, P.S., Lassmann, H., Turnbull, D.M., 2009. Mitochondrial changes within axons in multiple sclerosis. Brain 132, 1161–1174. Mannara, F., Valente, T., Saura, J., Graus, F., Saiz, A., Moreno, B., 2012. Passive experimental autoimmune encephalomyelitis in C57BL/6 with MOG: evidence of involvement of B cells. PLoS ONE 7, e52361. Marson, A., Kretschmer, K., Frampton, G.M., Jacobsen, E.S., Polansky, J.K., Macisaac, K.D., Levine, S.S., Fraenkel, E., Von Boehmer, H., Young, R.A., 2007. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature 445, 931–935. Martin, M., Cravens, P.D., Winger, R., et al., 2008. Decrease in the numbers of dendritic cells and CD4+ T cells in cerebral perivascular spaces due to natalizumab. Arch. Neurol. 65, 1596–1603. Martin, J.F., Perry, J.S.A., Jakhete, N.R., Wang, X., Bielekova, B., 2010. An IL-2 paradox: blocking CD25 on T cells induces IL-2-driven activation of CD56bright NK cells. J. Immunol. 185, 1311–1320.

9

Massacesi, L., Genain, C.P., Lee-Parritz, D., Letvin, N.L., Canfield, D., Hauser, S.L., 1995. Active and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis. Ann. Neurol. 37, 519–530. Mathey, E.K., Derfuss, T., Storch, M.K., Williams, K.R., Hales, K., Woolley, D.R., Al-Hayani, A., Davies, S.N., Rasband, M.N., Olsson, T., Moldenhauer, A., Velhin, S., Hohlfeld, R., Meinl, E., Linington, C., 2007. Neurofascin as a novel target for autoantibody-mediated axonal injury. J. Exp. Med. 204, 2363–2372. Matute, C., Alberdi, E., Domercq, M.A., Pérez-Cerdá, F., Pérez-Samartín, A., SánchezGómez, M.V., 2001. The link between excitotoxic oligodendroglial death and demyelinating diseases. Trends Neurosci. 24, 224–230. Mcgeachy, M.J., Stephens, L.A., Anderton, S.M., 2005. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4 + CD25 + regulatory cells within the central nervous system. J. Immunol. 175, 3025–3032. Mcgeer, P.L., Schwab, C., Parent, A., Doudet, D., 2003. Presence of reactive microglia in monkey substantia nigra years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration. Ann. Neurol. 54, 599–604. Medana, I.M., Gallimore, A., Oxenius, A., Martinic, M.M.A., Wekerle, H., Neumann, H., 2000. MHC class I-restricted killing of neurons by virus-specific CD8+ T lymphocytes is effected through the Fas/FasL, but not the perforin pathway. Eur. J. Immunol. 30, 3623–3633. Meuth, S.G., Bittner, S., Meuth, P., Simon, O.J., Budde, T., Wiendl, H., 2008. TWIK-related acid-sensitive K+ channel 1 (TASK1) and TASK3 critically influence T lymphocyte effector functions. J. Biol. Chem. 283, 14559–14570. Meuth, S.G., Herrmann, A.M., Simon, O.J., Siffrin, V., Melzer, N., Bittner, S., Meuth, P., Langer, H.F., Hallermann, S., Boldakowa, N., Herz, J., Munsch, T., Landgraf, P., Aktas, O., Heckmann, M., Lessmann, V., Budde, T., Kieseier, B.C., Zipp, F., Wiendl, H., 2009. Cytotoxic CD8 + T cell–neuron interactions: perforin-dependent electrical silencing precedes but is not causally linked to neuronal cell death. J. Neurosci. 29, 15397–15409. Micu, I., Jiang, Q., Coderre, E., Ridsdale, A., Zhang, L., Woulfe, J., Yin, X., Trapp, B.D., Mcrory, J.E., Rehak, R., Zamponi, G.W., Wang, W., Stys, P.K., 2006. NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia. Nature 439, 988–992. Mikita, J., Dubourdieu-Cassagno, N., Deloire, M.S., Vekris, A., Biran, M., Raffard, G., Brochet, B., Canron, M.-H., Franconi, J.-M., Boiziau, C., Petry, K.G., 2011. Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis. Amelioration of clinical status by M2 activated monocyte administration. Mult. Scler. J. 17, 2–15. Miklossy, J., Doudet, D.D., Schwab, C., Yu, S., Mcgeer, E.G., Mcgeer, P.L., 2006. Role of ICAM1 in persisting inflammation in Parkinson disease and MPTP monkeys. Exp. Neurol. 197, 275–283. Minagar, A., Alexander, J.S., 2003. Blood–brain barrier disruption in multiple sclerosis. Mult. Scler. 9, 540–549. Minagar, A., Toledo, E.G., Alexander, J.S., Kelley, R.E., 2004. Pathogenesis of brain and spinal cord atrophy in multiple sclerosis. J. Neuroimaging 14, 5S–10S. Mizuno, T., Zhang, G., Takeuchi, H., Kawanokuchi, J., Wang, J., Sonobe, Y., Jin, S., Takada, N., Komatsu, Y., Suzumura, A., 2008. Interferon-γ directly induces neurotoxicity through a neuron specific, calcium-permeable complex of IFN-γ receptor and AMPA GluR1 receptor. FASEB J. 22, 1797–1806. Moll, C., Mourre, C., Lazdunski, M., Ulrich, J., 1991. Increase of sodium channels in demyelinated lesions of multiple sclerosis. Brain Res. 556, 311–316. Molnarfi, N., Schulze-Topphoff, U., Weber, M.S., Patarroyo, J.C., Prod'homme, T., VarrinDoyer, M., Shetty, A., Linington, C., Slavin, A.J., Hidalgo, J., Jenne, D.E., Wekerle, H., Sobel, R.A., Bernard, C.C.A., Shlomchik, M.J., Zamvil, S.S., 2013. MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies. J. Exp. Med. 210, 2921–2937. Morsali, D., Bechtold, D., Lee, W., Chauhdry, S., Palchaudhuri, U., Hassoon, P., Snell, D.M., Malpass, K., Piers, T., Pocock, J., Roach, A., Smith, K.J., 2013. Safinamide and flecainide protect axons and reduce microglial activation in models of multiple sclerosis. Brain 136, 1067–1082. Mosser, D.M., Edwards, J.P., 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969. Neumann, H., Cavalie, A., Jenne, D.E., Wekerle, H., 1995. Induction of MHC class I genes in neurons. Science 269, 549. Neumann, H., Schmidt, H., Cavalié, A., Jenne, D., Wekerle, H., 1997. Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-γ and Tumor necrosis factor (TNF)-α. J. Exp. Med. 185, 305–316. Neumann, H., Medana, I.M., Bauer, J., Lassmann, H., 2002. Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases. Trends Neurosci. 25, 313–319. Newcombe, J., Uddin, A., Dove, R., Patel, B., Turski, L., Nishizawa, Y., Smith, T., 2008. Glutamate receptor expression in multiple sclerosis lesions. Brain Pathol. 18, 52–61. Nielsen, N., Ødum, N., Ursø, B., Lanier, L.L., Spee, P., 2012. Cytotoxicity of CD56bright NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A. PLoS ONE 7, e31959. Niesner, R., Siffrin, V., Zipp, F., 2013. Two-photon imaging of immune cells in neural tissue. Cold Spring Harb. Protoc. 3. http://dx.doi.org/10.1101/pdb.prot073528. Nikic, I., Merkler, D., Sorbara, C., Brinkoetter, M., Kreutzfeldt, M., Bareyre, F.M., Bruck, W., Bishop, D., Misgeld, T., Kerschensteiner, M., 2011. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat. Med. 17, 495–499. Nitsch, R., Bechmann, I., Deisz, R.A., Haas, D., Lehmann, T.N., Wendling, U., Zipp, F., 2000. Human brain-cell death induced by tumour-necrosis-factor-related apoptosisinducing ligand (TRAIL). Lancet 356, 827–828. Nitsch, R., Pohl, E.E., Smorodchenko, A., Infante-Duarte, C., Aktas, O., Zipp, F., 2004. Direct impact of T cells on neurons revealed by two-photon microscopy in living brain tissue. J. Neurosci. 24, 2458–2464.


10


Ouyang, W., Kolls, J.K., Zheng, Y., 2008. The Biological Functions of T Helper 17 Cell Effector Cytokines in Inflammation. Immunity 28, 454–467. Pannemans, K., Broux, B., Goris, A., Dubois, B., Broekmans, T., Van Wijmeersch, B., Geraghty, D., Stinissen, P., Hellings, N., 2013. HLA-E restricted CD8+ T cell subsets are phenotypically altered in multiple sclerosis patients. Mult. Scler. J. 0, 1–12. http://dx.doi.org/10.1177/1352458513509703. Pitt, D., Werner, P., Raine, C.S., 2000. Glutamate excitotoxicity in a model of multiple sclerosis. Nat. Med. 6, 67–70. Quintanilla, R.A., Johnson, G.V.W., 2009. Role of mitochondrial dysfunction in the pathogenesis of Huntington's disease. Brain Res. Bull. 80, 242–247. Raivich, G., Banati, R., 2004. Brain microglia and blood-derived macrophages: molecular profiles and functional roles in multiple sclerosis and animal models of autoimmune demyelinating disease. Brain Res. Rev. 46, 261–281. Ransohoff, R.M., Kivisakk, P., Kidd, G., 2003. Three or more routes for leukocyte migration into the central nervous system. Nat. Rev. Immunol. 3, 569–581. Rawji, K.S., Yong, V.W., 2013. The benefits and detriments of macrophages/microglia in models of multiple sclerosis. Clin. Dev. Immunol. 2013, 13. Reisberg, B., Doody, R., Stöffler, A., Schmitt, F., Ferris, S., Möbius, H.J., 2003. Memantine in moderate-to-severe Alzheimer's disease. N. Engl. J. Med. 348, 1333–1341. Saijo, K., Winner, B., Carson, C.T., Collier, J.G., Boyer, L., Rosenfeld, M.G., Gage, F.H., Glass, C.K., 2009. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 137, 47–59. Schattling, B., Steinbach, K., Thies, E., Kruse, M., Menigoz, A., Ufer, F., Flockerzi, V., Bruck, W., Pongs, O., Vennekens, R., Kneussel, M., Freichel, M., Merkler, D., Friese, M.A., 2012. TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat. Med. 18, 1805–1811. Shatz, C.J., 2009. MHC class I: an unexpected role in neuronal plasticity. Neuron 64, 40–45. Sheridan, J.P., Zhang, Y., Riester, K., Tang, M.T., Efros, L., Shi, J., Harris, J., Vexler, V., Elkins, J.S., 2011. Intermediate-affinity interleukin-2 receptor expression predicts CD56bright natural killer cell expansion after daclizumab treatment in the CHOICE study of patients with multiple sclerosis. Mult. Scler. J. 17, 1441–1448. Sierra, A., Abiega, O., Shahraz, A., Neumann, H., 2013. Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis. Front. Cell. Neurosci. 7. Siffrin, V., Brandt, A.U., Herz, J., Zipp, F., 2007. New insights into adaptive immunity in chronic neuroinflammation. In: Frederick, W.A. (Ed.), Advances in Immunology. Academic Press. Siffrin, V., Radbruch, H., Glumm, R., Niesner, R., Paterka, M., Herz, J., Leuenberger, T., Lehmann, S.M., Luenstedt, S., Rinnenthal, J.L., Laube, G., Luche, H., Lehnardt, S., Fehling, H.-J., Griesbeck, O., Zipp, F., 2010. In vivo imaging of partially reversible Th17 cell-induced neuronal dysfunction in the course of encephalomyelitis. Immunity 33, 424–436. Smith, T.R.F., Kumar, V., 2008. Revival of CD8+ Treg-mediated suppression. Trends Immunol. 29, 337–342. Srinivasan, R., Sailasuta, N., Hurd, R., Nelson, S., Pelletier, D., 2005. Evidence of elevated glutamate in multiple sclerosis using magnetic resonance spectroscopy at 3 T. Brain 128, 1016–1025. Stefferl, A., Brehm, U., Storch, M., Lambracht-Washington, D., Bourquin, C., Wonigeit, K., Lassmann, H., Linington, C., 1999. Myelin oligodendrocyte glycoprotein induces experimental autoimmune encephalomyelitis in the “Resistant” brown Norway rat: disease susceptibility is determined by MHC and MHC-linked effects on the B cell response. J. Immunol. 163, 40–49. Steinman, L., 2007. A brief history of TH17, the first major revision in the TH1/TH2 hypothesis of T cell-mediated tissue damage. Nat. Med. 13, 139–145. Stephens, L.A., Malpass, K.H., Anderton, S.M., 2009. Curing CNS autoimmune disease with myelin-reactive Foxp3+ Treg. Eur. J. Immunol. 39, 1108–1117. Stüve, O., Marra, C.M., Bar-Or, A., et al., 2006a. Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis. Arch. Neurol. 63, 1383–1387.

Stüve, O., Marra, C.M., Jerome, K.R., Cook, L., Cravens, P.D., Cepok, S., Frohman, E.M., Phillips, J.T., Arendt, G., Hemmer, B., Monson, N.L., Racke, M.K., 2006b. Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann. Neurol. 59, 743–747. Takahashi, K., Aranami, T., Endoh, M., Miyake, S., Yamamura, T., 2004. The regulatory role of natural killer cells in multiple sclerosis. Brain 127, 1917–1927. Tansey, M.G., Mccoy, M.K., Frank-Cannon, T.C., 2007. Neuroinflammatory mechanisms in Parkinson's disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp. Neurol. 208, 1–25. Tischner, D., Weishaupt, A., Brandt, J.V.D., Müller, N., Beyersdorf, N., Ip, C.W., Toyka, K.V., Hünig, T., Gold, R., Kerkau, T., Reichardt, H.M., 2006. Polyclonal expansion of regulatory T cells interferes with effector cell migration in a model of multiple sclerosis. Brain 129, 2635–2647. Trapp, B.D., Stys, P.K., 2009. Virtual hypoxia and chronic necrosis of demyelinated axons in multiple sclerosis. Lancet Neurol. 8, 280–291. Trapp, B.D., Peterson, J., Ransohoff, R.M., Rudick, R., Mörk, S., Bö, L., 1998. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338, 278–285. Venters, H.D., Dantzer, R., Kelley, K.W., 2000. A new concept in neurodegeneration: TNFα is a silencer of survival signals. Trends Neurosci. 23, 175–180. Vergo, S., Craner, M.J., Etzensperger, R., Attfield, K., Friese, M.A., Newcombe, J., Esiri, M., Fugger, L., 2011. Acid-sensing ion channel 1 is involved in both axonal injury and demyelination in multiple sclerosis and its animal model. Brain 134, 571–584. Villoslada, P., Arrondo, G., Sepulcre, J., Alegre, M., Artieda, J., 2009. Memantine induces reversible neurologic impairment in patients with MS. Neurology 72, 1630–1633. Vogt, J., Paul, F., Aktas, O., Müller-Wielsch, K., Dörr, J., Dörr, S., Bharathi, B.S., Glumm, R., Schmitz, C., Steinbusch, H., Raine, C.S., Tsokos, M., Nitsch, R., Zipp, F., 2009. Lower motor neuron loss in multiple sclerosis and experimental autoimmune encephalomyelitis. Ann. Neurol. 66, 310–322. Waxman, S.G., 2006. Axonal conduction and injury in multiple sclerosis: the role of sodium channels. Nat. Rev. Neurosci. 7, 932–941. Weber, M.S., Prod'homme, T., Patarroyo, J.C., Molnarfi, N., Karnezis, T., Lehmann-Horn, K., Danilenko, D.M., Eastham-Anderson, J., Slavin, A.J., Linington, C., Bernard, C.C.A., Martin, F., Zamvil, S.S., 2010. B-cell activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity. Ann. Neurol. 68, 369–383. Wemmie, J.A., Chen, J., Askwith, C.C., Hruska-Hageman, A.M., Price, M.P., Nolan, B.C., Yoder, P.G., Lamani, E., Hoshi, T., Freeman Jr., J.H., Welsh, M.J., 2002. The acidactivated ion channel ASIC contributes to synaptic plasticity, learning, and memory. Neuron 34, 463–477. Xiong, Z.-G., Pignataro, G., Li, M., Chang, S.-Y., Simon, R.P., 2008. Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases. Curr. Opin. Pharmacol. 8, 25–32. Ye, Z.-C., Wyeth, M.S., Baltan-Tekkok, S., Ransom, B.R., 2003. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J. Neurosci. 23, 3588–3596. Zambonin, J.L., Zhao, C., Ohno, N., Campbell, G.R., Engeham, S., Ziabreva, I., Schwarz, N., Lee, S.E., Frischer, J.M., Turnbull, D.M., Trapp, B.D., Lassmann, H., Franklin, R.J.M., Mahad, D.J., 2011. Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis. Brain 134, 1901–1913. Zarate, C., Machado-Vieira, R., Henter, I., Ibrahim, L., Diazgranados, N., Salvadore, G., 2010. Glutamatergic modulators: the future of treating mood disorders? Harv. Rev. Psychiatry 18, 293–303. Ziemssen, T., Kümpfel, T., Klinkert, W.E.F., Neuhaus, O., Hohlfeld, R., 2002. Glatiramer acetate‐specific T‐helper 1‐ and 2‐type cell lines produce BDNF: implications for multiple sclerosis therapy. Brain 125, 2381–2391.


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