Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd(mdx) mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca(2+) influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome--autophagy--and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.

The P2RX7 receptor is a unique member of a family of extracellular ATP (eATP)-gated ion channels expressed in immune cells, where its activation triggers the inflammatory cascade. Therefore, P2RX7 has been long investigated as a target in the treatment of infectious and inflammatory diseases. Subsequently, P2RX7 signaling has been documented in other physiological and pathological processes including pain, CNS and psychiatric disorders and cancer. As a result, a range of P2RX7 antagonists have been developed and trialed. Interestingly, the recent crystallization of mammalian and chicken receptors revealed that most widely-used antagonists may bind a unique allosteric site. The availability of crystal structures allows rational design of improved antagonists and modeling of binding sites of the known or presumed inhibitors. However, several unanswered questions limit the cogent development of P2RX7 therapies. Firstly, this receptor functions as an ion channel, but its chronic stimulation by high eATP causes opening of the non-selective large pore (LP), which can trigger cell death. Not only the molecular mechanism of LP opening is still not fully understood but its function(s) are also unclear. Furthermore, how can tumor cells take advantage of P2RX7 for growth and spread and yet survive overexpression of potentially cytotoxic LP in the eATP-rich environment? The recent discovery of the feedback loop, wherein the LP-evoked release of active MMP-2 triggers the receptor cleavage, provided one explanation. Another mechanism might be that of cancer cells expressing a structurally altered P2RX7 receptor, devoid of the LP function. Exploiting such mechanisms should lead to the development of new, less toxic anticancer treatments. Notably, targeted inhibition of P2RX7 is crucial as its global blockade reduces the immune and inflammatory responses, which have important anti-tumor effects in some types of malignancies. Therefore, another novel approach is the synthesis of tissue/cell specific P2RX7 antagonists. Progress has been aided by the development of p2rx7 knockout mice and new conditional knock-in and knock-out models are being created. In this review, we seek to summarize the recent advances in our understanding of molecular mechanisms of receptor activation and inhibition, which cause its re-emergence as an important therapeutic target. We also highlight the key difficulties affecting this development.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease, leading to severe disability and death in young men. Death is caused by the progressive degeneration of striated muscles aggravated by sterile inflammation. The pleiotropic effects of the mutant gene also include cognitive and behavioral impairments and low bone density. Current interventions in DMD are palliative only as no treatment improves the long-term outcome. Therefore, approaches with a translational potential should be investigated, and key abnormalities downstream from the absence of the DMD product, dystrophin, appear to be strong therapeutic targets. We and others have demonstrated that DMD mutations alter ATP signaling and have identified P2RX7 purinoceptor up-regulation as being responsible for the death of muscles in the mdx mouse model of DMD and human DMD lymphoblasts. Moreover, the ATP–P2RX7 axis, being a crucial activator of innate immune responses, can contribute to DMD pathology by stimulating chronic inflammation. We investigated whether ablation of P2RX7 attenuates the DMD model mouse phenotype to assess receptor suitability as a therapeutic target. Using a combination of molecular, histological, and biochemical methods and behavioral analyses in vivo we demonstrate, to our knowledge for the first time, that genetic ablation of P2RX7 in the DMD model mouse produces a widespread functional attenuation of both muscle and non-muscle symptoms. In dystrophic muscles at 4 wk there was an evident recovery in key functional and molecular parameters such as improved muscle structure (minimum Feret diameter, p < 0.001), increased muscle strength in vitro (p < 0.001) and in vivo (p = 0.012), and pro-fibrotic molecular signatures. Serum creatine kinase (CK) levels were lower (p = 0.025), and reduced cognitive impairment (p = 0.006) and bone structure alterations (p < 0.001) were also apparent. Reduction of inflammation and fibrosis persisted at 20 mo in leg (p = 0.038), diaphragm (p = 0.042), and heart muscles (p < 0.001). We show that the amelioration of symptoms was proportional to the extent of receptor depletion and that improvements were observed following administration of two P2RX7 antagonists (CK, p = 0.030 and p = 0.050) without any detectable side effects. However, approaches successful in animal models still need to be proved effective in clinical practice. These results are, to our knowledge, the first to establish that a single treatment can improve muscle function both short and long term and also correct cognitive impairment and bone loss in DMD model mice. The wide-ranging improvements reflect the convergence of P2RX7 ablation on multiple disease mechanisms affecting skeletal and cardiac muscles, inflammatory cells, brain, and bone. Given the impact of P2RX7 blockade in the DMD mouse model, this receptor is an attractive target for translational research: existing drugs with established safety records could potentially be repurposed for treatment of this lethal disease. Muscular dystrophies are inherited diseases in which the body’s muscles gradually weaken and waste away. The most common and severe muscular dystrophy—Duchenne muscular dystrophy (DMD)—also includes cognitive (thinking) and behavioral impairments and low bone density as well as chronic inflammation. DMD affects about 1 in 3,500 boys; girls can be carriers of DMD but rarely have any symptoms. At birth, boys who carry a mutation (genetic change) in the gene that makes the protein dystrophin seem normal, but the symptoms of DMD soon begin to appear. Affected children may initially have difficulty walking or may find it hard to sit or stand independently. As they age, their muscle strength progressively declines, a process that is aggravated by sterile inflammation (an immune system response to tissue damage that occurs in the absence of an infectious agent), and most affected boys are confined to a wheelchair by the time they are 12 years old. The diaphragm and other muscles involved in breathing also weaken, and the heart muscle becomes enlarged. Consequently, few boys with DMD live beyond their early 20s, usually dying from breathing or heart problems. At present, there is no cure for DMD. However, physical therapy and treatment with steroids (intended to reduce sterile inflammation) can prolong the ability of patients to walk, and assisted ventilation can help with their breathing. One way to treat DMD under investigation is replacement of the defective dystrophin in muscles using gene therapy. Dystrophin normally forms structural scaffolds that sit in the membranes that surround muscle fibers and protect the fibers from damage during muscle contraction. In DMD, the loss of dystrophin, dystrophin-associated proteins, and specific signaling processes causes progressive muscle loss. Although gene therapy approaches that target dystrophin hold some promise, achieving sufficient dystrophin expression in all the crucial muscle groups to prevent progressive muscle damage is hard. Moreover, gene therapy targeted at muscles will not treat the non-muscle-related characteristics of DMD. Targeting an abnormality downstream of dystrophin might therefore be a better approach to the treatment of DMD. One such target is P2RX7. This purinoceptor was originally identified as a sensor of ATP released from damaged cells and is an activator of innate immune responses. Because upregulation of P2RX7 is responsible for muscle death in the mdx mouse model of DMD and for the death of human DMD lymphoblasts, in this study, the researchers investigate whether genetic ablation of P2RX7 can attenuate the DMD symptoms of the mdx mouse model. The researchers mated mdx mice and mice that lack the gene for P2RX7 to obtain Pf-mdx/P2RX7^−/− mice, which make no functional dystrophin or P2RX7. They then compared the structure and function of the muscles (dystrophic pathology) in these mice with those in mdx mice. They also examined specific aspects of the behavior of the mice. At four weeks, there was improved muscle structure and strength, decreased inflammation, and decreased fibrosis (thickening and scarring of the connective tissue covering the muscles) in the Pf-mdx/P2RX7^−/− mice compared to the mdx mice. P2RX7 ablation also reduced blood levels of creatinine kinase (a marker of muscle, heart, and brain injury), cognitive impairment, and bone structure alterations. Importantly, the reduction in inflammation and fibrosis was still evident at 20 months in the leg, diaphragm, and heart muscles of the Pf-mdx/P2RX7^−/− mice compared to the mdx mice. Finally, the dystrophic pathology in mdx mice could also be reduced by treating these mice with P2RX7 antagonists (molecules that bind to P2RX7 and prevent its function). These findings show that genetic ablation of P2RX7 can improve muscle function in the short and long term and can also correct cognitive impairment and bone loss in a mouse model of DMD. Thus, in mdx mice, P2RX7 ablation affects multiple disease mechanisms that affect skeletal and heart muscles, inflammatory cells, brain, and bone. Other preliminary findings suggest that P2RX7 blockade in mdx mice also improves DMD symptoms. These are promising results, but results in animals do not necessarily translate into effective clinical treatments. Nevertheless, these findings identify P2RX7 as an attractive target for the treatment of DMD, particularly since it might be possible to repurpose P2RX7 antagonists originally developed for the treatment of chronic pain for the treatment of DMD. This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001888.

P2X7 purinoceptor promotes survival or cytotoxicity depending on extracellular adenosine triphosphate (ATP) stimulus intensity controlling its ion channel or P2X7-dependent large pore (LP) functions. Mechanisms governing this operational divergence and functional idiosyncrasy are ill-understood. We have discovered a feedback loop where sustained activation of P2X7 triggers release of active matrix metalloproteinase 2 (MMP-2), which halts ion channel and LP responses via the MMP-2-dependent receptor cleavage. This mechanism operates in cells as diverse as macrophages, dystrophic myoblasts, P2X7-transfected HEK293, and human tumour cells. Given that serum-born MMP-2 activity also blocked receptor functions, P2X7 responses in vivo may decrease in organs with permeable capillaries. Therefore, this mechanism represents an important fine-tuning of P2X7 functions, reliant on both cell-autonomous and extraneous factors. Indeed, it allowed evasion from the ATP-induced cytotoxicity in macrophages and human cancer cells with high P2X7 expression levels. Finally, we demonstrate that P2X7 ablation eliminated gelatinase activity in inflamed dystrophic muscles in vivo. Thus, P2X7 antagonists could be used as an alternative to highly toxic MMP inhibitors in treatments of inflammatory diseases and cancers.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle disorder that causes severe disability and death of young men. This disease is characterized by progressive muscle degeneration aggravated by sterile inflammation and is also associated with cognitive impairment and low bone density. Given that no current treatment can improve the long-term outcome, approaches with a strong translational potential are urgently needed. Duchenne muscular dystrophy (DMD) alters P2RX7 signaling in both muscle and inflammatory cells and inhibition of this receptor resulted in a significant attenuation of muscle and non-muscle symptoms in DMDmdx mouse model. As P2RX7 is an attractive target in a range of human diseases, specific antagonists have been developed. Yet, these will require lengthy safety testing in the pediatric population of Duchenne muscular dystrophy (DMD) patients. In contrast, Nucleoside Reverse Transcriptase Inhibitors (NRTIs) can act as P2RX7 antagonists and are drugs with an established safety record, including in children. We demonstrate here that AZT (Zidovudine) inhibits P2RX7 functions acting via the same allosteric site as other antagonists. Moreover, short-term AZT treatment at the peak of disease in DMDmdx mice attenuated the phenotype without any detectable side effects. Recovery was evident in the key parameters such as reduced sarcolemma permeability confirmed by lower serum creatine kinase levels and IgG influx into myofibres, decreased inflammatory cell numbers and inflammation markers in leg and heart muscles of treated mice. Moreover, this short-term therapy had some positive impact on muscle strength in vivo and no detrimental effect on mitochondria, which is the main side-effect of Nucleoside Reverse Transcriptase Inhibitors (NRTIs). Given these results, we postulate that AZT could be quickly re-purposed for the treatment of this highly debilitating and lethal disease. This approach is not constrained by causative DMD mutations and may be effective in alleviating both muscle and non-muscle abnormalities.