The P2X7 purine receptor has come under intense scrutiny of late, amid claims that under conditions of tissue stress its activation may trigger numerous CNS pathologies. In fact, this ATP-gated ion channel is rapidly gaining a reputation as being thoroughly pervasive in its contributions to cellular dysfunction. A quick survey of the scientific literature reveals that in only the first 3 months of 2013, the P2X7 receptor was cited as a critical player in the pathogenesis of Alzheimer's disease, epilepsy, tumor-genesis, CNS ischemic damage, depression, and autism, as well as peripheral neuropathic pain, rheumatoid arthritis, and glaucoma. Within the CNS this apparent fecundity arises, in large part, from the P2X7 receptor's position as a gatekeeper between glia and neurons in inflammatory cascades, coupled with physiologic characteristics that set it apart from other ATP-sensing P2 receptors. While the role of the P2X7 receptor subtype in neuroinflammation is well documented, evidence for direct cytotoxic actions on neurons has been sparse. In the current issue of the Journal of Neurochemistry, however, Gandelman and colleagues turn the spotlight back from glia to neurons as they demonstrate a direct role for P2X7 receptors in motor neuron cell death pertinent to amyotrophic lateral sclerosis (ALS).
Extracellular nucleotides regulate numerous physiologic processes throughout the body via the activation of purinergic P1 (G-protein coupled, adenosine-preferring), P2X (ionotropic, ATP-preferring), and P2Y (G-protein coupled, ATP and metabolite-preferring) nucleotide receptors. The seven identified P2X receptor subtypes are differentially distributed throughout the CNS, where they contribute to the regulation of a diverse range of critical homeostatic and trophic processes in normal tissue, including inflammatory responses and microglial proliferation, phagocytosis, neurotransmission, and pain perception. In this regard, P2X7 receptors are predominantly localized to microglia throughout the CNS, but are also found on astrocytes and oligodendrocytes, and pre-synaptically on neurons in the cerebral cortex, hippocampus, brainstem, spinal cord, and at neuromuscular junctions (Weisman et al. 2012). Accordingly, their normal CNS functions encompass the control of inflammatory processes (including microglial proliferation, cytokine release, and autolysosome generation), and modulating the release of neurotransmitters including GABA, glutamate, acetylcholine, and ATP (Volonté et al. 2012). P2X7 receptors possess one particular trait, however, that set them apart from their P2X kin, namely the ability to switch between two open channel states depending on extracellular conditions during activation. Basal P2X7 receptor activation leads to opening of membrane channels permeable to small cations (e.g., Ca2+, K+, Na+). During prolonged exposure to high concentrations of ATP, for example following trauma or inflammation-induced cell damage or lysis, P2X7 receptors show little change in current over several minutes, while other P2X receptors desensitize rapidly (in milliseconds to seconds). Instead, prolonged activation leads to opening of a dilated membrane pore permeable to molecules up to 900 Da, rendering the cell vulnerable to necrosis or apoptosis triggers or osmotic lysis. Abnormal fluctuations in local concentrations of ATP can effectively turn a P2X7 receptor from a homeostatic regulator to a cell death trigger (Volonté et al. 2012).
As ATP is released in the CNS following both relatively minor inflammatory events, as well as major responses to trauma and ischemia, a complex balancing act is required to maintain the inflammatory machinery, including P2X7 receptors, in neuroprotection mode. As a result, P2X7 receptors have garnered a great deal of interest as potential therapeutic targets for disorders involving neuroinflammation. One example is ALS, where inflammatory responses and the astroglial environment appear to be key to deciding motor neuron fate. While the mechanism of neurodegeneration underlying muscle loss in ALS is still not clear, over the last few years there has been a realization that a special relationship exists between glial cells and motor neurons in the disease. In this regard, several of the prevalent hypotheses of disease etiology are intrinsically linked with astrocyte and microglial functions, including glutamate excitotoxicity, oxidative damage, energetic dysfunction, and decreased trophic support (Fig. 1). Microglial and astrocytic activation in the spinal cords of ALS patients is a hallmark of the disease, and is also prominent in transgenic mouse models expressing familial ALS gene mutations (most commonly Cu/Zn-superoxide dismutase (SOD1) mutations). As well as providing structural, metabolic, and trophic support to motor neurons, it is proposed that non-neuronal cells may be critical for motor neuron toxicity in ALS models (reviewed by Vargas and Johnson 2010). This concept initially arose from observations in SOD1 mutant mice that segregating mutant SOD1 expression into either neurons or astrocytes precluded motor neuron pathology.
Against this backdrop of increased neuroinflammation in ALS, findings of up-regulation of P2X7 receptors in spinal cord microglia and astrocytes from ALS patients, and in SOD1 animal models, led to the hypothesis that ATP signaling may trigger cytotoxic events in astroglial cells that result in damage to proximal motor neurons. A mechanistic bridge between glial cell activation and motor neuron death was proposed in 2010, when Gandelman and colleagues reported that normal spinal cord astrocytes could initiate a neurotoxic phenotype in co-cultured wild type motor neurons when the astrocytes were repeatedly exposed to high ATP concentrations (Gandelman et al. 2010). This phenomenon involved release of diffusible factors from the astrocytes and induction of peroxynitrite-induced neuronal damage that was blocked by P2X7 receptor-selective inhibitors. Furthermore, they found that P2X7 receptors on astrocytes expressing mutant SOD1 from a familial ALS mouse model were basally active without the need for excessive ATP priming, and that P2X7 receptor activation was required to maintain their toxic phenotype. A similar phenomenon was previously demonstrated in a cortical neuron/microglia co-culture (Volonté et al. 2012). Hence, it appears that ATP release in ALS spinal cord may, via activation of P2X7 receptors, create the conditions necessary to transform astroglial cells from a trophic to a cytotoxic phenotype.
In the present issue, Gandelman and colleagues ask if ATP might also directly target motor neurons, as P2X7 receptors are located on motor neuron axons in addition to surrounding astroglial cells (Gandelman et al. 2013) (Fig. 1). Using cultured wild type rat spinal cord motor neurons, in the absence of any glial cell support, the group demonstrate that a subset of motor neurons were susceptible to relatively low concentrations of the selective P2X7 agonist 3-O-(4-benzoyl)-ATP. A ‘peroxynitrite-fueled apoptotic cascade’ ensued, involving activation of neuronal p38 MAPK, nitric oxide synthase, and Fas signaling, resulting in neuronal death. The authors go on to show that Ca2+ influx following P2X7 receptor activation underlies the nitric oxide synthase activation, and the subsequent cell death mechanism corresponds with the peroxynitrite/Fas death pathway previously identified in motor neuron death following mutant SOD1 expression or trophic factor deprivation. Together, the studies in mixed cultures and in primary motor neuron cultures suggest that P2X7 receptor activation can mediate motor neuron damage both indirectly as a result of stimulation of astroglial cells, and directly via activation of neuronal receptors, with both routes involving activation of downstream signaling pathways associated with oxidative damage (Gandelman et al. 2010, 2013). Several questions remain unanswered, however, including how the consequences of an ALS-associated gene mutation might impact neuronal receptor activation, plus whether P2X7 receptors on motor neurons contribute to cell death in intact ALS models in vivo. This latter point is particularly pertinent because, as Gandelman and colleagues describe, the natural endogenous agonist, ATP, can have opposing beneficial effects in situ as a result of actions of the ATP degradation product adenosine (Gandelman et al. 2013). In addition, P2X7 receptor activation can induce expression of other purinergic receptor subtypes, including neuroprotective P2Y2 receptors (reviewed by Weisman et al. 2012).
The observations of a neuronal role for P2X7 receptors in a motor neuron degeneration model add to the rapidly accumulating evidence that P2X7 receptor antagonists may be effective therapeutic targets for multiple neurodegenerative disorders, not limited to ALS. For example, in Huntington's disease mouse models expression of mutant huntingtin protein is linked with an up-regulation of P2X7 receptors at presynaptic sites on cortico-striatal projection neurons (Díaz-Hernández et al. 2009). Treatment with a P2X7 inhibitor abrogated behavioral deficits and neuronal apoptosis in the Huntington's disease mice. In Alzheimer's disease, the microglial P2X7 receptor has taken center stage following reports that receptor expression is up-regulated in microglia around β-amyloid plaques and regulates amyloid precursor protein processing in brains of amyloid precursor protein over-expressing mice (Diaz-Hernandez et al. 2012). Furthermore, inhibition of P2X7 is linked to increased α-secretase activity through inhibition of glycogen synthase kinase 3, which translated to a reduction in numbers of hippocampal amyloid plaques in a mouse model. Another recent study reports that silencing P2X7 receptor expression increased microglial phagocytic activity, leading to increased β-amyloid clearance in vitro (Ni et al. 2013).
The P2X7 receptors possess several features that make them reasonable candidates as drug targets, as North and Jarvis describe in a recent review (North and Jarvis 2013). These include their relatively restricted tissue distribution, and structural differentiation from other P2 receptors. With respect to this latter point, one characteristic feature of the P2X7 receptor that could be targeted is its long intracellular C-terminus ‘tail’, as disruption of this region has been shown to impact multiple properties of the receptor, including the capacity for large pore formation, activation of intracellular signaling pathways, and non-exocytotic glutamate release (Cervetto et al. 2013). Despite increasing interest in targeting P2X7 receptors, drug development has been relatively slow because of a dearth of small molecule tools. The potential for successful drug development increased substantially, however, following the recent solution of the P2X4 receptor atomic structure, opening the door for molecular modeling approaches. Currently, P2X7 inhibitors are being tested preclinically in animal models of numerous CNS disorders, while antagonists have reached clinical trials for treatment of chronic inflammatory diseases, including rheumatoid arthritis, Crohn's disease, and pain.
In conclusion, in circumstances where neuroinflammation is present, P2X7 receptor activation by ATP can play an early role in the propagation of cytotoxic insults, including oxidative damage cascades. This positioning of the P2X7 receptor early in the ATP signaling pathway, plus its localization to multiple cell types, makes it an attractive target for drug manipulation with the potential to impact multiple CNS and peripheral disorders. The success of drug development efforts for CNS disorders, however, will be critically dependent on the ability to modulate the receptor's cytotoxic actions in specific tissues, while maintaining its normal physiologic activities elsewhere.