Báo cáo khoa học: Membrane compartments and purinergic signalling: P2X receptors in neurodegenerative and neuroinflammatory events pdf

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-

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 (P2X1–7) 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 signallallow-ing, 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 micromonanomo-lar ranges, depending on the subunit composition Common to all P2X subtypes is a direct influx of extracellular Ca2+ promoted by purines via the receptor channel, which constitutes a significant source of intracellular Ca2+ This leads to a secondary activation of voltage-gated Ca2+ channels, which probably make the primary contribution to the total intracellular Ca2+ 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.

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, oligodendroastro-cytes, Schwann cells and microglia

[5,6] In particular, P2X1 receptors mediate the

puri-nergic component of sympathetic and parasympathetic

nerve-mediated smooth muscle contraction in a

multi-plicity of tissues P2X2 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 P2X2 receptors are also

significantly localized in the peripheral nervous system

on both sensory and autonomic ganglion neurons

Thus, P2X2 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 P2X3 protein subunit was originally cloned

from rat dorsal root ganglion sensory neurons and, in

the adult, P2X3 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] P2X3 receptors are now recognized as

play-ing a major role in mediatplay-ing the primary sensory

effects of ATP and, as such, are of major importance

in nociception and mechanosensory transduction The

gene encoding the P2X4 protein was originally cloned

from rat brain, where P2X4receptors 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, P2X4 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] P2X5 mRNA and immunoreactivity are found in a wide variety of tissues including brain, spinal cord and eye P2X6mRNA and immunoreactivity are present throughout the CNS, particularly in portions of the cerebellum (Purkinje cells) and hippocampus (pyramidal cells) In addition, P2X6 receptors have been reported in sensory ganglia The P2X7 receptor is predominantly localized on vari-ous types of glia within the peripheral nervvari-ous 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 P2X7 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

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 neuroinflammaneurodegenera-tion, 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 Ca2+ 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] P2X2 and P2X4 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 P2X7 receptor subtype is an apparently important component of the mechanisms of cell dam-age induced by hypoxia/ischaemia After a prolonged ischaemic insult, P2X7 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 P2X1 P2X2 P2X3 P2X4 P2X6 P2X7

Neuropathic pain – [66] [66] [66] – [66]

stimulus fails to induce changes in P2X7 mRNA and

immunoreactivity, whereas serum deprivation

aug-ments P2X7 receptor immunoreactivity only in

astro-cytic, and not in neuronal populations Nevertheless,

presynaptic P2X7 receptor exhibited an increased

response to ATP and 2¢,3¢-O-(4-benzoyl)-benzoyl-ATP

(BzATP) after ischaemic insult, despite no changes in

P2X7 mRNA and P2X7 immunoreactivity [25] In

microglia, increased P2X7 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 P2X7receptor 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 [3H]GABA, an effect inhibited by the selective

P2X7 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 [3H]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 P2X1receptor subtype was also

proved to be potentially disadvantageous in the path of

in vitro ischaemia during oxygen/glucose deprivation

The P2X1 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 P2X1,2,4,7 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 Ca2+ 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 P2X7

receptor subtype, because IL-1b release was stimulated

by the specific agonist BzATP and reversed by the P2X7 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 P2X7 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 P2X7 receptor subtype were reported to stimulate the production and release of copious amounts of superoxide (O2)·), 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 P2X7 receptor, and thus stimulate reactive oxygen species production from the microglia in an autocrine manner [36] Both ATP and

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 P2X7 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 Ca2+ responses induced in these cells

by the selective P2X7 receptor agonist BzATP were

moreover increased by 145% after b-amyloid

(frag-ment 1–42) pretreat(frag-ment They were largely blocked if

the P2X7 receptor inhibitor oATP was added with the

b-amyloid peptide in pretreatment solution [37]

These results suggest novel key roles for the P2X7

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 Cu2+ of the ATP-gated

cur-rents evoked through the P2X4 receptor This was

interpreted as an involvement of P2X4receptors 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 P2X2 mRNA alone was detect-able in undifferentiated PC12 cells, the mRNAs for all P2X1–7 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 P2X2–4 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 P2X7

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 P2X7

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

P2X2,4,6 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 P2X1,3,4,6 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 P2X4 and P2X7 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 P2X7 receptor, were directly studied in ALS post-mortem human spinal cord tissue All ALS cases showed not only increased numbers of P2X7-immunoreactive microglia with respect to control spinal cords, but also a marked upregulation of P2X7 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 P2X7 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 P2X7 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 P2X4 receptor subtype was suggested by the observation that strong P2X4 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

the death of motor neurons in ALS It was recently

shown that preincubation of motor neurons with the

P2X4receptor modulator ivermectin, or with the P2X7

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

P2X4 receptor, and to inhibition of the P2X7 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

P2X7receptor 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 P2X7 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 P2X7 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 P2X7 receptor that can mediate cell death in vitro and

in vivo; (b) activation of P2X7 receptor contributes to tissue damage in experimental autoimmune encephalo-myelitis (EAE) pathology (an animal model for study-ing MS); and (c) finally that P2X7 receptor expression

is increased in human MS tissue before lesion forma-tion Moreover, it was demonstrated that mice defi-cient in P2X7receptor 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 P2X4 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 P2X4receptor in macrophages paralleled those of infil-tration and disease severity, suggesting a role for the P2X4 receptor in immunoregulation occurring during CNS inflammation [62]

Finally, the pattern of P2X1–4,6 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 P2X1 protein

is present in blood vessels on cells of haematopoietic origin, whereas atypical immunohistochemistry signals for P2X2,4receptors seem to be localized in grey mat-ter neuronal nuclei A strong signal for P2X3protein 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 P2X6 rece-ptor seems to be absent from both white and grey mat-ter MS frontal cortex, whereas the human P2X5

receptor protein could not be detected by lack of appropriate immunoreactive antiserum

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 P2X2 and P2X4 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 P2X3,2/3,4,7subtypes, 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

20 µµm

Merged

50 µm DAB-P2X

3

Fig 1 P2X3receptor 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-P2X3serum (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.

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 In addition to

the new and more effective agonists and antagonists

for P2X receptors, or to the direct control of their

phenotypic expression in the brain, the most

innova-tive therapeutic strategies should include the genetic/

pharmacologic manipulation of the extracellular

release, breakdown, reuptake of ATP metabolites, and

of P1 and P2Y receptors

Acknowledgements

Studies from the authors’ laboratory described in this

paper were supported by Cofinanziamenti MIUR

‘Purinoceptors and Neuroprotection’, and by grant

from Ministero della Salute RF05.105V

References

1 Roberts JA, Vial C, Digby HR, Agboh KC, Wen H, Atterbury-Thomas A & Evans RJ (2006) Molecular properties of P2X receptors Pflugers Arch 452, 486– 500

2 Egan TM, Samways DS & Li Z (2006) Biophysics of P2X receptors Pflugers Arch 452, 501–512

3 Volonte´ C, Amadio S, Cavaliere F, D’Ambrosi N,

Vac-ca F & Bernardi G (2003) Extracellular ATP and neu-rodegeneration Curr Drug Targets CNS Neurol Disord

2, 403–412

4 Franke H, Kru¨gel U & Illes P (2006) P2 receptors and neuronal injury Pflugers Arch 452, 622–644

5 Gever JR, Cockayne DA, Dillon MP, Burnstock G & Ford AP (2006) Pharmacology of P2X channels Pflu-gers Arch 452, 513–537

6 Burnstock G & Knight GE (2004) Cellular distribution and functions of P2 receptor subtypes in different sys-tems Int Rev Cytol 240, 31–304

7 Koshimizu TA & Tsujimoto G (2006) Functional role

of spliced cytoplasmic tails in P2X2-receptor-mediated cellular signaling J Pharmacol Sci 101, 261–266

8 Brederson JD & Jarvis MF (2008) Homomeric and het-eromeric P2X3 receptors in peripheral sensory neurons Curr Opin Invest Drugs 9, 716–725

9 Inoue K, Tsuda M & Koizumi S (2004) ATP- and adenosine-mediated signaling in the central nervous sys-tem: chronic pain and microglia: involvement of the ATP receptor P2X4 J Pharmacol Sci 94, 112–114

10 Sperla´gh B, Vizi ES, Wirkner K & Illes P (2006) P2X7 receptors in the nervous system Prog Neurobiol 78, 327–346

11 Burnstock G (2008) Purinergic signalling and disorders

of the central nervous system Nat Rev 7, 575–590

12 Gao HM & Hong JS (2008) Why neurodegenerative diseases are progressive: uncontrolled inflammation drives disease progression Trends Immunol 29, 357– 365

13 Sayre LM, Perry G & Smith MA (2008) Oxidative stress and neurotoxicity Chem Res Toxicol 21, 172–188

14 Pankratov Y, Lalo U, Verkhratsky A & North RA (2006) Vesicular release of ATP at central synapses Pflugers Arch 452, 589–597

15 Sabirov RZ & Okada Y (2005) ATP release via anion channels Purinergic Signal 1, 311–312

16 Hussain MS & Shuaib A (2008) Research into neuro-protection must continue But with a different approach Stroke 39, 521–522

17 Hachinski V (2002) Stroke: the next 30 years Stroke

33, 1–4

18 Cavaliere F, D’Ambrosi N, Sancesario G, Bernardi G

& Volonte´ C (2001) Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antago-nist basilen blue Neurochem Int 38, 199–207

19 Cavaliere F, D’Ambrosi N, Ciotti MT, Mancino G,

Sancesario G, Bernardi G & Volonte´ C (2001) Glucose

deprivation and chemical hypoxia: neuroprotection by

P2 receptor antagonists Neurochem Int 38, 189–197

20 La¨mmer A, Gu¨nther A, Beck A, Kru¨gel U, Kittner H,

Schneider D, Illes P & Franke H (2006)

Neuroprotec-tive effects of the P2 receptor antagonist PPADS on

focal cerebral ischaemia-induced injury in rats Eur J

Neurosci 23, 2824–2828

21 Cavaliere F, Florenzano F, Amadio S, Fusco FR,

Vis-comi MT, D’Ambrosi N, Vacca F, Sancesario G,

Ber-nardi G, Molinari M et al (2003) Up-regulation of

P2X2, P2X4 receptor and ischemic cell death:

preven-tion by P2 antagonists Neuroscience 20, 85–98

22 Cavaliere F, Sancesario G, Bernardi G & Volonte´ C

(2002) Extracellular ATP and nerve growth factor

inten-sify hypoglycemia-induced cell death in primary

neu-rons: role of P2 and NGFRp75 receptors J Neurochem

3, 1129–1138

23 Cavaliere F, Amadio S, Sancesario G, Bernardi G &

Volonte´ C (2004) Synaptic P2X7 and oxygen/glucose

deprivation in organotypic hippocampal cultures

J Cereb Blood Flow Metab 24, 392–398

24 Franke H, Gu¨nther A, Grosche J, Schmidt R, Rossner

S, Reinhardt R, Faber-Zuschratter H, Schneider D &

Illes P (2004) P2X7 receptor expression after ischemia

in the cerebral cortex of rats J Neuropathol Exp Neurol

63, 686–699

25 Wirkner K, Kofalvi A, Fischer W, Gu¨nther A, Franke

H, Gro¨ger-Arndt H, No¨renberg W, Madarasz E, Vizi

ES, Schneider D et al (2005) Supersensitivity of P2X

receptors in cerebrocortical cell cultures after in vitro

ischemia J Neurochem 95, 1421–1437

26 Melani A, Amadio S, Gianfriddo M, Vannucchi MG,

Volonte´ C, Bernardi G, Pedata F & Sancesario G

(2006) P2X7 receptor modulation on microglial cells

and reduction of brain infarct caused by middle cerebral

artery occlusion in rat J Cereb Blood Flow Metab 26,

974–982

27 Le Feuvre RA, Brough D, Touzani O & Rothwell NJ

(2003) Role of P2X7 receptors in ischemic and

excito-toxic brain injury in vivo J Cereb Blood Flow Metab 23,

381–384

28 Sperlagh B, Zsilla G, Baranyi M, Illes P & Vizi ES

(2007) Purinergic modulation of glutamate release under

ischemic-like conditions in the hippocampus

Neurosci-ence 149, 99–111

29 Aihara H, Fujiwara S, Mizuta I, Tada H, Kanno T,

Tozaki H, Nagai K, Yajima Y, Inoue K, Kondoh T

et al.(2002) Adenosine triphosphate accelerates

recov-ery from hypoxic/hypoglycemic perturbation of guinea

pig hippocampal neurotransmission via a P(2) receptor

Brain Res 952, 31–37

30 Cavaliere F, Amadio S, Dinkel K, Reymann K &

Cin-zia Volonte´ C (2007) P2 receptor antagonist

trinitrophe-nyl-adenosine-triphosphate protects hippocampus from oxygen and glucose deprivation cell death J Pharmacol Exp Ther 323, 70–77

31 Blennow K, De Leon MJ & Zetterberg H (2006) Alzhei-mer’s disease Lancet 368, 387–403

32 McLarnon JG, Choi HB, Lue LF, Walker DG & Kim

SU (2005) Perturbations in calcium-mediated signal transduction in microglia from Alzheimer’s disease patients J Neurosci Res 81, 426–435

33 Rampe D, Wang L & Ringheim GE (2004) P2X7 recep-tor modulation of beta-amyloid- and LPS-induced cyto-kine secretion from human macrophages and microglia

J Neuroimmunol 147, 56–61

34 Fa¨rber K & Kettenmann H (2006) Purinergic signaling and microglia Pflugers Arch 452, 615–621

35 Parvathenani LK, Tertyshnikova S, Greco CR, Roberts

SB, Robertson B & Posmantur R (2003) P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer’s disease J Biol Chem 278, 13309–13317

36 Kim SY, Moon JH, Lee HG, Kim SU & Lee YB (2007) ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in

an autocrine fashion Exp Mol Med 39, 820–827

37 McLarnon JG, Ryu JK, Walker DG & Choi HB (2006) Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus

J Neuropathol Exp Neurol 65, 1090–1097

38 Estrada Sa´nchez AM, Mejı´a-Toiber J & Massieu L (2008) Excitotoxic neuronal death and the pathogenesis

of Huntington’s disease Arch Med Res 39, 265–276

39 Nakatsuka T, Tsuzuki K, Ling JX, Sonobe H & Gu JC (2003) Distinct roles of P2X receptors in modulating glutamate release at different primary sensory synapses

in rat spinal cord J Neurophysiol 89, 3243–3252

40 Volonte´ C & Merlo D (1996) Selected P2 purinoceptor modulators prevent glutamate-evoked cytotoxicity in cultured cerebellar granule neurons J Neurosci Res 45, 183–193

41 Nguyen T, Hamby A & Massa SM (2005) Clioquinol down-regulates mutant huntingtin expression in vitro and mitigates pathology in a Huntington’s disease mouse model Proc Natl Acad Sci USA 102, 11840–11845

42 Ferrada E, Arancibia V, Loeb B, Norambuena E, Olea-Azar C & Huidobro-Toro JP (2007) Stoichiometry and conditional stability constants of Cu(II) or Zn(II) clio-quinol complexes; implications for Alzheimer’s and Hun-tington’s disease therapy Neurotoxicology 28, 445–449

43 Cookson MR (2005) The biochemistry of Parkinson’s disease Annu Rev Biochem 74, 29–52

44 Sun JH, Cai GJ & Xiang ZH (2007) Expression of P2X purinoceptors in PC12 phaeochromocytoma cells Clin Exp Pharmacol Physiol 4, 1282–1286