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.
Methods and Findings
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.
Dariusz Gorecki and colleagues investigate the effect of P2RX7 ablation on muscle, brain, and bone pathology in a DMD model mouse.
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.
Why Was This Study Done?
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.
What Did the Researchers Do and Find?
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).
What Do These Findings Mean?
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.
The US National Institute of Neurological Disorders and Stroke provides information on muscular dystrophy (in English and Spanish)
The US National Human Genome Research Institute also provides basic information on Duchenne muscular dystrophy and links to additional resources
The US Centers for Disease Control and Prevention has information about muscular dystrophy
The not-for-profit Nemours Foundation provides information about muscular dystrophy for parents, children, and teenagers (in English and Spanish)
The US not-for-profit organization Parent Project Muscular Dystrophy provides detailed information about all aspects of Duchenne muscular dystrophy and parents’ stories about Duchenne muscular dystrophy
MedlinePlus provides links to further resources on muscular dystrophy and an encyclopedia page on Duchenne muscular dystrophy (in English and Spanish)
Wikipedia has pages about Duchenne muscular dystrophy and P2RX7 (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
TREAT-NMD is a network for the neuromuscular field that provides an infrastructure to ensure that the most promising new therapies reach patients as quickly as possible