NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - REVIEW ARTICLE

The role of glutamate and its receptors in multiple sclerosis

Ivana R. Stojanovic • Milos Kostic •

Srdjan Ljubisavljevic

Received: 31 October 2013 / Accepted: 27 February 2014

� Springer-Verlag Wien 2014

Abstract Glutamate is an excitatory neurotransmitter of

the central nervous system, which has a central role in a

complex communication network established between

neurons, astrocytes, oligodendrocytes, and microglia.

Multiple abnormal triggers such as energy deficiency,

oxidative stress, mitochondrial dysfunction, and calcium

overload can lead to abnormalities in glutamate signaling.

Thus, the disturbance of glutamate homeostasis could

affect practically all physiological functions and interac-

tions of brain cells, leading to excitotoxicity. Excitotoxicity

is the pathological process by which nerve cells are dam-

aged or killed by excessive stimulation by glutamate.

Although neuron degeneration and death are the ultimate

consequences of multiple sclerosis (MS), it is now widely

accepted that alterations in the function of surrounding

glial cells are key features in the progression of the disease.

The present knowledge raise the possibility that the mod-

ulation of glutamate release and transport, as well as

receptors blockade or glutamate metabolism modulation,

might be relevant targets for the development of future

therapeutic interventions in MS.

Keywords Multiple sclerosis � Glutamate �Excitotoxicity � Neurodegeneration

Introduction

Multiple sclerosis (MS) is an inflammatory, demyelinating,

and neurodegenerative disease of the central nervous sys-

tem (CNS) (van Horssen et al. 2012; Gray et al. 2013;

Sinnecker et al. 2012). In the early phase of the disease,

inflammatory lymphocyte, macrophage, and activated

microglia infiltrates, followed by an excessive inflamma-

tory mediators’ production, lead to demyelination and

axonal conduction block. The hallmarks of the advanced

stage of the disease are diffuse degeneration and damage of

neurons. The disease affects young individuals, more

female than male. The general characteristics of the disease

include immunoregulatory deficit, blood–brain barrier

damage, and the consequent CNS parenchyma inflamma-

tory cell invasion, neurological disabilities, demyelination,

and MS plaque formation. Lesions, pathognomonic for

multiple sclerosis, are demyelination plaques in which

infiltration of immune cells, demyelination, oligodendro-

cyte death, and axonal degeneration have been observed. A

variety in MS lesions suggests multiple mechanisms in the

pathogenesis of this disease (Lucchinetti et al. 2000). The

inflammatory cytokines and glutamate neurotoxicity have

been proposed as major determinants accompanying the

demyelination and axonal degeneration observed during

the course of MS.

Glutamate excitotoxicity has been emerged as a poten-

tial mechanism involved in the pathogenesis of MS. In fact,

glutamate levels increase in the cerebrospinal fluid (CSF)

(Sarchielli et al. 2003) and in the brains of MS patients

(Cianfoni et al. 2007) and a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA), N-methyl-D-aspartate

(NMDA), and kainate receptors are upregulated (New-

combe et al. 2008). Using the animal model of MS, known

as experimental autoimmune encephalomyelitis (EAE),

Sulkowski et al. (2013) demonstrated that pharmacological

inhibition of ionotropic NMDA glutamate receptors

(GluRs) by their antagonists (amantadine and memantine)

suppressed neurological symptoms of the disease in EAE

rats and reduced the expression of pro-inflammatory

I. R. Stojanovic (&) � M. Kostic � S. Ljubisavljevic

Faculty of Medicine, University of Nis, Nis,

Serbia and Montenegro

e-mail: stojanovicivana38@gmail.com

123

J Neural Transm

DOI 10.1007/s00702-014-1188-0

cytokines in the brain. Conversely, antagonists of group I

metabotropic glutamate receptors, mGluRs (LY 367385

and MPEP), did not affect the inflammatory process and

the neurological condition of EAE rats. The beneficial

effects in EAE and in MS are not limited only to oligo-

dendrocytes, but also to neuronal damage (Centonze et al.

2009). Although excessive activation of GluRs triggers

uncontrolled intracellular signaling cascades involved in

neuronal toxicity, a family of astrocyte high-affinity glu-

tamate transporters efficiently controls glutamate concen-

tration in the synaptic cleft. Thus, excitotoxicity is also

associated with a significant impairment of glutamate

uptake. There is evidence of altered expression of gluta-

mate transporters in MS (Vallejo-Illarramendi et al. 2006)

and in animal models of the disease (Ohgoh et al. 2002).

Alterations of the mechanisms of glutamate reuptake and

the loss of glutamate transporters are found in MS lesions

in the presence of activated microglia and synaptic damage

suggestive of excitotoxicity (Vercellino et al. 2007).

Glutamate

Glutamate (Glu), a nonessential amino acid, is the major

excitatory neurotransmitter in the central and peripheral

nervous systems (CNS and PNS). As an excitatory neuro-

transmitter, a product of glutamine deamination, a critical

step in nitrogen metabolism, and energy source, this amino

acid has been well studied for its role in cellular homeostasis.

The large body of evidence demonstrated the importance of

glutamatergic signaling in long-term potentiation known to

be fundamental for the processes, such as neuronal plastic-

ity, learning, and memory (Alix and Domingues 2011).

Compared to all other neurotransmitters, the levels of

glutamate are extremely high about 1,000-fold higher than

those of many other important neurotransmitters. Yet,

under pathologic conditions, the glutamate concentration

levels in the brain interstitial space can increase 55-fold

(Bogaert et al. 2000). The extracellular concentrations of

glutamate and other endogenous excitatory amino acids

need to be kept low to limit tonic activation of receptors

and to ensure that the depolarization-evoked release of

glutamate is accompanied by a sufficient increase in GluR

activation and subsequent signaling. If enough ionotropic

Glu receptors are stimulated simultaneously, high con-

centrations of cation influx will result in an action poten-

tial—the fastest type of excitatory synaptic transmission

throughout the CNS and PNS. After the signal is received

by the target cell, excitatory amino acid transporters (EA-

ATs, also known as glutamate transporters) expressed on

postsynaptic and supporting glial cells are tasked with

emptying the synaptic cleft of Glu to effectively turn off

the signal and reset the system for generation and propa-

gation of new action potentials. In addition, it has long

been recognized that excessive activation of GluRs can kill

the cells that express these receptors.

Cerebral oxygen and glucose deprivation may result in

the excessive release of stored synaptic glutamate due to the

loss of ATP stores, leading to potassium efflux and mem-

brane depolarization and the opening of voltage-dependent

sodium channels. As a result, glutamate is released by

synaptic exocytosis and trapped in the interstitium due to

the reversal of the glutamate transporters. This leads to the

overstimulation of NMDA receptors, known as ‘‘glutamate

excitotoxicity’’ (Dempsey et al. 2000; Palmer 2001).

Increased extracellular glutamate may also contribute to

‘‘vasogenic edema’’ and the increase in microvascular

permeability (Abbott 2000). Upon overstimulation of its

ionotropic receptors, the consequent calcium influx leads to

the production of reactive oxygen species, causing a further

release of intracellular glutamate (Love 1999), representing

a vicious cycle in neurons. Moreover, it has been shown

that, in cerebral circulation, polymorphonuclear leukocytes,

upon stimulation by inflammatory stimuli, are capable of

releasing glutamate, which, through the stimulation of

mGluR, causes a breakdown of the endothelial barrier

(Collard et al. 2002; Sharp et al. 2003).

In neuroinflammation, the most probable sources of Glu

are activated microglial cells. Glial cells have the molec-

ular machinery to communicate with neurons (and among

themselves) using neurotransmitters (Agulhon et al. 2010;

Perea and Araque 2010), thus participating in synaptic

transmission via neuronal–glial networking in a manner

that contributes to brain function (Verkhratsky 2010).

Microglia and macrophage accumulation is a common

pathological feature of active MS lesions. Upon lipopoly-

saccharide (LPS) influence, which mimics an inflammatory

environment, they release a large amount of glutamate

through unpaired hemichannels that are openly exposed to

extracellular space (Yawata et al. 2008), or by the xc-

glutamate/cystine antiporter system (Piani and Fontana

1994). In these cells, glutamate is produced by the action of

glutaminase that converts glutamine to glutamate and

ammonia (Newsholme and Calder 1997). Excessive sig-

nalling by excitatory neurotransmitters, like glutamate and

ATP, can be deleterious to neurons and oligodendroglia.

Sustained activation of AMPA, kainate, and NMDA

receptors damages oligodendrocytes (Matute 2006). Thus,

overproduction of glutamate, as well as inhibition of glu-

tamate uptake by activated microglia, can compromise

glutamate homeostasis and induce oligodendrocyte ex-

citotoxicity and myelin destruction.

Glutamate receptors

Glutamate initiates signaling cascades upon binding to its

receptors—GluRs, divided into two major classes:

I. R. Stojanovic et al.

123

ionotropic and metabotropic (Traynelis et al. 2010; Julio-

Pieper et al. 2011). Glutamate ionotropic receptors (iG-

luRs) are classified into AMPA, kainate, and NMDA sub-

types according to their preferred agonist. Molecular

cloning has revealed that each receptors subtype is com-

posed of several subunits with high homology within each

receptor class. Overactivation of these receptors leads to

excitotoxicity, especially in brain regions that are devel-

opmentally and regionally vulnerable to this kind of injury.

The two receptor families differ in their mechanisms of

activation and downstream effectors: iGluRs are voltage-

gated ion channels that initiate Ca2? and/or K? influx and

downstream signaling while mGluRs are atypical G-protein-

coupled receptors (GPCRs), which activate second messen-

ger pathways, such as phospholipase C (PLC), phosphoino-

sitide 3 kinase/retrovirus AK thymoma/mTOR (PI3K/AKT/

mTOR), and mitogen-activated protein kinase (MAPK) sig-

naling. Metabotropic GluRs contain the classic seven-trans-

membrane domain structure and initiate signaling cascades or

cation influx upon Glu binding (Ribeiro et al. 2010). Both

receptor families are further classified into subgroups based

on amino acid sequence homology, pharmacology, second

messenger associations, and other signaling characteristics.

mGluRs are further categorized into the group I, II, and III

subfamilies (Willard and Koochekpour 2013).

The literature data about beneficial effects of NMDA,

AMPA, and kainate receptor antagonists in EAE (Bolton

and Paul 2006) point out the considerable involvement of

glutamate in the pathology of multiple sclerosis. Increased

activation of ionotropic receptors, associated with aberrant

glutamate transporter mechanisms in resident cells of the

CNS, contribute to excess glutamate levels the disturbance

of normal homeostasis and nerve function. Figure 1

summarizes the involvement of glutamate, its receptors,

and cell membrane transporters in neuroinflammation. The

release of glutamate as a consequence of inflammatory

mediators action, as well as direct discharge from resident

or infiltrating cell, lead to an increase of glutamate con-

centrations in CNS.

Activated microglia are present in multiple sclerosis

lesions. Incubation of primary cultured rat microglia with

rat-brain derived myelin for 24-h-induced microglial acti-

vation, inducing the consequent expression of neuronal

caspases and neuronal death in cultured cerebellar granule

cell neurons induced by microglial-derived soluble toxins.

Co-incubation of microglia with agonists or antagonists of

different metabotropic glutamate receptor (mGluR) sub-

types ameliorated microglial neurotoxicity by inhibiting

soluble neurotoxin production. Activation of microglial

mGluR2 exacerbated myelin-evoked neurotoxicity while

activation of mGluR3 was protective as was activation of

group III mGluRs. These data show that myelin-induced

microglial neurotoxicity can be prevented by the regulation

of mGluRs and suggest these receptors on microglia may

be promising targets for therapeutic intervention in multi-

ple sclerosis. (Pinteaux-Jones et al. 2008).

Regulation of glutamate transporters by inflammatory

mediators

In the late 1960s, it was recognized that, when present in

excess, glutamate has the potential to be excitotoxic. That

is why, glutamate uptake is the mechanism responsible for

the long-term maintenance of low, nontoxic concentrations

of glutamate in extracellular space, including the synaptic

cleft. Glutamate transporter proteins, which use the

Fig. 1 Mechanisms of neuronal

damage in neuroinflammation

and neurodegeneration

The role of glutamate

123

electrochemical gradients across the plasma membranes,

uptake glutamate from extracellular space into neurons and

glial cells. In humans, there are five different subtypes of

high-affinity glutamate transporters, named sodium- and

potassium-coupled glutamate transporters or, preferably,

excitatory amino acid transporters 1–5 (EAAT 1–5)

(Danbolt 2001). The flux of transported molecules depends

on the density of transporters in the cell plasma membrane,

which determines whether synaptic independence is com-

promised by the synaptic transmitter cross talk. Glial cells

express GLT-1 (GAAT2) and GLAST (GAAT1), which

facilitate the uptake of glutamate and its accumulation in

synaptic vesicles (Kanai et al. 1997). The dysfunction of

these transporter systems in both neural and glial cells may

result in excitotoxic damage, which has been proved in

EAE (Ohgoh et al. 2002).

Under pathologic conditions, these transporters could

become either inoperative or acting reversibly and raise

extracellular glutamate concentrations. Numerous studies

indicate that in neuroinflammation, proinflammatory cyto-

kines (TNFa and IL-1b) mediate negative regulation of the

expression and activity of glutamate transporters (Tilleux

and Hermans 2007).

Besides, in pathological conditions, Na?-independent,

high-affinity glutamate transport system that carries cystine

into the cell in exchange for internal glutamate, named the

glutamate/cystine exchanger or xc- antiporter system, is of

great importance. In human brain, it is present mostly in

neurons, but it could be also found in glial cells (Burdo et al.

2006). In pathological occasions, astrocytes can release

glutamate using multiple mechanisms, both Ca2?-dependent

and Ca2?-independent. The second one includes reversed

action of glutamate reuptake carriers, exchange with cystine,

the essential substrate for astrocytic production of glutathi-

one, mediated by cystine–glutamate antiporter. The study of

Domercq et al. (2007) provided evidence that the concom-

itant glutamate release from activated microglia by the

cystine/glutamate antiporter and the inhibition of Na?-

independent glutamate uptake by activated microglia could

induce a local increase in extracellular glutamate, leading to

excitotoxic oligodendrocyte death. During the clinically

active phase of MS, activated monocytes, macrophages, and

microglia infiltrating the CNS overexpress the xc- antiporter

system and intensively release glutamate (Pampliega et al.,

2011). In high extracellular glutamate concentrations, xc-

antiporter function is inhibited, inducing a decrease in cys-

tine uptake and intracellular glutathione level, thus favoring

oxidative stress in neurons and oligodendrocytes.

Glutamate and neuroinflammation

Neuroinflammation is a complex process with multiple

mediators, signaling pathways, and feedback loops. It

comprises the activation of glial cells, recruitment of

peripheral immune cells, and production of cytokines, such

as interferon-gamma (IFN-c) and tumor necrosis factor-

alpha (TNF-a) (Neumann 2001). TNF-a mediates cyto-

toxic damage to glial cells and neurons, while IFN-cinduces cell surface molecules important in immune and

brain cells interactions between (Imai et al. 2007).

Reactive glia shift toward proinflammatory phenotype,

thus releasing cytokines, chemokines, and neurotoxic

molecules (Zindler and Zipp 2010). Activated glia affects

neuronal injury and death through the production of neu-

rotoxic factors like glutamate, S100B, TNF-a, IL-1b,

prostaglandins, and reactive oxygen and nitrogen species.

As disease progresses, inflammatory secretions engage

neighboring cells, including astrocytes and endothelial

cells, resulting in autocrine and paracrine amplification of

inflammation, leading to neurodegeneration.

Bender et al. (2005) documented that the increased

levels of TNF-a and IL-1b could alter the activity of

neurons. Using primary rat and human neuronal cultures,

Ye et al. (2013) proved that these two proinflammatory

cytokines induced cell death and apoptosis in vitro. By

binding to neuronal TNF receptors, TNFa can cause cell

death directly linked to death domains that activate cas-

pase-dependent apoptosis (Zhao et al. 2001). This cytokine

can induce additional release of ROS, by inducing NADPH

oxidase activity (Li et al. 2005).

Both intra- and extracellular glutamate levels were

increased upon TNF-a and IL-1b treatment (Ye et al. 2013).

In this study, pretreatment with NMDA receptor antagonist

MK-801 blocked cytokine-induced glutamate production

and alleviated neurotoxicity, indicating that these cytokines

induced neurotoxicity through glutamate. It was documented

that TNF-a was higher in CSF of progressive MS subjects

(Rossi et al. 2014). In murine brain slices, incubated in the

presence of CSF from progressive MS patients, these authors

observed increased spontaneous excitatory postsynaptic

currents and glutamate-mediated neuronal swelling through

a mechanism dependent on enhanced TNF-a signaling,

pointing out TNF-a as a primary neurotoxic molecule in

progressive forms of MS. They also suggested a pathogenic

role of B cells in TNF-a CSF increase, associated with

exacerbation of glutamatergic transmission and neuronal

damage. Besides, Ye et al. (2013) indentified glutaminase as

an important player in glutamate overproduction during

inflammatory cytokine stimulation, inducing dysregulation,

translocation or release of glutaminase isoforms, which

consequently induced neurotoxicity and apoptosis.

In mouse cortical astrocytes, Fang et al. (2012) reported

that the chemokine macrophage inflammatory protein-2c(MIP-2c) increased significantly upon stimulation with LPS

or TNF-a in vitro. They suggested that MIP-2c mediated the

pathogenesis of CNS disorders associated with neutrophil

I. R. Stojanovic et al.

123

infiltration in the brain and decreased GLT-1 activity. This

chemokine reduces the expression of glutamate transporter-1

on astrocytes and increases neuronal sensitivity to glutamate

excitotoxicity. Astrocytes overexpressing MIP-2c down-

regulated the expression of GLT-1 at the mRNA and protein

level and caused redistribution of GLT-1 out of the lipid

rafts that mediate glutamate uptake.

The data of Tolosa et al. (2011) link two important

pathogenic mechanisms, excitotoxicity and neuroinflamma-

tion, proving that TNF-a-induced nuclear factor-kappaB

(NF-jB) activation potentiates glutamate excitotoxicity on

spinal cord motononeurons. The authors reported that

chronic glutamate excitotoxicity, induced by the glutamate

uptake inhibitor threohydroxyaspartate (THA), resulted in

motoneuron loss that was associated with a neuroinflam-

matory response, which was potentiated with TNF-a and

mediated by the downregulation of the astroglial glutamate

transporter-1 (GLT-1), which were prevented by NF-jB

inhibition. Furthermore, TNF-a and THA also cooperated in

the induction of oxidative stress in a mechanism involving

the NF-jB signalling pathway as well.

Furthermore, the treatment with TNF-a inhibitors

showed a beneficial effect on EAE (Lim and Constan-

tinescu 2010), while in MS patients this treatment had

opposite effects with even worsening of the disease proved

by magnetic resonance imaging (van Oosten et al. 1996).

Also, the studies showed that anti-TNF-a agents may ini-

tiate or unmask and underly demyelinating disease

(Kaltsonoudis et al. 2014).

Glutamate excitotoxicity is known to contribute to

autoimmune neuroinflammation (Melzer et al. 2008; Pitt

et al. 2000). In inflammation, activated microglia and

astrocytes release and maintain high level of extracellular

glutamate (Takeuchi et al. 2006). Also, glutamate leakage

from serum across the compromised BBB in CNS inflam-

mation, plus infiltrating inflammatory leukocytes and acti-

vated resident microglia with the potential to synthesise and

release glutamate provides continuous, local supply of this

neurotransmitter. Microglia are known to generate reactive

oxygen and nitrogen species that impair glutamate uptake

mechanisms. The permanent increased availability of glu-

tamate would induce upregulation of its receptors and the

synthesis of molecules responsible for neuronal dysfunction

(Ohgoh et al. 2002). In CNS, activated cells release proin-

flammatory cytokines, which may reinforce local glutamate

excitotoxicity, such as TNFa, known to reduce the

expression of EAATs and detoxifying enzymes in glial

cells, thus limiting their capacity for glutamate uptake.

Astrocytes play direct, active, and critical roles in

mediating neuronal survival and function. The removal of

glutamate from the extracellular space by astrocytes con-

fers neuroprotection, while astrocyte release of potentially

toxic molecules promotes neurodegeneration (Hauser and

Cookson 2011). Still, the exact mechanism of inflamma-

tory mediators in the disease progression is still poorly

understood.

The key cells in neurodegeneration, formed on the

pathogenic substrate of inflammation, are activated mi-

croglial cells. When stimulated by proinflammatory sig-

nals, microglia may undergo a reaction that includes a

morphological transformation into ameboid shape, pro-

ducing prostanoids, cytokines, chemokines, nitric oxide,

inducing surface markers, including members of the major

histocompatibility complex family and initiating oxidative

burst (Block and Hong 2005; Decoursey and Ligeti 2005).

Activated microglia and the release of molecules that are

detrimental to oligodendrocyte have been suggested as

mechanisms by which innate immunity causes demyelin-

ation in MS. In early inflammation, microglia initiate

immune responses by enhancing the expression of toll-like

receptors (TLR) and a wide range of proinflammatory

mediators (TNFa, IL-1, and IL-6 for the removal of the

CNS threat (Floden et al. 2005). They proliferate, migrate,

phagocyte, produce oxidants, and induce gene expression

(iNOS, COX-2, MHC class II, complement, etc.).

Microglial cells are likely to play a dual role in MS,

depending on signals present in their microenvironment.

While early microglial activation could represent a bene-

ficial response (promoting tissue repair and removal of

misfolded protein), in chronic phase, microglia express

detrimental effects and promote neuronal death, thus con-

tributing to the progression of the disease.

It has been hypothesized that microglial proliferation is

followed by programmed cell death (Gehrmann and Banati

1995), possibly as a compensatory feedback phenomenon.

Whether or not activation of microglia during the acute

phase of EAE is sufficient to induce neuronal cell death is

still a matter of debate (Aarum et al. 2003; Butovsky et al.

2006, Walton et al. 2006). Nevertheless, the morphological

and functional changes of microglial cells observed in mice

with EAE (Centonze et al. 2010) clearly support the idea

that activated microglia exert a key role in synaptic alter-

ations and possibly neuronal damage. The consequent

damage to oligodendrocytes, neurons, and the BBB, plus

inflammatory cytokine release from microglia, contribute

considerably to the pathology of EAE and MS and could be

considered as one of the major disturbances in neuroin-

flammation. Although it is obvious that neuroinflammation

by itself is not the cause of neuronal cell death, nowadays,

there is strong evidence that neuroinflammation contributes

to neurodegeneration.

Glutamate neurotoxicity and neurodegeneration in MS

Chronic inflammation and the consequent damage of neurons

are considered as the key processes in neurodegenerative

The role of glutamate

123

diseases (Huang et al. 2005). Neuroinflammation in MS is

considered as one of the constitutive components of the

disease pathogenesis and the lesions generation. Multiple

sclerosis has been considered for a long time only as an

inflammatory demyelinating disease. The evidence is now

increasing that excessive glutamate is released at the site of

demyelination and axonal degeneration in MS plaques, and

the most probable candidates for this cellular release are

infiltrating leukocytes and activated microglia. The molecu-

lar mechanisms linking systemic inflammation and neuronal

excitotoxicity are still poorly understood. The study of Degos

et al. (2013) provides experimental support that group I

mGluRs are involved in the mechanisms underlying

inflammation-mediated sensitization to excitotoxic neuro-

degeneration. The mechanisms of neurodegeneration in MS

are likely multifactorial and include direct damage by T cells

and humoral immunity as well as oxidative stress, glutamate-

mediated excitotoxicity, and neuronal and oligodendrocyte

apoptosis.

Excitotoxicity is the pathological process by which

nerve cells are damaged or killed by abnormal stimulation

by excitatory neurotransmitters. Dysregulation of gluta-

mate signaling leads to neurodegeneration (Maragakis and

Rothstein 2006). Excitotoxic neuronal death can be direct,

as the result of excessive stimulation of NMDA receptor or

indirect. The hypothesis of indirect excitotoxic death

pathway suggests that bioenergetic deficit causes depolar-

ization when the nontoxic levels of glutamate become

lethal (Kroemer et al. 2007).

The numerous triggers, such as oxidative stress, calcium

overload, mitochondrial dysfunction, and energy depletion,

can lead to changes in neuronal excitation process, which

has been proved to be involved also in multiple sclerosis

pathogenesis (van Horssen et al. 2012). These mechanisms

lead to the damage of proteins, nucleic acids, and lipids and

mitochondrial disruption followed by overproduction of

free radicals, energy depletion, and pro-apoptotic factors

activation, resulting in death of neuronal cells (Farooqui

and Farooqui 2009).

Oxidative stress and excitotoxicity

Glutamate receptor overstimulation is the main mediator to

intracellular oxidative stress (Kumar et al. 2011). The

recent studies suggest strong relationship between exces-

sive calcium influx and glutamate-triggered neuronal injury

(Sendrowski et al. 2013). The prolonged elevation of

intracellular calcium concentration occurs due to an

excessive depolarization of neurons as well as due to

release from internal stores, mitochondria, and endoplas-

mic reticulum, or the malfunction of receptors and chan-

nels present in their membranes. The increased

intracellular calcium concentration can trigger a range of

downstream neurotoxic cascades, with mitochondria play-

ing a central role in cell biology as ATP producer and

regulators of calcium signal, resulting in increased forma-

tion of ROS and activation of both caspase-dependent and

caspase-independent apoptotic-like cell death (Lipton

2008). Reactive oxygen species can exert multiple dam-

aging reactions to proteins, lipids, carbohydrates, and

nucleic acids, thereby disrupting cellular functions (Mar-

tindale and Holbrook 2002). Lipid peroxidation results

from the chemical attack by free radicals on fatty acids in

membranes (Allen et al. 2012) and leads to membrane

damage and cell lyses.

NO and other reactive nitrogen species

Nitric oxide (NO), the free radical and intra- and inter-

cellular messenger molecule, is synthesized from L-argi-

nine in the reaction catalyzed by nitric oxide synthases

(NOSs) family: neuronal (nNOS, NOS1), endothelial

(eNOS, NOS3), and inducible (iNOS, NOS2) isoforms

(Steinert et al. 2010, West and Tseng 2011). In the brain,

eNOS is expressed in cerebral vascular endothelial cells,

while iNOS is expressed in astrocytes and microglia cells

in response to inflammatory stimuli. Due to specific cel-

lular distribution, they play different roles in both physio-

logic and pathologic processes.

Neuronal NOS activity increases upon intracellular

calcium increase, so overactivation of NMDA receptors

and the consequent flux of calcium provide a link between

an excitotoxic insult and NO-mediated cell damage. Ex-

citotoxicity is further intensified by NO, which stimulates

glutamate release from astrocytes. The recent studies

document nitric oxide involvement in the pathology of

many neurodegenerative diseases (Kumar et al. 2011).

There is evidence that NO and iNOS are elevated in

CNS and plasma in both MS (Stojanovic et al. 2012; Ba-

gasra et al. 1995) and experimental allergic EAE, an

experimental model of MS (Ljubisavljevic et al. 2012).

iNOS expression, by itself, is not able to induce cell death.

The double key in the mechanism of neurodegeneration is

simultaneous activation of iNOS and phagocyte NADPH

oxidase with the consequent effects mediated by perox-

ynitrite consequent to the initiation of an oxidative burst

that produces superoxide from an orchestrated mechanism

involving NADPH oxidase (Block and Hong 2005; De-

coursey and Ligeti 2005). Although free radical NO is not

as reactive as some other ROS, so the predominant con-

tribution of NO to excitotoxicity depends on increased

superoxide ion (O2_) production. It rapidly reacts with NO

forming peroxynitrite (ONOO-) (Kumar et al. 2012). This

exposure to NO/O2_ with resultant ONOO- formation

results in necrosis of neurons or apoptosis, depending on its

intensity (Rossi et al. 2014).

I. R. Stojanovic et al.

123

Rose et al. (2004) have suggested a mechanism that

operates through the actions of two enzymes, cyclooxy-

genase-2 (COX-2) and inducible nitric oxide synthase

(iNOS), both of which have been documented to be present

in MS lesions. COX-2-derived prostanoids, present at high

concentrations in EAE and MS CNS, stimulate glutamate

release. Additionally, nitric oxide (NO), from iNOS, can

increase COX-2. It also reacts with reactive oxygen species

(ROS), forming peroxynitrite (ONOO-) that inactivates the

glutamate transporters (Gurwitz and Kloog 1998) and

directly damages myelin, oligodendrocytes, and axons

(Mattle et al. 2004).

Calcium overload

Although it is known that every class of GluRs is involved

in excitotoxic cell death, it is broadly accepted that NMDA

receptors play the most important role, due to their per-

meability for calcium (Kroemer et al. 2007). Upon their

activation, a massive influx of extracellular Ca2? leads to

the activation of a number of Ca2?-dependent enzymes,

such as proteases, protein kinases, phosphatases, phos-

pholipases, nitric oxide synthase, and endonucleases, that

influence a wide variety of cellular components involved in

a number of cellular processes (Nicholls 2004) that are

associated with neuritic degeneration and cell death

through different pathways, including membrane break-

down, cytoskeleton alterations, and nitric oxide-derived

free radicals formation (Norenberg and Rao 2007).

One of the hallmarks of glutamate excitotoxicity is the

degradation of the neuronal cytoskeleton mediated by

NMDA receptor activity. Chung et al. (2005) suggested

that excitotoxicity triggered a progressive pathway of

cytoskeletal degeneration within axons, initially charac-

terized by the loss of neurofilament proteins. The state of

axonal integrity depends on the adequate phosphorylation

of cytoskeleton proteins, especially microtubule-associated

proteins, and neurofilaments. An important second mes-

senger system that regulates the phosphorylation of cyto-

skeletal proteins is Ca2?. Upon activation of glutamatergic

receptors, the consequent intracellular Ca2? increase

results in changes in microtubules and neurofilaments

phosphorylation state. Chung et al. (2005) found that

treatment with even nontoxic levels of glutamate resulted

in dramatic alterations in the axonal cytoskeleton that

ultimately led to the total degradation of axonal structure.

Mitochondrial dysfunction

There is an emerging evidence that mitochondrial dys-

function actively contributes to neurodegeneration and the

damage of axons (Witte et al. 2013; Fiebiger et al. 2013;

Mahler et al. 2012; Virgili et al. 2013). As ATP producers

and regulators of calcium signal, mitochondria play a

central role in cell biology. The brain consumes about

20 % of total oxygen and, due to great energy demands,

has an intensive oxidative metabolism. More than half of

the energy in the brain is used to restore resting potential in

excitatory cells (Ames, 2000). In physiological conditions,

calcium induced depolarization and calcium uptake regu-

late activation of pyruvate, a-ketoglutarate, and isocitrate

dehydrogenases (Yacoubian et al. 2010), as well as mito-

chondrial dehydrogenases and mitochondrial ATP synthase

(Brandon et al. 2006). However, during overactivation of

NMDA receptor and excessive influx of Ca2?, there is also

Ca2? release from intracellular compartments (mitochon-

dria and endoplasmic reticulum), which together over-

whelm Ca2?-regulatory mechanisms, leading to these

enzymes inhibition, as well as respiratory chain complex I

and the subsequent spread of excitation and neuronal death

(Sendrowski et al. 2013).

There is accumulated evidence that mitochondrial dys-

function contributes to axonal degeneration in inflamma-

tory phase of MS, as well as that it is an important

mechanism of neuron degeneration in the chronic stage of

the disease (Witte et al. 2010; Mahad et al. 2009; Trapp

and Stys 2009). In active demyelinating MS lesions, in

both axons and neurons, the increased mitochondrial den-

sity was associated with the enhanced expression of

mtHSP70, the marker of mitochondrial stress (Witte et al.

2009; Mahad et al. 2008). In inflammatory MS lesions,

macrophages and activated microglia produce ROS, which

damage proteins, lipids, and DNA. Lu et al. (2000) docu-

mented that in these conditions, the most damage was

found in mitochondrial DNA (mtDNA). It has been known

that mtDNA is 10 times more vulnerable to oxidative

damage than nuclear one (Mecocci et al. 1993). This was

associated with decreased activity of mitochondrial respi-

ratory chain complex I and the decrease in oxidative

phosphorylation. Mahad et al. (2008) found that the

decreased respiratory chain complex IV activity in ful-

minant MS lesions correlated with the number of activated

microglia and infiltrated macrophages. The consequent

decreased ATP production, together with an increased ROS

production by respiratory chain itself, contributes to the

degeneration of axons in MS (Browne et al. 1997). Fur-

thermore, mitochondria in nondemyelinated MS gray

matter neurons were also found to have decreased activity

of complexes I and III (Dutta et al. 2006). Increased

mitochondrial ROS production and decreased activity of

complexes I and III strengthen each other, leading to an

accumulation of oxidative damage and mitochondria-dri-

ven degenerative mechanisms of axonal injury.

Apart from the inhibition of enzyme activity and

respiratory chain disturbances, the opening of a large

nonselective pore in the inner mitochondrial membrane,

The role of glutamate

123

termed ‘‘mitochondrial permeability transition pore’’

(MPTP), has also been suggested as a key mechanism in

neuronal excitotoxicity (Choi et al. 2013). The induction of

MPTP can consecutively trigger apoptotic cascades via

liberation of apoptosis-induced factor or cytochrome C,

leading to cell death due to mitochondrial depolarization

(Veto et al. 2010). Forte et al. (2007) reported that cyclo-

philin D (a key regulator of the MPTP) knockout mice

were less sensitive to oxidative and nitrosative axonal

damage, indicating that their axonal mitochondria are more

resistant to inflammation-derived ROS and subsequent

mitochondrial dysfunction, resulting in less axonal

degeneration.

The mechanism of excitotoxicity is now accepted in

terms of glutamate role in the pathogenesis of neurode-

generative diseases. The two key pathways that trigger

excitotoxicity involve glutamate neurotoxicity and intra-

cellular calcium overload. In MS, almost all aspects of

glutamate homeostasis are pathologically altered, which

point out glutamate excitotoxicity as an important mecha-

nism in the pathogenesis of the disease. The present body

of evidence suggests that immunoinflammatory and neu-

rodegenerative processes coexist in MS and that glutamate

excitotoxicity is a link between them.

These observations have already been partially con-

firmed, beside in animal models, by postmortem studies

and in vivo analyses in MS patients, thus raising the pos-

sibility that modulation of glutamate release and transport,

as well as receptors blockade or glutamate metabolism

modulation, might be relevant targets for the development

of future therapeutic interventions (Frigo et al. 2012; Rahn

et al. 2012). Positive outcomes (decreased neuronal loss,

improved cognition) that have been demonstrated suggest

that glutamate metabolism and transport modulation could

be a promising target in the prevention and delay of neu-

rodegeneration and cognitive impairment in multiple

sclerosis.

Acknowledgments This paper was supported by The Ministry of

Education and Science of the Republic of Serbia under the project

number 41018.

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