MINIREVIEW Membrane compartments and purinergic signalling: P2X receptors in neurodegenerative and neuroinflammatory events Savina Apolloni, Cinzia Montilli, Pamela Finocchi and Susanna Amadio Santa Lucia Foundation, Rome, Italy P2X purinergic receptors are ion channels possessing tertiary structures with two transmembrane domains. Seven distinct P2X subtypes (P2X 1–7 ) have been cloned from mammalian species, and all can form homo- or heteromultimer combinations, of which the minimum stoichiometric ratio is a trimer. Different subtype com- binations yield different receptor characteristics, allow- ing diversity in transmission signalling, in agonist and antagonist selectivity, channel and desensitization properties [1]. Among the different P2X receptors, the potencies of ATP can vary enormously, from nanomo- lar to micromolar ranges, depending on the subunit composition. Common to all P2X subtypes is a direct influx of extracellular Ca 2+ promoted by purines via the receptor channel, which constitutes a significant source of intracellular Ca 2+ . This leads to a secondary activation of voltage-gated Ca 2+ channels, which probably make the primary contribution to the total intracellular Ca 2+ influx and accumulation. These transduction mechanisms do not depend on the Keywords Alzheimer’s disease; amyotrophic lateral sclerosis; ATP; cell death; extracellular ATP; Huntington’s disease; ischaemia; multiple sclerosis; nervous system; P2 receptors; Parkinson’s disease Correspondence S. Amadio, Santa Lucia Foundation, Via del Fosso di Fiorano 65, 00143 Rome, Italy Fax: +3906 50170 3321 Tel: +3906 50170 3060 E-mail: s.amadio@hsantalucia.it (Received 15 July 2008, revised 10 October 2008, accepted 5 November 2008) doi:10.1111/j.1742-4658.2008.06796.x ATP is a potent signalling molecule abundantly present in the nervous system, where it exerts physiological actions ranging from short-term responses such as neurotransmission, neuromodulation and glial communi- cation, to long-term effects such as trophic actions. The fast signalling targets of extracellular ATP are represented by the ionotropic P2X recep- tors, which are broadly and abundantly expressed in neurons and glia in the whole central and peripheral nervous systems. Because massive extra- cellular release of ATP often occurs by lytic and non-lytic mechanisms, especially after stressful events and pathological conditions, purinergic sig- nalling is correlated to and involved in the aetiopathology and/or progres- sion of many neurodegenerative diseases. In this minireview, we highlight the contribution of the subclass of ionotropic P2X receptors to several dis- eases of the human nervous system, such as neurodegenerative disorders and immune-mediated neuroinflammatory dysfunctions including ischae- mia, Parkinson’s, Alzheimer’s and Huntington’s diseases, amyotrophic lat- eral sclerosis and multiple sclerosis. The role of P2X receptors as novel and effective targets for the genetic/pharmacological manipulation of purinergic mechanisms in several neuropathological conditions is now well estab- lished. Nevertheless, any successful therapeutic intervention against these diseases cannot be restricted to P2X receptors, but should take into consid- eration the whole and multipart ATP signalling machinery. Abbreviations AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; BzATP, 2¢,3¢-O-(4-benzoyl)-benzoyl-ATP; CNS, central nervous system; COX-2, cyclooxygenase-2; EAE, experimental autoimmune encephalomyelitis; HD, Huntington’s disease; MND, motor neuron disease; MS, multiple sclerosis; oATP, periodate oxidized ATP; PD, Parkinson’s disease; SN, substantia nigra; SOD1, superoxide dismutase Cu/Zn. 354 FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS production and diffusion of second messengers within the cytosol or the membrane and the cellular response time is generally very rapid. Electrophysiological mea- sures demonstrate that P2X receptor stimulation can produce two types of current: fast desensitizing and non-desensitizing, thus suggesting different functional phenotypes for these receptors [2]. In the nervous system, P2X receptors have an estab- lished role in neurotransmission, co-transmission, neu- romodulation, glial communication and trophic actions (neurite outgrowth and the proliferation of glial cells). More recently, they were found to be involved in biological tasks ranging from survival, repair and remodelling during development, to contri- butions in injury, metabolism impairment, excitotoxic- ity, acute and chronic neurodegenerative conditions [3,4]. All subunits of P2X receptors are expressed in the nervous system in both neuronal cells and in astro- cytes, oligodendrocytes, Schwann cells and microglia [5,6]. In particular, P2X 1 receptors mediate the puri- nergic component of sympathetic and parasympathetic nerve-mediated smooth muscle contraction in a multi- plicity of tissues. P2X 2 receptors [7] are expressed in the central nervous system (CNS) in cortex, cere- bellum, hypothalamus, striatum, hippocampus and the nucleus of the solitary tract, as well as in the dorsal horn of the spinal cord, where they act in ATP-medi- ated fast synaptic transmission at both nerve terminals and interneuronal synapses. P2X 2 receptors are also significantly localized in the peripheral nervous system on both sensory and autonomic ganglion neurons. Thus, P2X 2 receptors have wide-ranging functions in the regulation of many neuronal processes including memory and learning, motor function, autonomic coordination and sensory integration. The gene encod- ing the P2X 3 protein subunit was originally cloned from rat dorsal root ganglion sensory neurons and, in the adult, P2X 3 proteins are predominantly expressed on small-to-medium diameter C-fibre and Ad sensory neurons within the dorsal root, trigeminal and nodose sensory ganglia. Moreover, they are present on both the peripheral and central terminals of primary sensory afferents projecting to somatosensory and visceral organs [8]. P2X 3 receptors are now recognized as play- ing a major role in mediating the primary sensory effects of ATP and, as such, are of major importance in nociception and mechanosensory transduction. The gene encoding the P2X 4 protein was originally cloned from rat brain, where P2X 4 receptors may be the most widely distributed among all P2X receptors. Localiza- tion studies indicate that this receptor subunit is found in cerebellar Purkinje cells, spinal cord, autonomic and sensory ganglia. Moreover, P2X 4 receptors are abun- dantly expressed in microglia, where they become upregulated during chronic inflammatory and neuro- pathic pain, and are an important target for pharma- cological approaches [9]. P2X 5 mRNA and immunoreactivity are found in a wide variety of tissues including brain, spinal cord and eye. P2X 6 mRNA and immunoreactivity are present throughout the CNS, particularly in portions of the cerebellum (Purkinje cells) and hippocampus (pyramidal cells). In addition, P2X 6 receptors have been reported in sensory ganglia. The P2X 7 receptor is predominantly localized on vari- ous types of glia within the peripheral nervous system and CNS, including microglia, astrocytes, oligoden- drocytes and Schwann cells [10]. Currently, there is compelling electrophysiological, pharmacological and immunological evidence for the presence of and role for P2X 7 receptors also in neuronal functions and injury. Given the general widespread and abundant occur- rence of P2X receptors in the nervous system, it is fea- sible to imagine that extracellular ATP arising from injury and/or deregulated release, can confer to all the P2X protein subunits a central role in neuropatholo- gical conditions, even identifying these receptors as potential tools for effective pharmacological approaches [11]. Neurodegenerative, neuroinflammatory conditions and ATP release Neurodegeneration is the progressive loss of structure and/or function of neurons, eventually culminating in death. Neurodegenerative diseases are the subset of neurological disorders sharing neurodegeneration, uncontrolled inflammation [12] and additional features, but which exclude diseases due to cancer, trauma, poi- soning, ethanol, drug abuse, etc. The most frequent diseases that involve several common paths of neu- rodegeneration include Alzheimer’s (AD), Hunting- ton’s (HD) and Parkinson’s (PD) diseases and amyotrophic lateral sclerosis (ALS). Among the com- mon features, AD-like dementia and/or the character- istic histopathological markers of plaques and tangles may occur in PD as well; PD-like movement dysfunc- tion and/or accompanying Lewy body histopathology have been reported in notable numbers of AD patients too. Many of these features can be extended to motor neuron diseases (MND) and ALS, which can in fact co-exist, for example, with AD-like properties, because mRNA for amyloid protein precursor is found to be upregulated in dying motor neurons. By contrast, a disease not strictly classified as a neurodegenerative condition is multiple sclerosis (MS), which meets the S. Apolloni et al. P2XR in neurodegenerative and neuroinflammatory events FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS 355 requirements for a neuroinflammatory disease. It usu- ally commences with an autoimmune inflammatory reaction to myelin components, and then progresses to a chronic phase in which oligodendrocytes, myelin and axons degenerate. Nevertheless, because neuroinflam- mation exerted by activated microglia and astrocytes in the proximity of degenerating neurons is a patholog- ical hallmark generally seen in MND and in models of ALS, the line between neurodegenerative and neuro- inflammatory diseases is somehow very subtle [13]. Among the characteristics of both neurodegenera- tion and neuroinflammation, we can certainly enu- merate the extracellular release of ATP (or additional purine/pyrimidine molecules) [14,15] from both neurons and glia. Many of the properties of extracel- lular ATP described to date make it in fact an ideal molecule to deliver cell-to-cell signals under patholog- ical conditions. Besides acting alone as a neurotrans- mitter, neuromodulator, growth or toxic factor, ATP is often co-released, for example, with the neurotrans- mitters acetylcholine, noradrenalin, glutamate and GABA, depending on the specific transmitter reper- toire of each neuron. By interacting with other neuro- or gliotransmitters at both the receptor and signal transduction levels, ATP thus modifies and/or amplifies their mutual physiopathological effects. Any alteration of these well-tuned systems is then involved in several human diseases such as neurodegenerative disorders and immune-mediated neuroinflammatory dysfunction. P2X receptors and neurodegenerative/ neuroinflammatory diseases A tight molecular interplay exists among all the com- ponents of the purinergic signalling machinery, which comprises purinergic ligands, ectonucleotide meta- bolizing enzymes, P2/P1 receptors, nucleoside trans- porters and extracellular nucleotide release. This has implications for the response of almost any cell to acute or chronic neurodegenerative insults, ischaemia and neuroinflammatory conditions. Nevertheless, without neglecting the involvement of the entire puri- nergic signalling machinery, we now set our emphasis on the role exerted by ionotropic P2X receptors dur- ing neurodegenerative and neuroinflammatory events (Table 1). Ischaemia Cerebral ischaemia is one of the most common causes of death in aged people, being responsible for 10–12% of deaths worldwide per year [16,17]. Ischaemic injury involves a marked reduction in intracellular oxygen and glucose, which leads to fast cell death associated with an increase in intracellular Ca 2+ influx. This in turn directly controls the activation of proteolytic enzymes, of apoptotic genes, and the production of reactive oxygen species with concomitant oxidative stress. In this context, purine/pyrimidine nucleotides are actively released or passively extruded from healthy/ damaged cells, and ATP may reach high concentra- tions in the extracellular space. Therefore, the direct participation of extracellular ATP in ischaemic stress becomes manifest, to the point of exerting a significant direct excitotoxic effect mediated by P2 receptors in various cellular systems (without excluding a concomi- tant role also for ectonucleotide hydrolyzing enzymes, P1 receptors and ectonucleoside transporters) [3,4]. Accordingly, in different cell culture models of CNS and peripheral nervous system cell culture, the P2 receptor antagonists Reactive Blue-2, suramin and pyridoxal-phosphate-6-azophenyl-2¢,4¢-disulfonate were shown to prevent neuronal death under hypoglycaemia and chemically induced hypoxia [18,19]. Moreover, the inhibition of P2 receptors can also partially reduce the in vivo functional and morphological deficits occurring in rat after acute cerebral ischaemic events [20]. P2X 2 and P2X 4 receptors are upregulated in vitro after oxygen and glucose deprivation in organotypic slice cultures, and in vivo after ischaemia in gerbil in CA1–CA3 pyramidal cell layers [21]. Also the P2X 7 receptor subtype is an apparently important component of the mechanisms of cell dam- age induced by hypoxia/ischaemia. After a prolonged ischaemic insult, P2X 7 receptor mRNA and protein become upregulated in cultured cerebellar granule neurons, organotypic hippocampal cultures and both neurons and glial cells from in vivo tissues [22–24]. By contrast, in primary cortical cultures, a short ischaemic Table 1. P2X receptors and neuropathological conditions. Evidence is presented about the involvement of different P2X receptor sub- types in several neurodegenerative/neuroinflammatory conditions. ALS, amyotrophic lateral sclerosis. Disease P2X 1 P2X 2 P2X 3 P2X 4 P2X 6 P2X 7 AD – – – – – [33,35–37] ALS – – – [54,55] – [35,53–55] Epilepsy – [64] – [64] – – HD –––– –– Ischaemia [30] [21] – [21] – [22–28] MS – – – [62] – [53,60,61] Neuropathic pain – [66] [66] [66] – [66] PD [49] [48] – [48] [48] [47] P2XR in neurodegenerative and neuroinflammatory events S. Apolloni et al. 356 FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS stimulus fails to induce changes in P2X 7 mRNA and immunoreactivity, whereas serum deprivation aug- ments P2X 7 receptor immunoreactivity only in astro- cytic, and not in neuronal populations. Nevertheless, presynaptic P2X 7 receptor exhibited an increased response to ATP and 2¢,3¢-O-(4-benzoyl)-benzoyl-ATP (BzATP) after ischaemic insult, despite no changes in P2X 7 mRNA and P2X 7 immunoreactivity [25]. In microglia, increased P2X 7 receptor protein expression appears to contribute to the mechanisms of cell death caused in vivo by ischaemia [26]. It was finally sug- gested that activation of the P2X 7 receptor might regu- late the release of neurotransmitters from astrocytes and neurons, as well as the cleavage and release of interleukin-1b (IL-1b) from macrophages and micro- glia [27]. In neuronal-enriched primary cortical cultures, a short ischaemic stimulus increased the ATP- and BzATP-induced release of previously incor- porated [ 3 H]GABA, an effect inhibited by the selective P2X 7 receptor antagonists Brilliant Blue G and perio- date oxidized ATP (oATP) [25]. Finally, in a recent study on rat hippocampal slices, the P2 receptor antag- onists pyridoxal-phosphate-6-azophenyl-2¢,4¢-disulfo- nate (0.1–10 lm) and Brilliant Blue G (1–100 nm), were shown to decrease the long-term oxygen/glucose deprivation-evoked [ 3 H]glutamate efflux. This indicated that endogenous ATP released from the hippocampus upon energy deprivation can activate various subtypes of P2X receptors to elicit glutamate overflow, therefore facilitating ischaemia-evoked glutamate excitotoxicity [28]. An opposing protective role for ATP against hyp- oxic/hypoglycaemic perturbation of hippocampal neurotransmission was conversely demonstrated by inhibition of neuronal activity through enhancement of GABA release via P2X receptors [29]. Using the organotypic model of rat hippocampus, the involvement of the P2X 1 receptor subtype was also proved to be potentially disadvantageous in the path of in vitro ischaemia during oxygen/glucose deprivation. The P2X 1 receptor was strongly and transiently upreg- ulated within 24 h of an ischaemic insult on structures likely corresponding to mossy fibres and Schaffer col- laterals of CA1–CA3 and dentate gyrus. It was consis- tently downregulated by pharmacological treatment with the antagonist trinitrophenyl-adenosine-triphos- phate, which was also found to be neuroprotective against ischaemic cell damage and death [30]. In conclusion, this experimental evidence demon- strating a post-ischaemic time- and space-dependent modulation of P2X 1,2,4,7 receptor subtypes on both neurons and glia, clearly suggests a direct role for these same receptors in the physiopathology of cere- bral ischaemia both in vitro and in vivo (Table 1). Alzheimer’s disease AD, among the most common causes of dementia, is a neurodegenerative disorder for which there is currently no cure. It is characterized by global cognitive decline including a progressive loss of memory, orientation and reasoning. The cause and progression of AD is not well understood, but at the microscopic level the disease is associated with senile or neuritic plaques composed of b-amyloid, and with neurofibrillary tan- gles composed of hyperphosphorylated tau protein [31]. At the macroscopic level, AD is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical areas. Three major hypotheses exist to explain the cause of this disease. The oldest, on which most currently available drug therapies are based, is known as the cholinergic hypothesis, which suggests that AD is due to reduced biosynthesis of the neurotransmitter acetylcholine. In 1991, the amyloid hypothesis was instead formulated, which considered that the aggregates of b-amyloid assume major respon- sibility in AD neuronal impairment. Research after 2000, became aware of the additional role played by tau proteins as causative factors in this disease. Little is still known regarding the potential contribu- tion of purinergic mechanisms to AD, although it has been reported that extracellular ATP diminishes Ca 2+ release from endoplasmic reticulum stores in AD microglia [32]. Moreover, extracellular ATP modulates b-amyloid peptide-induced cytokine IL-1b secretion from human macrophages and microglia, likely playing a direct role in the neuroimmunopathology of AD. This last effect was apparently mediated by the P2X 7 receptor subtype, because IL-1b release was stimulated by the specific agonist BzATP and reversed by the P2X 7 antagonist oATP [33]. This is consistent with both the general biological response that ATP is known to evoke in microglia [34] and with the general contribution that microglia cells, releasing pro-inflam- matory substances and inducing neurotoxicity, have make to the progression of AD. In addition, the P2X 7 receptor subtype was found to be specifically upregu- lated in microglia around b-amyloid plaques in a mouse model of AD. In primary rat microglia, both ATP and BzATP acting on the P2X 7 receptor subtype were reported to stimulate the production and release of copious amounts of superoxide (O 2 ) ·), through activation of NADPH oxidase [35]. In this regard, it was also reported that b-amyloid can induce the release of ATP itself, which in turn can activate NADPH oxidase via the P2X 7 receptor, and thus stimulate reactive oxygen species production from the microglia in an autocrine manner [36]. Both ATP and S. Apolloni et al. P2XR in neurodegenerative and neuroinflammatory events FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS 357 BzATP stimulated microglia-induced cortical cell death in a mouse model of AD (Tg2576), indicating that this specific pathway may contribute to AD-associated neurodegeneration [37]. Enhanced expression (70% increase) of the P2X 7 receptor was also seen in both adult microglia obtained from AD brains (compared with control non-demented microglia) and in cultured fetal human microglia exposed to b-amyloid [37]. Amplitudes of Ca 2+ responses induced in these cells by the selective P2X 7 receptor agonist BzATP were moreover increased by 145% after b-amyloid (frag- ment 1–42) pretreatment. They were largely blocked if the P2X 7 receptor inhibitor oATP was added with the b-amyloid peptide in pretreatment solution [37]. These results suggest novel key roles for the P2X 7 receptor in mediating purinergic inflammatory responses in AD brain. Although indirectly, this evi- dence supports a direct contribution of extracellular ATP and a likely contribution of additional P2X receptors to the features and mechanisms of AD (Table 1). Huntington’s disease HD, caused by polyglutamate expansions in the huntingtin protein, is a progressive neurodegenerative disease resulting in motor and cognitive impairments and death. Neuronal dysfunction and degeneration both contribute to progressive physiological, motor, cognitive and emotional disturbances typical of HD. Nevertheless, the relationship between expression of the huntingtin protein and the death of the neurons in the neostriatum (resulting in the appearance of gener- alized involuntary movements), is not fully understood. According to experimental evidence indicating that neurons in the neostriatum are selectively vulnerable to glutamate, excitotoxic neuronal death was suggested to be directly involved in neurodegeneration associated with HD [38]. Extracellular ATP acting on P2, and particularly on P2X receptors, is known to interfere with the release of glutamate, for example, in primary synapses in the CNS [39]. Moreover, P2 receptor antagonists were reported to directly prevent glutamate release and glu- tamate-evoked excitotoxicity in CNS primary neuronal cultures [40]. In addition, the metal chelator clioquinol has been shown to mitigate HD neuropathological symptoms in a mouse model of HD [41]. It was accordingly reported that clioquinol can prevent the inhibition by neurotoxic Cu 2+ of the ATP-gated cur- rents evoked through the P2X 4 receptor. This was interpreted as an involvement of P2X 4 receptors in the neurotoxic effects exerted by metals in HD [42]. From this perspective, a correlation between HD and P2X receptors is likely, although there is as yet no undeniable experimental evidence on the topic (Table 1). Parkinson’s disease PD is an idiopathic chronic and progressive neurode- generative disorder of the CNS that often impairs motor skills (provoking tremor, rigidity, bradykinesia and postural instability), and causes mood, cognitive, speech, sensation and sleep disturbances. It is charac- terized by selective cell death of dopaminergic neurons in the substantia nigra. The primary symptoms are the results of a decreased stimulation of the motor cortex by the basal ganglia, normally caused by the insuffi- cient formation and action of dopamine. The symp- toms only become apparent when > 50% of the dopaminergic neurons in the substantia nigra pars compacta are lost, which then leads to an > 80% reduction in dopamine levels in the striatum. Second- ary symptoms may include high cognitive dysfunction and subtle language problems. Although many forms of parkinsonism are ‘idiopathic’, ‘secondary’ cases may result from toxicity, most notably caused by drugs, head trauma or other medical disorders. Recessive juvenile-onset form of PD is the most frequent type of familial PD, associated to mutations in the parkin gene, now accepted as one of eight genes responsible for PD [43]. The evidence available on a potential involvement of purinergic receptors in PD is still scarce (Table 1). Concerning P2X receptors, in particular, recent work was performed with the pheochromocytoma PC12 cell line, a cellular model system frequently used in vitro for PD. These cells are capable of differentiating into dopaminergic-like neurons following stimulation with the neurotrophin nerve growth factor. RT-PCR showed that whereas P2X 2 mRNA alone was detect- able in undifferentiated PC12 cells, the mRNAs for all P2X 1–7 receptor subtypes were highly increased after dopaminergic differentiation of PC12 cells [44]. These results are in accordance with previous studies per- formed by western blot analysis showing that P2X 2–4 receptor proteins were induced by nerve growth factor in these same cells [45,46]. In an additional cellular model system for PD, consisting of SN4741 inducible dopaminergic neurons derived from substantia nigra, it was moreover demonstrated that the ionotropic P2X 7 subtype is functionally expressed and responsible for ATP-induced cell swelling and necrotic cell death [47]. Although this would indicate that degeneration of dopaminergic neurons can be accelerated by P2X 7 P2XR in neurodegenerative and neuroinflammatory events S. Apolloni et al. 358 FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS receptor activation (potentially induced by excess amount of ATP released from damaged cells or acti- vated astrocytes), the in vivo role of this receptor sub- type in the progression of PD remains to be proved. Regarding the juvenile-onset form of PD, Sato and co-workers demonstrated that parkin produces a very substantial increase in the maximum currents induced by extracellular ATP in PC12 cells after dopaminergic differentiation, without a significant change in sensitiv- ity to ATP [48]. This was not apparently associated to an increased number and/or affinity of ionotropic P2X 2,4,6 receptor subtypes, but rather involved an increase in the gating of these same receptors. Finally, a topographical analysis was performed in rat brain slices from striatum and substantia nigra for the pres- ence of all P2 receptor proteins identified to date and cloned from mammalian tissues [49]. Various different P2X subtypes (but also metabotropic P2Y subunits) were found in vivo at the protein level in dopaminergic, GABAergic neurons or astrocytes. Moreover, dopa- mine denervation obtained by unilateral injections in the rat brain of 6-hydroxydopamine (used as animal model of PD), generated a significant rearrangement of several P2X receptor proteins. Most P2X subunits were found to be decreased respectively on GABAergic and dopaminergic neurons in the lesioned striatum and substantia nigra, most likely as a consequence of dopa- minergic denervation and/or neuronal degeneration. Conversely P2X 1,3,4,6 proteins were augmented on GABAergic neurons in the lesioned substantia nigra pars reticulata, as a probable compensatory reaction to dopamine shortage [49]. These studies in their whole contribute to disclose a potential direct participation of P2X receptors to the lesioned nigro-striatal circuit. Amyotrophic lateral sclerosis ALS is a late-onset neurodegenerative disorder charac- terized by the death of motor neurons in the cerebral cortex and spinal cord. The familial form of ALS accounts for  10% of all cases, and is usually trans- mitted as an autosomal dominant trait. Known muta- tions in the Cu/Zn superoxide dismutase (SOD1) gene (an ubiquitously expressed and highly conserved metal- loenzyme involved in the detoxification of free radi- cals), are responsible for  15% of familial forms of ALS. A pathological hallmark lately seen in mutated- SOD1 models of ALS is neuroinflammation exerted by activated microglia and astrocytes in the proximity of degenerating motor neurons. Mutant SOD1 may thus cause neurotoxicity not only directly in motor neurons, but also indirectly by perturbing the function of non-neuronal cells such as microglia. Several studies in genetically engineered mouse models have indeed indi- cated that expression of mutant SOD1 in neurons alone is insufficient to cause motor neurons degeneration, and that participation of non-neuronal cells may be required [50,51]. Clearly, microglia has a great potential to drastically modify neuropathological events. How- ever, the role of microglia is dual, being neuroprotec- tive as well as neurotoxic, with the final outcome likely depending on the intensity of the microglia reaction, the kind of stimuli received and other local factors, including cross-talk with neighbouring neuronal cells, or induction of downstream effectors. Molecules directly secreted from or activating micro- glia could thus be prime candidates for the propaga- tion of motor neuron injury in ALS and, among these, also extracellular ATP might have a pivotal role. Other than expressing a wide range of P2X (but also P2Y) receptors, microglia cells are well known to release ATP and respond to extracellular nucleotides that, for example, induce migration and initiation of the phago- cytotic process. ATP acting on microglia, and particu- larly on P2X 4 and P2X 7 receptors, stimulates cytokine release [52]. Therefore, molecules known to be expressed in activated microglial cells/macrophages, and to play a role in inflammatory cascades, such as cyclooxygenase-2 (COX-2) and the P2X 7 receptor, were directly studied in ALS post-mortem human spinal cord tissue. All ALS cases showed not only increased numbers of P2X 7 -immunoreactive microglia with respect to control spinal cords, but also a marked upregulation of P2X 7 protein/cell in activated micro- glia/macrophages [53]. A biological cascade of degener- ation was then postulated: cell death would increase extracellular ATP that would activate P2X 7 receptor expressed by microglia/macrophages; the latter would induce the release of IL-1b, which in turn would induce COX-2, leading to further cell death and ATP release, therefore perpetuating a death cycle [53]. Accordingly, it was also demonstrated that expression of P2X 7 receptor is more abundant in end-stage trans- genic rodents carrying the SOD1 G93A mutation, concomitantly with activated microglia [54]. A possible role for the P2X 4 receptor subtype was suggested by the observation that strong P2X 4 immu- noreactivity was selectively associated with degenerat- ing motor neurones in spinal cord ventral horns, in the rodent models of ALS expressing G93A mutated human SOD1. Moreover, this receptor provided to be a unique and valuable tool for revealing sick neurons in these ALS models [54]. Alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid receptor-mediated excitotoxicity is also well known to contribute to S. Apolloni et al. P2XR in neurodegenerative and neuroinflammatory events FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS 359 the death of motor neurons in ALS. It was recently shown that preincubation of motor neurons with the P2X 4 receptor modulator ivermectin, or with the P2X 7 receptor antagonist Cibacron Blue, protects from alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death, thus suggesting that defensive mechanisms might be due to both potentiation of the P2X 4 receptor, and to inhibition of the P2X 7 subtype. Moreover, treatment of SOD1 G93A-mice with iver- mectin also resulted in an extension of the animal life span of almost 10% [55]. These notions, coupled with the production and release of superoxide directly from microglia following P2X 7 receptor activation [35], clearly suggest that puri- nergic signalling is central to microglia functioning in the brain, with potentially far-reaching consequences for pathological conditions also associated to ALS (Table 1). Multiple sclerosis A distinct pathology thought to usually commence with an autoimmune inflammatory condition in which the immune system attacks myelin components of the CNS, and then to progress to a chronic phase in which oligodendrocytes, myelin and axons degenerate is MS, causing numerous physical and mental symp- toms and often progressing to physical and cognitive disability. Almost any neurological symptom can accompany this disease. MS patients may be affected by a relapsing–remitting early form of the disease, but a large proportion of the patients soon evolve into pri- mary and secondary progressive phases, which result in a gradual loss of neurological functions [56]. MS does not have a cure, but several therapies have pro- ven helpful. The treatments usually adopted aim to return the general functions to normal after an attack, to prevent new attacks, and to prevent disability. Although MS is still widely regarded as a white matter disease, according to the most recent studies the occur- rence of demyelination and oligodendrocyte lesions in grey matter appears to be prominent and widespread too [57]. Little is still known regarding purinergic P2X recep- tors and MS (Table 1). It was recently established that the P2X 7 receptor subtype is predominantly expressed in differentiated oligodendrocytes [58] and that ATP signalling can directly trigger migration, differentiation and proliferation of oligodendrocyte progenitor cells via activation of several P2 receptors [59]. On the basis of these results, we proposed a model in which ATP released in vivo by damaged or dying tissue, might act as an early signal to mobilize both innate immune cells like dendritic cells and monocytes/macrophages (that are essential for host defense and tissue remodeling), and oligodendrocyte progenitors (that contribute to trigger tissue repair mechanisms). Nevertheless, multi- focal oligodendrocyte death and demyelination occur- ring in all CNS parenchymal areas, very often coexist with oligodendrocyte migration, proliferation, differen- tiation and remyelination efforts. From this perspec- tive, a recent study hypothesized that extracellular ATP might directly contribute to MS lesion-associated release of IL-1b, via P2X 7 receptor-dependent induc- tion of COX-2 protein and downstream pathogenic mediators [53]. These studies were further corroborated by Matute and co-workers [60], showing that (a) oligo- dendrocytes and myelin indeed express functional P2X 7 receptor that can mediate cell death in vitro and in vivo; (b) activation of P2X 7 receptor contributes to tissue damage in experimental autoimmune encephalo- myelitis (EAE) pathology (an animal model for study- ing MS); and (c) finally that P2X 7 receptor expression is increased in human MS tissue before lesion forma- tion. Moreover, it was demonstrated that mice defi- cient in P2X 7 receptor function are more susceptible to EAE than wild-type mice, also showing enhanced inflammation in the CNS [61]. Regarding additional ionotropic P2X receptors, it was also reported that the P2X 4 subtype is probably involved in EAE pathology, being expressed by macro- phages infiltrating the brain and spinal cord, from the early and asymptomatic phase, to the recovery phase of EAE. Moreover, the kinetics of accumulation of P2X 4 receptor in macrophages paralleled those of infil- tration and disease severity, suggesting a role for the P2X 4 receptor in immunoregulation occurring during CNS inflammation [62]. Finally, the pattern of P2X 1–4,6 receptor protein expression and cell distribution was described by immunohistochemistry and immunofluorescence confo- cal microscopy in frontal cortex sections from human MS brain (Amadio and Montilli, personal communica- tion). A clear immunoreactive signal for P2X 1 protein is present in blood vessels on cells of haematopoietic origin, whereas atypical immunohistochemistry signals for P2X 2,4 receptors seem to be localized in grey mat- ter neuronal nuclei. A strong signal for P2X 3 protein is found only in degenerating cortical pyramidal neurons in grey matter, as confirmed by confocal colocalization with the nonphosphorylated epitope of the heavy chain neurofilament protein (Fig. 1). Finally, the P2X 6 rece- ptor seems to be absent from both white and grey mat- ter MS frontal cortex, whereas the human P2X 5 receptor protein could not be detected by lack of appropriate immunoreactive antiserum. P2XR in neurodegenerative and neuroinflammatory events S. Apolloni et al. 360 FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS These and the previously described results unequivo- cally correlate selected P2X receptors to the extent of demyelination and pathologic alterations occurring in MS. Other pathological conditions Of course P2X receptors are implicated in additional neurological disease, such as epilepsy (a common chronic neurological disorder characterized by recur- rent unprovoked seizures due to abnormal, excessive or synchronous neuronal activity in the brain and loss of astrocytic organization [63]), and neuropathic pain (initiated or caused by a primary lesion or dysfunction in the peripheral and/or CNS) (Table 1). Whereas the expression of P2X 2 and P2X 4 receptor subtypes is apparently decreased in the hippocampus of seizure- prone gerbils [64], and a positive relationship between P2X and GABA receptors is well established [65], we still do not know if these effects are only due to compensatory responses to the modulation of GABA functions. Likewise, evidence from a variety of experi- mental strategies, including genetic manipulation and the synthesis of selective antagonists, has clearly indi- cated that the activation of several P2X receptors including P2X 3,2/3,4,7 subtypes, can also modulate neu- ropathic pain [66]. Because of the copious literature available on these specific pathological conditions, and also on other disorders such as trauma, mood altera- tions, schizophrenia and migraine, the reader is addressed to authoritative reviews for a detailed survey of these specific issues [11,67]. Future perspectives Considering that a plethora of differences indeed exists among the various P2X receptor subtypes simulta- neously expressed on any cell phenotype under both normal and/or neurodegenerative or neuroinflammato- ry conditions, full understanding of their role is chal- lenging for both biology and medicine. The design of selective pharmacological compounds potentially ame- liorating pathological conditions involving P2X recep- tors must necessarily take into account these complex and subtle discriminative properties, together with receptor abundance and multiple and composite recep- tor interactions. Thanks to new chemical synthesis, molecular modelling technologies and single molecule biology approaches, novel and more potent and effective tools for P2X receptors are continuously SMI32 P2X 3 20 µ µ m Merged 50 µ m DAB-P2X 3 Fig. 1. P2X 3 receptor expression in human MS frontal cortex tissue. The tissue was supplied by UK Multiple Sclerosis Tissue Bank at Imperial College London, UK. Cryo- stat-obtained frontal sections of human MS cerebral cortex (40 lm thick) were incu- bated with rabbit anti-P2X 3 serum (Alomone, Jerusalem, Israel, red signal); mouse anti- dephosphorylated neurofilament-H protein serum (SMI 32-Sternberger Monoclonals, Inc. Baltimore, MD, green signal), and processed for double immunofluorescence confocal analysis (yellow merged signal). Immunohistochemistry analysis (DAB) was also performed with anti-P2X 3 serum. S. Apolloni et al. P2XR in neurodegenerative and neuroinflammatory events FEBS Journal 276 (2009) 354–364 ª 2008 The Authors Journal compilation ª 2008 FEBS 361 generated. However, several fundamental questions remain to be answered. From a drug discovery pro- spective, we do not yet know the precise structural basis for ligand specificity to a particular P2X receptor subtype, and how the general structure of P2X recep- tors can be finely discriminated to bind such a large and chemically diverse spectrum of different ligands. From a cellular prospective, we are unaware of how to manage the mutual and consistent interactions of so many different P2X receptor subtypes in triggering the biological properties/functions that result distorted during pathological conditions. It is without doubt that P2X receptors, and P2/P1 receptors in general, are more than the sum of their single entities, and that he purinergic functions in which they are involved require a high level of molecular complexity, fine-tuning and coordination. Concluding remarks We have illustrated the implications and/or corre- lations of P2X purinergic signalling with several nervous system dysfunctions. As reported, this is a well-consolidated field for insults such as ischaemia, although it represents an intriguing new challenge for neurodegenerative diseases such as PD, AD, HD and ALS and for neuroinflammatory/neurodegenerative pathologies as MS. Only preliminary studies and cor- relative data highlight the potential role of P2X recep- tors and extracellular ATP in these new and unexpected areas and spheres of intervention. Never- theless, P2X receptors constitute the tip of the iceberg in purinergic physiopathological mechanisms. Under- standing the entire purinergic signalling machinery, also comprising additional P2/P1 receptors, enzymes and transporters for purinergic ligands [68], thus rep- resents a major task and improvement in trying to ameliorate the neurodegenerative and neuroinflamma- tory conditions that we have described. 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MINIREVIEW Membrane compartments and purinergic signalling: P2X receptors in neurodegenerative and neuroinflammatory events Savina Apolloni, Cinzia Montilli, Pamela Finocchi and Susanna. iceberg in purinergic physiopathological mechanisms. Under- standing the entire purinergic signalling machinery, also comprising additional P2/P1 receptors, enzymes and transporters for purinergic. immune-mediated neuroinflammatory dysfunction. P2X receptors and neurodegenerative/ neuroinflammatory diseases A tight molecular interplay exists among all the com- ponents of the purinergic signalling machinery,