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NeuroToxicology

Perspectives on neuroinflammation and excitotoxicity: A neurotoxic conspiracy? Barbara Viviani *, Mariaserena Boraso, Natalia Marchetti, Marina Marinovich Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita` degli Studi di Milano, Milan, Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 November 2013 Received in revised form 11 March 2014 Accepted 11 March 2014 Available online xxx

Emerging evidences underline the ability of several environmental contaminants to induce an inflammatory response within the central nervous system, named neuroinflammation. This can occur as a consequence of a direct action of the neurotoxicant to the CNS and/or as a response secondary to the activation of the peripheral inflammatory response. In both cases, neuroinflammation is driven by the release of several soluble factors among which pro-inflammatory cytokines. IL-1b and TNF-a have been extensively studied for their effects within the CNS and emerged for their role in the modulation of the neuronal response, which allow the immune response to integrate with specific neuronal functions, as neurotransmission and synaptic plasticity. In particular, it has been evidenced a potential detrimental link between these cytokines and the glutamatergic system that seems to be part of increased brain excitability and excitotoxicity occurring in different pathological conditions. Aim of this mini-review will be to present experimental evidence on the way IL-1b and TNF-a impact neurons, focusing on the glutamatergic signalling, to provide a perspective on novel pathways possibly involved in environmental contaminants neurotoxicity. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Neuroinflammation Glutamatergic system IL-1b TNF-a Environmental contaminants

1. Introduction In these very last years neuroinflammation has been an emerging topic in neuroscience and neurotoxicology due to its relevance in the progression of neurodegeneration and neuronal dysfunction. The term neuroinflammation define an inflammatory response organized within the nervous system, which rises following the exposure to a trigger directly acting within the central nervous system or via a systemic (‘‘peripheral’’) inflammatory response. As such, this process contemplates the contribution of microglia, regarded as the principal cells of the innate brain immune system. In addition astrocytes, neurons, oligodendrocytes and vascular pericytes participate in cytokines related neuroinflammatory processes (Colton and Wilcock, 2010; Ransohoff and Engelhardt, 2012; Ransohoff et al., 2003). Neuroinflammation has been initially implicated in the progression and exacerbation of neurodegenerative diseases (Block et al., 2007; Wang and Shuaib, 2002), in which overactivation and

* Corresponding author at: Department of Pharmacological and Biomolecular Sciences, Universita` degli Studi di Milano, via Balzaretti 9, 20133 Milan, Italy. Tel.: +39 0250318241. E-mail address: [email protected] (B. Viviani).

dysregulation of glial cells actively contributes to neuronal damage by the excessive production of a large array of cytotoxic factors among which pro-inflammatory cytokines such as interleukin-b (IL-1b) and tumour necrosis factor-a (TNF-a) (Allan and Rothwell, 2001; Montgomery and Bowers, 2012). Currently available evidence shows that cytokines play also a role in cognitive processes and may be implicated in the pathogenesis of psychiatric disorders such as major depression, dementia and schizophrenia (McAfoose and Baune, 2009; Meyer et al., 2011). It is now becoming clear that activation of microglia and astrocytes along with the release of inflammatory cytokines is a feature that often accompanies exposure to several environmental contaminants (Kraft and Harry, 2011) linked to the development of both neurodegenerative diseases (i.e. organophosphates, Mn) (Freire and Koifman, 2012) and cognitive disturbances (i.e. environment particulate matter) (Chen and Schwartz, 2009; Fonken et al., 2011) (Table 1). These experimental evidences support the possibility that release and production of inflammatory cytokines by neurotoxicants might represent a link with behavioural deficits and the progression of neurological diseases. Indeed, drugs that extinguish the inflammatory response may ameliorate the neurotoxic outcome in different in vivo models of neurotoxicity (Chung et al., 2011; Dutta et al., 2010; Ferger et al., 2004; Mangano et al., 2012; Zhang et al., 2010) (Table 1).

http://dx.doi.org/10.1016/j.neuro.2014.03.004 0161-813X/ß 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Viviani B, et al. Perspectives on neuroinflammation and excitotoxicity: A neurotoxic conspiracy? Neurotoxicology (2014), http://dx.doi.org/10.1016/j.neuro.2014.03.004

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Table 1 Neurotoxicants whose effect has been associated to an increase of IL-1b and TNF-a within different brain areas. The neurotoxic effect and, when evaluated, the relevance of the neuroinflammatory response on the observed effect have been reported. Hp, hippocampus; CX, cortex; PM, particulate matter; OB, olfactory bulb; a-syn, a-synuclein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SN, substantia nigra; SNc, substantia nigra pars compacta; FC, frontal cortex. Toxicant

Organophospates Soman From 30 min to 7 d

172 mg/kg (+oxime)1 180 mg/kg (+oxime, +atropine)2,3 s.c Sarin From 2 h to 30 d1 108 mg/kg (+oxime, +atropine) i.m. Dimethoate 5 weeks/3 times week 1.4 mg/kg i.p. Dichlorvos 12 weeks 2.5 mg/kg sc Others Manganese 50–1000 mM2 1–30 mM3 1 mmol/ml Intra-striatal1

MPTP 4 injection/each 2 h

IL-1b and/or TNF-a production

Effect related to cytokines increase/ relevance of inflammation Possible involvement of glutamate

References

Hp, pyriform cortex, Thalamus

NMDA-dependent seizures1,2, neuronal damage and glial activation both reduced by ketamine (NMDAR-antagonist and anti-inflammatory)+ atropine1

1

mRNA1,2 and protein1,3

(Dhote et al., 2012, 2007) (in vivo)

2

(Dillman et al., 2009) (in vivo) (Johnson and Kan, 2010) (in vivo)

3

Hp and Cx Protein

Seizures, brain damage

(Chapman et al., 2006) (in vivo)

Hp, striatum

Increased TNF-a per se, potentiate LPS (5 mg/kg) effect

(Astiz et al., 2013) (in vivo)

Ventral midbrain, Corpus striatum Protein and mRNA

Concomitant selective loss of DA neurons

(Binukumar et al., 2011) (in vivo)

Microglia: N92, primary3 Striatum1 mRNA1 and protein2,3

In vitro potentiate LPS (10–500 ng/ml2; 0.5–2 ng/ml3) effect Minocycline1,3 and Naloxone3 reduce IL-1, TNF-a and dopaminergic neurotoxicity3 circumstantial evidences (see text)

1

SN

Effectivness of fluoxetine in reducing IL1 and TNF-a production, microglia derived neurotoxicity and promoting partial motor recovery

(Chung et al., 2011) (in vivo)

Hp mRNA

Development of depressive-like responses and impairment in spatial learning and memory as compared to mice exposed to filtered air

(Fonken et al., 2011) (in vivo).

CX mixed glia (primary) mRNA

Altered expression of subunits of glutamate receptors. Increase NMDAinduced neuronal death in hippocampal slices. Impaired neurite outgrowth in embryonic neuron cultures

(Morgan et al., 2011) (in vivo, in vitro)

mRNA

(Zhao et al., 2009) (in vivo) (Filipov et al., 2005) (in vitro) (Zhang et al., 2010) (in vitro)

2 3

20 mg/kg i.p. Particle Matter PM2.5 10-months/6 h day/5 days a week 16.85 mg/m3 normalized over 10 month

nPM 10-week/5 h day/3 day week 0.3 L/min Vitro: 10 mg/ml 24 h

Thus, neuroinflammation represents a potential new mechanism to revisit classical neurotoxicants suggesting novel approaches to develop most effective countermeasure for neurotoxicity. Nevertheless, we have to take advantage from the knowledge accumulating in the treatment of neurodegenerative diseases showing that the use of anti-inflammatory drugs to prevent or slow their progression has yielded controversial results (Schwartz and Shechter, 2010). This suggests that a better understanding of neuroinflammatory dynamics is mandatory in order to identify new targets for a most successful approach. Although glial and immune cells play a central role in the onset of neuroinflammation, the final output of this process relies on a

delicate balance between the production of inflammatory mediators and the ability of neurons to sense them through the expression of specific receptors. This latter point in particular has received little attention to date. Thus, while referring to relevant studies of glial and peripheral cells participation in organizing both inflammatory states and neurotoxicity, this mini-review will emphasize the way neurons sense, adapt and respond to pro-inflammatory cytokines. In this context, we will take into account the signalling system of the two most studied cytokines, interleukin-b (IL-1b) and tumour necrosis factor-a (TNF-a) and the interaction with the glutamatergic system, also discussing the possible impact on neurotoxicity to suggest novel pathways worth to be investigated.

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2. Cytokines and cellular mediators of neuroinflammation Inflammatory cytokines represent key elements in the communication existing between immune vs. surrounding cells, regulating tissue responses to infection, inflammation and stress. Cytokine within the CNS have two possible origins: the peripheral immune organs and/or the CNS itself. Despite the existence of the blood brain barrier (BBB), peripherally produced cytokines may enter the CNS by passive diffusion at the circumventricular organs (brain areas lacking the blood brain barrier) or active transport across the blood brain barrier (Banks, 2006; Banks et al., 2001; Maier, 2003). Another possibility is the synthesis by resident and/ or peripheral immune cells invading the CNS (Block et al., 2007; Ransohoff and Engelhardt, 2012). Microglia are the major resident immune cells in the brain, where they constantly survey the microenvironment and produce factors, among which cytokines, that influence surrounding astrocytes and neurons. Pathogen invasion, tissue damage, neurotoxic exposure are implicated in the switch of microglia to an activated phenotype and thereby promote an inflammatory response that serves to further engage the immune system. Inflammatory responses to infectious agents are initiated by pattern recognition receptors (PRRs) that bind pathogen-associated molecular patterns (Block et al., 2007; Kraft and Harry, 2011; Saı¨d-Sadier and Ojcius, 2012). Several members of the PRRs family and multiple additional receptors (i.e. neurotransmitter receptors, purinergic recptors) also recognize endogenously derived molecules that are generated as a consequence of tissue injury or other pathological processes (i.e. protein aggregates, components of dead neurons, extracellular nucleotides) (For extensive review see: (Biber et al., 2007; Block et al., 2007; Kraft and Harry, 2011)). These signals have been termed ‘‘On’’ signals for their ability to instruct microglia activation (Biber et al., 2007). CNS injury or neuronal demise also activate microglia through the disappearance of the so called ‘‘Off’’ signals (Biber et al., 2007). The ‘‘Off’’ signals are carried (membrane bound, i.e. CD200, CD22, and CD47) or released (i.e. TGF-b, CD22, CX3CL1, growth factors) by healthy neurons and constitutively keep microglia in their resting state (Biber et al., 2007). Both the loss of ‘‘Off’’ and the appearance of ‘‘On’’ signals indicate a threat to the structural and functional integrity of the CNS leading to a robust production of proinflammatory mediators such as TNF-a, IL-1b, IL-6, ROS (Biber et al., 2007; Lehnardt, 2010). In addition to these biological stimuli, some in vitro evidences suggest the possibility that chemicals and environmental compounds might directly activate microglia (Dutta et al., 2010; Ni et al., 2011; Wang et al., 2011). Astrocytes are the major glial cell type in the CNS and also participate in inflammatory responses. Astrocytes express a more limited PRRs repertoire, which probably stems from the fact that they are not classical immune cells but can contribute to inflammation if necessary. Cytokines and other soluble products released by microglia contribute to recruit astrocytes in the inflammatory response. Like microglia, astrocytes become activated, a process known ‘astrogliosis’, which is characterized by altered gene expression, increased expression of marker molecules (i.e. GFAP and vimentin), hypertrophy, and proliferation (Ridet et al., 1997). Activated astrocytes release a wide array of immune mediators, contributing either to the response raised by microglia (Farina et al., 2007). The neuroinflammatory response is not only confined to glial cells. Peripheral immune cells also, and no less crucially, participate (Ransohoff and Engelhardt, 2012). Macrophages, perivascular monocytes, and lymphocytes (T and B cells) may be recruited within the CNS and participate in magnifying the local response to an acute or chronic CNS insult. This easily occurs both through the compromised blood–brain barrier (BBB), a feature

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accompanying several CNS diseases, and intact BBB during peripheral inflammation thanks to cytokine-driven expression of adhesion factors (D’Mello et al., 2009; Ransohoff et al., 2003). While short-term and thus controlled neuroinflammatory response is generally accepted to serve a neuroprotective role, chronic activation has been implicated as a potential mechanism in neurodegenerative disorders. As such, release of pro-inflammatory cytokines is intended to prevent further damage to CNS tissue, but may also be detrimental to neurons and other glial cells when dysregulated (Smith et al., 2012; Wang and Shuaib, 2002). 3. Cytokines and the glutamatergic system: a shared pathway to neuronal dysfunction and death Among the pro-inflammatory mediators that can be produced and released within the CNS, a special emphasis has been placed on inflammatory cytokines like IL-1 (mainly IL-1b) and TNF-a, possibly for their involvement in a plethora of CNS diseases and neurotoxic conditions. IL-1 and TNF-a expression at both messenger and protein levels, are rapidly up-regulated in response to clinical or experimental damage in the brain. Both these cytokines raise in brain or CSF of patients affected by acute and chronic neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, stroke, traumatic brain injury and HIVassociated dementia (Griffin et al., 1995; Shiozaki et al., 2005; Tarkowski et al., 2003, 1999; Zhao et al., 2001), as well as in animal models mimicking those diseases or exposed to environmental neurotoxicants (Table. 1) (Astiz et al., 2013; Binukumar et al., 2011; Chapman et al., 2006; Chung et al., 2011; Dhote et al., 2007; Dillman et al., 2009; Fonken et al., 2011; Henderson et al., 2002; Liu et al., 2009; Mangano et al., 2012; Mitra et al., 2011; Morgan et al., 2011; Zhang et al., 2010; Zhao et al., 2009). The presence of IL-1 and TNF-a at detectable levels along with the development of diseases or neurotoxicity does not prove them either beneficial or detrimental. Nevertheless, an amelioration of several forms of neuronal damage has been observed as a result of the neutralization of TNF-a or IL-1b response such as in KO animals for these cytokines (Boutin et al., 2001) or for their receptors (He et al., 2007; Sriram et al., 2002), as well as in the presence of (i) interleukin-1 receptor antagonist (Bagetta et al., 1999; Relton and Rothwell, 1992), (ii) soluble TNF inhibitors (McCoy et al., 2006) or (iii) specific antibodies (Yamasaki et al., 1995). This suggests that an increased expression of this cytokines is indeed harmful. In accordance, intracerebral injection of recombinant IL-1 markedly exacerbates ischaemic and excitotoxic brain damage (Yamasaki et al., 1995) as over-expression of IL-1b in the substantia nigra of rats results in dopaminergic cell death (Ferrari et al., 2006). Finally, the observation that human IL-1 and/or TNF-a gene polymorphisms – associated with their increased production – increase the relative risk for Alzheimer’s and Parkinson’s Disease (Di Bona et al., 2009, 2008; Wahner et al., 2007) is also consistent with a detrimental role. Since the pharmacological modulation of TNF-a and IL-1 production and response is effective in counteracting also neuronal damage induced by exposure to neurotoxicants, it has been assumed that the connection between cytokines production and occurrence of harmful effects extends beyond pathology (Chung et al., 2011; Dutta et al., 2010; Ferger et al., 2004; Harry et al., 2008; Levesque et al., 2011b; Sriram et al., 2002; Viviani et al., 1998, 2006; Zhao et al., 2009). Although evidence supporting TNF-a and IL-1b contribution to neurotoxicity are considerable, some studies also indicate a neuroprotective role (Bernardino et al., 2005; Bruce et al., 1996; Shaftel et al., 2007; Strijbos and Rothwell, 1995). Due to the pleiotropic actions of cytokines and the complexity of the considered models this is not surprising, but the coexistence of a dualistic effect underline the necessity to better define the

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specific changes in the CNS environment responsible for enhanced detrimental effects. This approach might also reveal new druggable targets for the treatment of neurodegenerative diseases or amelioration of neurotoxicity. IL-1 and TNF-a may contribute to neuronal injury through the activation of microglia, the induction of nitric oxide and arachidonic acid release, the generation of prostaglandins and other inflammatory eicosanoids, the expression of adhesion molecules and the stimulation of immune cell invasion [see (Allan and Rothwell, 2001; Montgomery and Bowers, 2012) for extensive review]. Another mechanism of neuronal dysfunction and injury recruited by both IL-1b and TNF-a is represented by the disturbance of the glutamatergic transmission, a mechanism shared by different pathological and toxicological states and considered a basis for disorders ranging from cognitive and memory impairment to neuronal death (Galic et al., 2012; McAfoose and Baune, 2009; Yirmiya and Goshen, 2011). Indeed, glutamate is essential for fast synaptic transmission, and learning and memory, but a steep amount leads to excitotoxic cell death through excessive calcium entry into neurons, release of free radicals, arachidonic acid and nitric oxide (Choi, 1988; Meldrum and Garthwaite, 1990). A link between cytokines and glutamate toxicity has been initially observed in models of excitotoxicity. Excitotoxic brain damage induced by local administration of NMDA increases both TNF-a and IL-1b expression (Hagan et al., 1996; Jander et al., 2000; Pearson et al., 1999), in turn IL-1b exacerbates NMDA-induced neuronal death (Lawrence et al., 1998). The observation that both IL-1b and TNF-a expression is reduced by an NMDA receptor antagonist independently on neuronal death (Jander et al., 2000) and excitotoxicity is reduced by administration of interleukin-1 receptor antagonist (Relton and Rothwell, 1992) further strengthens the concept that excitotoxcity and pro-inflammatory cytokines are not mutually exclusive (Fogal and Hewett, 2008). Furthermore, IL-1b and TNF-a driven enhancement of glutamate transmission and its involvement in neurodegenerative processes have been clearly assessed in pathological models of multiple sclerosis (Centonze et al., 2009; Rossi et al., 2012), epilepsy (Noe et al., 2013; Vezzani et al., 1999), spinal cord injury (Ferguson et al., 2008) and ischaemia (Loddick and Rothwell, 1996). Microglia, astrocytes, infiltrating peripheral immune cells and neurons, all differently contributes to the cytokine-glutamate interplay. Glia and immune cells are involved by (i) releasing IL-1b and TNF-a, which can be stimulated by glutamate release itself (Biber et al., 2007). Furthermore, astrocytes express high-affinity glutamate transporter GLAST/EAAT1 and GLT1/EAAT2 and thus influence synaptic activity by clearing glutamate from the synaptic cleft, failure in doing so results in altered synaptic activity and excitotoxicity (Rothstein et al., 1996). The expression of both these transporters is reduced as a consequence of neuroinflammation (Mandolesi et al., 2013; Takaki et al., 2012), both IL-1b (Mandolesi et al., 2013) and TNF-a (Carmen et al., 2009; Tolosa et al., 2011) are involved. In addition, TNF-a favours accumulation of extracellular glutamate, owing to PGE2 and Ca2+-induced glutamate release from astrocytes (Bezzi et al., 2001), while IL-1b increases glutamate export via the cysteine/glutamate exchanger (Fogal et al., 2007). Part of these mechanisms is also recruited by constitutive IL-1b and TNF-a, but massive elevation of their levels could cause neuronal dysfunction (Santello and Volterra, 2012; Yirmiya and Goshen, 2011). Several in vitro evidences based on the use of primary cultures suggest that IL-1b and TNF-a/glutamate cross-talk might directly involve neurons and not only be a reflection of the effect exerted on glia. In purified culture of rat hippocampal neurons, IL1b (i) enhances glutamate and NMDA-receptor-mediate calcium increase (Huang et al., 2011; Viviani et al., 2003) and currents (Yang

et al., 2005), (ii) modulates both NMDA and AMPA receptors phosphorylation, expression and distribution (Gardoni et al., 2011; Lai et al., 2006; Viviani et al., 2003, 2006) and (iii) increases levels of intracellular and extracellular glutamate through the upregulation of neuronal glutaminase (Ye et al., 2013). These effects relies on the activation of the IL-1 receptor type I (IL-1RI), being prevented by IL-1Ra, and converge to an enhanced glutamate and NMDA-induced synaptic loss and neuronal death (Kim et al., 2011; Mishra et al., 2012; Viviani et al., 2003, 2006; Ye et al., 2013). Similarly, TNF-a modulates neuronal functions and excitotoxicity influencing neuronal hyperxcytability and death through the recruitment of AMPA receptors and upreguation of neuronal glutaminase 1 (Balosso et al., 2009; Ferguson et al., 2008; Ye et al., 2013). Excitatory ionotropic glutamate receptors (AMPA and NMDA receptors) represent the front line in the molecular mechanisms of cognition, synaptic plasticity and neuronal demise. These receptors are regulated through phosphorylation/dephosphorylation, are components of the postsynaptic density and are transported in and out of the synapses to strengthen or weaken their action during plasticity, modulate cognitive functions and neuronal death (Choquet, 2010; Groc et al., 2009; Hardingham and Bading, 2010; Salter and Kalia, 2004). Substantial evidence points to these mechanism being disrupted in several disorders (Keifer and Zheng, 2010; Lau and Zukin, 2007). The ability of cytokines to modulate ionotropic glutamatergic receptors is an emergent and still underestimated mechanism to explain the interplay between neuroinflammation and excitotoxicity. Indeed, this effect could represent a unique advantage in modulating the neuroinflammatory response recruited in several adverse conditions, providing an alternative to the classical approach aimed at extinguish cytokine production in toto and in such a way acting on both the neurotoxic and the neuroprotective component of inflammation. We will thus now focus upon the ways TNF-a and IL-1b modulate glutamatergic response through the recruitment of ionotropic receptors. 3.1. Glutamate ionotropic receptors and TNF-a TNF-a signals through two main receptors, TNF-R1 (TNFp55R) and TNF-R2 (TNFp75R). TNFRs as well are constitutively expressed throughout the CNS (Kinouchi et al., 1991) by neurons, astrocytes, microglia and oligodendrocytes (Dopp et al., 1997; Yang et al., 2002). Historically, TNF-R1 engagement is believed to promote cytotoxicity while cell survival and protective responses induced by TNF-a are attributed to TNF-R2 (Fontaine et al., 2002; Yang et al., 2002). This dichotomy is evident also on neuronal excitability, where mice lacking TNFR1 display reduced seizure susceptibility in the hippocampus while mice lacking TNFR2 were more susceptible to seizures (Balosso et al., 2009). When TNF-a binds to TNFR1, the cytoplasmic domain (called ‘‘death domain’’) recruits several adaptor proteins to form an initial signalling protein. The type of adaptor protein recruited and the downstream molecules expressed will determine the type of process to be activated. TNFR2 lacks the death domain but anyway signals through different adaptor proteins partially overlapping with that of TNFR1 (Montgomery and Bowers, 2012). Central to TNF-a action is the AMPA receptor (AMPAR), also relevant to synaptic plasticity, excitability and in particular cases excitotoxicity. Evidence for a synergistic effect of TNF-a and AMPAR in excitotoxicity comes from mouse organotypic hippocampal slice cultures exposed to toxic concentrations of AMPA in the presence of high concentration of recombinant TNF-a (10 ng/ ml) (Bernardino et al., 2005) and from a model of spinal cord injury (Ferguson et al., 2008). Starting from the observation that synaptic strength and excitability are altered by AMPAR trafficking (Beattie et al., 2002), these authors evaluated whether TNF-a could

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influence this pathway. Application of exogenous TNF-a to mature dissociated hippocampal neuron cultures induces a rapid increase in AMPAR surface expression coupled to changes in AMPAR excitatory postsynaptic currents (Beattie et al., 2002; Stellwagen et al., 2005). Endogenously produced TNF-a contributes to this effect too (Stellwagen et al., 2005). In particular, many of the newly exocytosed AMPAR lack GluA2 and are thus permeable to Ca2+ when activated. This leads to excitotoxicity as observed both in vitro and in vivo in a model of spinal cord injury (Ferguson et al., 2008). All these described effects raised by TNF-a rely on neuronal TNFR1, but not TNFR2, recruitment (He et al., 2012; Pribiag and Stellwagen, 2013; Stellwagen et al., 2005). Controversial indications are reported regarding the ability of TNF-a to directly affect NMDAR trafficking (Beattie et al., 2002; Wheeler et al., 2009), while IL1b was less efficacious than TNF-a in increasing surface AMPAR and no evidence were found for a constitutive regulation of AMPAR in culture, suggesting the possibility that the two cytokines modulate glutamatergic sensitivity of neurons through distinct pathways. Finally, besides increasing surface expression of glutamate receptors, TNF-a also induces endocytosis of GABAA receptors, thus reducing the inhibitory drive (Pribiag and Stellwagen, 2013; Stellwagen et al., 2005). TNF-a data support the idea of an highly controlled immune signalling system in neurons relevant for the development of an altered and exaggerated response to glutamate. In this scenario, neurons might allow the immune response to be integrated with specific functions, such as neurotransmission and synaptic plasticity possibly leading in patho/toxicological conditions to hyper-activity and/or, in extreme situation to excitotoxicity. 3.2. Glutamate ionotropic receptors and IL-1b IL-1, a and b, exert similar biological effect by binding the membrane bound type I IL-1 receptor (IL-1R1) (Sims et al., 1988), leading to its association with the IL-1R accessory protein (IL1RAcP) (Korherr et al., 1997) and the myeloid differentiation primary response protein 88 (MyD88) (Burns et al., 1998) to form the core of the IL-1/IL-1R signalling complex and activate downstream signalling pathways. A second receptor, the IL-1 receptor type II (IL-1RII), can bind IL-1 but since it lacks the intracellular domain, it cannot signal. IL1RI is expressed throughout the brain, with highest levels found in cerebral cortex and hippocampus (Farrar et al., 1987; French et al., 1999) along with IL1RAcP (Liu et al., 1996) and MyD88 (Gardoni et al., 2011) and their expression is also attributable to neurons (French et al., 1999; Friedman, 2001; Gardoni et al., 2011). This expression pattern further support the possibility that IL-1, besides mediating the neuroinflammatory response involving both astrocytes and microglia, might directly act on neurons to modulate their activity. Regarding the involvement of ionotropic glutamate receptors, exposure of primary hippocampal neurons to recombinant IL-1b enhances NMDA-induced intracellular Ca2+ increase (Huang et al., 2011; Viviani et al., 2003) and currents (Yang et al., 2005). The enhancement of NMDA-induced Ca2+ increase is triggered by the activation of the src family of kinases (Huang et al., 2011; Viviani et al., 2003) and results in tyr-phosphorylation of the GluN2B subunit of the NMDAR and in an exacerbation of NMDA induced neuronal death (Viviani et al., 2003). These data point to IL-1RI as a coordinating factor of the functional interaction between IL-1b and NMDA. Furthermore, IL-1RI interacts specifically and physically with the GluN2B subunit of the NMDAR (Gardoni et al., 2011). This interaction is particularly evident at the post-synapse, where IL-1RI is enriched together with GluN2B. Both the distribution of IL-1RI and its interaction with the GluN2B subunit are dynamically modulated (Fig. 1). Thus, exposure of primary hippocampal neurons alone to recombinant IL-1b (at the same concentration

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that enhance NMDAR function) or NMDA enriches IL1RI at the post-synaptic spine (Gardoni et al., 2011). In addition, NMDA increases the interaction between IL-1RI and the GluN2B subunit of the NMDAR (Gardoni et al., 2011). The relationship between IL-1RI and GluN2B described in vitro extends also to an in vivo situation, as observed in a model of perinatal stress. A single 24 h-episode of maternal separation occurring at postnatal day (PND) 9 redistributes IL-1RI enriching the post-synapses and increases the interaction with the GluN2B subunit of the NMDAR at PND45 (Viviani et al., 2014). This effect specifically occurs in the hippocampus and not in the cortex, in male and not female rats, revealing a long-term, sex-dependent modification in IL-1RI receptor organization that might contribute to sensitize hippocampal synapses to the action of IL-1b in the adulthood as a consequence of an early-life stress. With respect of glutamate receptors, IL-1b decreases surface expressed AMPAR in primary hippocampal neurons in a dose dependent manner, through the recruitment of NMDAR and independently of synaptic transmitter release (Lai et al., 2006), strengthening the idea of a distinct role of IL-1b and TNF-a in relation to glutamatergic ionic receptors. Finally, as previously observed for TNF-a, acutely applied IL-1b decreases GABAA receptor mediated currents in primary hippocampal neurons through the recruitment of IL-1RI (Wang et al., 2000). This effect as well might contribute to the induction of neuronal hyperexcitabilty. All together these data reveal a sophisticated neuronal immune phenotype that renders neurons sensitive to cytokines like TNF-a and IL-1b and through the recruitment of the glutamatergic response contributes to neuronal dysfunction and demise. To do so, both TNF-a and IL-1b modulate expression, distribution and phosphorylation of distinct subunits of AMPA and NMDA receptors. 4. Cytokines/Glutamate cycle a bridge between neuroinflammation and neurotoxicity? Emerging data obtained both in animal models and in vitro reveal that some neurotoxicants upregulate pro-inflammatory cytokines and disturb glutamatergic transmission. This lead to the hypothesis that a cross-talk between the glutamatergic and inflammatory response might also represent a valuable mechanism to explain their neurotoxicity. So far the impact of the proinflammatory response raised by neurotoxicants on the dysregulation of the glutamatergic response has not been directly addressed, rather is based on circumstantial evidences with the exception of the glycoprotein of the HIV-1 virus Gp120. Nevertheless, several data seem to converge towards this possibility, revealing an interplay between glia and neurons in which the involvement of ionotropic glutamate receptors is also envisaged. In this chapter we will comment this hypothesis providing some examples of neurotoxicants who share both glutamate disturbance and cytokines production. 4.1. GP120 Gp120 is possibly the best example to illustrate the complex interplay exsiting between IL-1b, TNF-a, the dysregulation of the glutamatergic system and its relevance in neurotoxicity. The HIV-1 envelope glycoprotein Gp120 is shed by virus infected macrophages and microglia (Schneider et al., 1986) and is a potent neurotoxin whose toxicity is mediated through NMDAR mechanisms via direct action on neurons (Pattarini et al., 1998) and indirect mechanisms involving both macrophages and microglia and the release of inflammatory mediators (Kaul et al., 2001). Enhancement of NMDA functions may represent an important part of the complex cascade of events in AIDS dementia complex (HAD)

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Fig. 1. IL-1b vs. NMDA functional relationship. Exposure of primary hippocampal neurons to recombinant IL-1b results in an increased phosphorylation of the GluN2B subunit of the NMDAR. IL-1b does not alter Ca2+ homeostasis or neuronal survival unless NMDA is added. Addition of NMDA immediately after IL-b results in an enhancement of NMDA response both in terms of intra-neuronal Ca2+ increase and neuronal death. Treatment with IL-1b or NMDA enriches the post-synaptic spine with IL1RI. The same pathway is activated exposing primary hippocampal neurons to Gp120 in the presence of mixed glia. Gp120 induces IL-1b production and release together with glutamate (both endogenously produced), as a consequence GluN2B phosphorylation increases. This leads to increase of intra-neuronal Ca2+, reduction in the number of spines and neuronal death. Gp120 through the release of IL-1b and the activation of IL-1RI enriches the post-synapses (TIF: Triton Insoluble Fraction) specifically with the NMDAR sharing the GluN2B subunit and IL-1RI, no changes in the distribution of NMDAR sharing the GluN2A subunit and of PSD95 (Post-Synaptic Density 95) occur (see Viviani et al., 2003, 2006; Gardoni et al., 2011 for methodological details).

(Kaul et al., 2001; Potter et al., 2013). Both TNF-a and IL-1b are two of the many mediators released from glia by Gp120 involved in its pathogenic effect. Levels of IL-1b and TNF-a are elevated in the cerebrospinal fluid of AIDS patients and at post-mortem brain examination (Gallo et al., 1989; Tyor et al., 1992), as well as in several rat brain areas following intracerebroventricular infusion of Gp120 (Milligan et al., 2001; Pugh et al., 2000). Pharmacological manipulations to neutralize IL-1b or TNF-a reverted part of the Gp120 induced neurological effects in vivo (Milligan et al., 2001; Pugh et al., 2000). In vitro, Gp120 600 pM induces an increased expression (Viviani et al., 2001) and release of IL-1b (Kim et al., 2011; Viviani et al., 2006) and TNF-a (Bezzi et al., 2001) from primary glial cells. TNF-a released by Gp120 stimulates the release of glutamate by glial cells through the activation of the receptor

CXCR4 (Bezzi et al., 2001). Thus, exposure of primary hippocampal neurons in the presence of glia to Gp 120 allows neuronal stimulation by both endogenously produced IL-1b and glutamate (Fig. 1). In this culture condition Gp120 increases Ca2+ levels within neurons, this effect is evident only in the presence of glia (Viviani et al., 2006) and is inhibited by (i) ifenprodil (a selective antagonist of the GluN2B subunit of NMDAR), by (ii) the loading of neurons alone with pYEEIE (an inhibitor of the family of src-kinases) and by (iii) IL-1Ra (Viviani et al., 2006), thus suggesting the requirement of glia and the involvement of IL-1b, its receptor IL-1RI and the NMDAR (Fig. 1). As observed with recombinant IL-1b plus NMDA, Gp120 induces neuronal demise, which develops from the reduction in the number of synaptic spine to a frank neuronal death (Kim et al., 2011; Viviani et al., 2006). Intriguingly, Gp120

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redistributes both the GluN2B subunit of the NMDAR and IL-1RI enriching the post-synapses of primary hippocampal neurons (Fig. 1) (Viviani et al., 2006). Again, both synapse loss and neuronal death occurs only in neurons exposed to Gp120 in the presence of glia and are prevented by inhibitors of the src-family of kinases, of IL-1RI and CXCR4 (Kim et al., 2011; Viviani et al., 2006). All together, these data suggest a complex interplay between TNF-a and IL-1b signalling that by favouring glutamate release by glia and NMDAR over-activation in neurons leads to an increased neuronal susceptibility to glutamate towards excitotoxicity. The involvement of AMPAR in Gp120-induced neurotoxicity seems to be ruled out since only NMDAR antagonists, but not CNQX an AMPAR antagonist, prevent its effect (Potter et al., 2013).

2012), the exact mechanism in this process has not been identified. Owing to the potential importance of IL-1b and TNF-a in facilitating both the development of seizure and/or excitotoxic neural death (Fogal and Hewett, 2008; Galic et al., 2008), the dysregulation of the glutamatergic response might represent a possible target worth to be investigated. Indeed, the observation that a combination of ketamine (an NMDAR antagonist which also has anti-inflammatory properties (Shibakawa et al., 2005)) and atropine limits soman-induced neuroinflammation and afford neuroprotection even when the treatment is initiated during refractory status epilepticus (Dhote et al., 2012) supports this possibility.

4.2. Organophosphates (OP-chemical warfare agents)

Glutamate contributes to the neurotoxicity of contaminants other than OP, whose toxicity is also characterized by an inflammatory response coupled with IL-1b and TNF-a production. Mn affects dopaminergic neuronal circuitry, primarily damaging the basal ganglia, the globus pallidus, and the substantia nigra (Aschner et al., 2007). At the neuronal level, Mn preferentially accumulates in mitochondria, where it causes mitochondrial dysfunction (Aschner and Aschner, 1991; Gavin et al., 1999) driving oxidative/nitrosative stress (Erikson et al., 2004) also implemented by depletion of glutathione and glutathione peroxidase (Liccione and Maines, 1988). Emerging evidences underline a possible involvement of inflammatory mediators, among which cytokines, released by activated glia in dopaminergic neuronal damage (Zhang et al., 2010; Zhao et al., 2009). In vivo, Mn induces microglia activation and dopaminergic neurons degeneration that is suppressed by the use of minocycline, which inhibits microglia activation and afford neuroprotection (Liu et al., 2009; Zhang et al., 2010; Zhao et al., 2009). In vitro, Mn activate microglia cells only at high concentration (Filipov et al., 2005), but potentiate LPS induced release of IL-1b and TNF-a from both microglia and astrocytes (Filipov et al., 2005; Zhang et al., 2010). Although the mechanism involved in Mn-induced glial activation has not been identified, the comparison between in vivo and in vitro data suggest that Mn sensitizes glial cells to a subsequent stimuli that in vitro is reproduced by LPS and in vivo has yet to be determined. Besides the release of pro-inflammatory cytokines, Mn-induced glial activation results also in nitric oxide and reactive oxygen species production (Filipov et al., 2005; Zhang et al., 2010), which are considered so far the main candidate to drive dopaminergic neuronal death. Nevertheless, compelling evidences suggest that Mn by altering astrocytic functions lead to extracellular glutamate accumulation and activation of an excitotoxic state [see (SidorykWegrzynowicz and Aschner, 2013) for extensive review]. Indeed, glutamate-mediated mechanisms play a role in the development of Mn-induced neurotoxicity in vivo (Brouillet et al., 1993; Verity, 1999). Mn toxicity following injection into the rat striatum, in fact, is blocked by prior removal of the corticostriatal glutamatergic inputs or by treatment with an NMDA glutamate receptor antagonist (Brouillet et al., 1993), strongly implying excitotoxic processes in the neuronal damage produced by Mn in this brain area. Mechanisms contributing to Mn toxicity are (i) an increased sensitivity of post-synaptic receptors to glutamate leading to abnormal activation, as it has been selectively detected in globus pallidus cells dissociated from Mn2+-treated rats (Spadoni et al., 2000), and (ii) the disruption of glutamate transporting system, leading to both a reduction in glutamate uptake and elevation in extracellular glutamate level (Lee et al., 2009). Although these observations suggest the involvement of the cytokine/glutamate cycle in Mn neurotoxicity, so far the direct contribution of the inflammatory response raised in the glutamate-mediated effect has not been investigated.

The primary mechanism mediating the neurotoxicity of these compounds is the inhibition of acetylcholinesterase (AChE), resulting in peripheral parasympathomimetic effects as well as seizures and respiratory arrest. However, different evidences suggest that, in some cases, OP neurotoxicity does not entirely rely on disturbances of the cholinergic systems (Pope et al., 2005; Rohlman et al., 2011), as observed in induced delayed cell death, cognitive and neurobehavioural deficits (Chen, 2012; Collombet, 2011). For instance, intoxication with some members of the OP family (OP-chemical warfare agents) is characterized by seizures associated with neuronal damage (Chapman et al., 2006; Zimmer et al., 1997). The involvement of the glutamatergic system is likely. Initiation and early expression of seizures following OP are cholinergic in nature but may be reinforced by a subsequent unbalance of the glutamatergic (favoured) and the GABAergic (reduced) activity, due to overstimulation of muscarinic receptors (Kozhemyakin et al., 2010; Santos et al., 2003). Repeated seizures and prolonged NMDAR activation, by favouring excessive Ca2+ influx, might be the ultimate cause of a delayed secondary damage (Chen, 2012; McDonough and Shih, 1997). As such, antagonist of the muscarinic receptors has been proved useful for seizures management early after OP exposure, while NMDAR antagonists terminate OP seizures even if the treatment is delayed after 40 min of epileptic activity and are currently among the few compounds able to provide neuroprotection even during refractory status epilepticus (Lallement et al., 1998; McDonough and Shih, 1997; Zaja-Milatovic et al., 2009). Cortex, amygdala, hippocampus and thalamus are the brain areas predominantly affected by delayed secondary damage that has been recognized as a major contributor to memory loss and behavioural problems (Grauer et al., 2008). OPinduced seizures are associated with cytokines, chemokines elevation and glia activation in these same areas (Table. 1) (Chapman et al., 2006; Dhote et al., 2007; Johnson and Kan, 2010; Svensson et al., 2001). This response involves the increase in both mRNA (Dhote et al., 2012, 2007; Dillman et al., 2009; Svensson et al., 2001) and protein levels of cytokines (Chapman et al., 2006; Dhote et al., 2012; Johnson and Kan, 2010; Svensson et al., 2005), as well as glial activation and neutrophil infiltration (Dhote et al., 2012; Johnson and Kan, 2010). Cytokines, among which IL-1 and TNF-a are always present, are detected shortly after postintoxication to peak in between 6 and 48 h in dependence of the cytokine and the brain area considered (Dhote et al., 2007; Dillman et al., 2009; Svensson et al., 2001). After peaking, a decline but not always a complete recovery has been observed (Dhote et al., 2007; Dillman et al., 2009; Svensson et al., 2001), suggesting a persistent alteration of the neuroinflammatory background as a consequence of OP intoxication. Although it has been hypothesized that increases in neurotoxic cytokines likely play an active role in the progression of OP-induced neuropathology (Banks and Lein,

4.3. Manganese (Mn)

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4.4. Air pollution Air pollution is a multifaceted environmental toxin, in which particulate matter (PM) and ground level ozone are the most widespread health treats. A growing list of studies implicate air pollution as a relevant CNS health concern (Block et al., 2012; Block and Calderon-Garciduenas, 2009) The two fractions of PM predominantly implicated in effect in the CNS are PM25 and nanosized particulate matter (nPM). Mechanisms driving airpollution effect within the CNS are unclear although inflammation has been identified as a common feature at the base of air pollution and PM neurotoxicity (Block and Calderon-Garciduenas, 2009). Both (i) peripheral immune activation, in tissue such as those that comprise lung (Tamagawa and van Eeden, 2006), liver (Folkmann et al., 2007) and cardiovascular systems (Swiston et al., 2008), and (ii) a direct activation of CNS resident cells, once particles reach the brain, may contribute to air pollution induced neuroinflammation. As previously described, peripherally produced cytokines may enter the CNS or peripheral activated immune cells may invade the CNS giving rise to a local inflammatory response. As such brain tissues of individuals residing in highly polluted areas showed increased markers of infiltrating monocytes (or resident microglia) and of inflammation, among which IL-1 and TNF-a (CalderonGarciduenas et al., 2008). Local increase of both IL-1b and TNF-a mRNA has been found in the olfactory bulb, considered the way of entrance of air pollution components, following intranasal instillation of nano-sized carbon black (Tin Tin Win et al., 2008). In vitro experiments on brain rat capillaries suggest that these cells as well contribute to local production of cytokines in response to air pollution components (Hartz et al., 2008), possibly together with perivascular microglia surrounding the circumventricular organs. This response may thus propagate within the brain parenchyma. Independently on the pathway recruited, increase both expression and production of IL-1 and TNF-a has been observed in rodents brain exposed by inhalation to DE or airborn particulate matter in the midbrain and cortex (Campbell et al., 2005; Levesque et al., 2011a; Morgan et al., 2011). CNS damage associated to air-pollution exposure range from an enhanced risk for ischaemic stroke (Kettunen et al., 2007; Lisabeth et al., 2008) to mild cognitive impairments in humans (Chen and Schwartz, 2009; Power et al., 2011; Weuve et al., 2012); similar effects have been observed in rodents exposed to diesel exhaust or ambient fine airborne PM25 (Fonken et al., 2011; Win-Shwe et al., 2012). The basis for CNS disturbances from inhalation of urban air pollutants might thus also envisage a dysregulation of the glutamatergic system, critical to neuronal plasticity, memory processes and relevant in the progression of ischaemic damage. Indeed, nano carbon-black together with increased expression of IL-1 and TNF-a, increases extracellular glutamate levels in the olfactory bulb of instilled mice (Tin Tin Win et al., 2008). Furthermore, air pollution affects expression and phosphorylation of ionotropic glutamate receptors. 10-week inhalation exposure of mice decreased hippocampal levels of the GluA1 subunit of AMPA receptors with no changes in the GluA2 in concomitance with both glial activation and increased mRNA expression of both TNF-a and IL-1 (Morgan et al., 2011). Although expression of NMDARs subunits was not evaluated in this in vivo study, increased levels of both GluN2A and GluN2B, as well as serine dephosphorylation of GluN2B and GluA1 subunits, have been observed in hippocampal slices following 2 h exposure to aqueous nPM (

Perspectives on neuroinflammation and excitotoxicity: a neurotoxic conspiracy?

Emerging evidences underline the ability of several environmental contaminants to induce an inflammatory response within the central nervous system, n...
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