A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

<div><p>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 <i>Dmd^mdx</i> 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 Ca²⁺ 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 <i>p2rx7</i> 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.</p></div

Neuronal glycogen synthesis contributes to physiological aging

Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora's disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans

P2RX7 Purinoceptor: A Therapeutic Target for Ameliorating the Symptoms of Duchenne Muscular Dystrophy

open access articleDuchenne 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

Analysis of axonal transport and molecular chaperones during neurodegeneration in drosophila

Neuronal dysfunction and cell death occurs during neurodegeneration. Animal models that express human disease genes and show neurodegenerative-like pathologies are widely used to study particular molecular systems in early neurodegenerative changes. Axonal transport (AT) is perturbed in several prevalent neurodegenerative diseases. The development of a Huntington’s Disease (HD) model in Drosophila melanogaster larvae is described, in which disease gene expression is directed to motor neurons (Chapter 2). This results in stalling and accumulation of AT vesicles in live animals and a locomotion defect after additional environmental stress. The cause of AT disruption and neuronal dysfunction in most cases of neurodegeneration is unknown, but it is associated with protein misfolding and aggregation that overrides cellular defences such as the heat shock protein (HSP) molecular chaperone system. In addition to HD, this applies to human tauopathies such as Alzheimer’s Disease (AD), which involve axonal misfolding and aggregation of tau. Increased throughput assays to test larval locomotion are developed (Chapter 3) in a Drosophila larval model of tauopathy, in which locomotion defects are detectable under normal environmental conditions. Candidate chemical modulation of this locomotion phenotype is described that targets HSP induction (Chapter 4). The chemicals used result in no detectable change in hsp70 level, lower total tau levels, and worsening of the locomotion defect phenotype. Tissue-specific elevation of hsp70 after hypoxic stress (Chapter 5) protects from acute behavioural disability and reduced survival in aged adult Drosophila expressing human tau in the nervous system. These studies indicate some therapeutic potential for HSP elevation in tau mediated pathology. Nevertheless, further work is required if chemical chaperone induction, and the roles of HSPs in axonal transport and homeostasis during chronic neurodegenerative and acute environmental stress, are to be further explored in these model

P2X receptor signalling in skeletal muscle health and disease

Skeletal muscle (SM) is a heterogeneous and dynamic tissue that changes significantly in its form and function in response to external and internal stimuli and throughout life, from development right through to aging. The fully differentiated SM fiber is a highly specialized, complex and metabolically active cell containing finely tuned assemblies of contractile force-generating proteins, while its growth and repair are maintained by a resident stem cell population. There is increasing evidence that extracellular ATP (ATPe) released during physiological activity and acting on P2 purinoceptors is involved in a number of muscle functions. Furthermore, very high levels of ATPe released from injured muscles can trigger further damage either by altered activation of P2 purinoceptors on muscle cells or by promoting inflammatory cell infiltration. Therefore, the effects of activation of specific P2 purinoceptors in SM can vary from physiologically beneficial to pathologically catastrophic. The most studied in SM so far have been the P2X purinoceptors, which are a family of homo/heterotrimeric ATP-gated ion channels comprised of seven subtypes. Of these P2X1, P2X2, P2X4, P2X5, P2X6, and P2X7 have been identified, so far, predominantly in mouse SM and shown to influence cell growth, differentiation, death, and regeneration in health and disease. There is, however, a considerable diversity in expression of these receptors between different muscle groups and fiber types, which is not fully recognized yet. Understanding the roles of specific P2X purinoceptors in the physiology and pathology of different muscle groups might offer new opportunities for targeted pharmacological intervention in SM diseases

Using Drosophila models of neurodegenerative diseases for drug discovery

Introduction: Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease are increasing in prevalence as our aging population increases in size. Despite this, currently there are no disease-modifying drugs available for the treatment of these conditions. Drosophila melanogaster is a highly tractable model organism that has been successfully used to emulate various aspects of these diseases in vivo. These Drosophila models have not been fully exploited in drug discovery and design strategies.Areas covered: This review explores how Drosophila models can be used to facilitate drug discovery. Specifically, we review their uses as a physiologically-relevant medium to high-throughput screening tool for the identification of therapeutic compounds and discuss how they can aid drug discovery by highlighting disease mechanisms that may serve as druggable targets in the future. The reader will appreciate how the various attributes of Drosophila make it an unsurpassed model organism and how Drosophila models of neurodegeneration can contribute to drug discovery in a variety of ways.Expert opinion: Drosophila models of human neurodegenerative diseases can make a significant contribution to the unmet need of disease-modifying therapeutic intervention for the treatment of these increasingly common neurodegenerative conditions. <br/

Purinergic receptors in skeletal muscles in health and in muscular dystrophy

The P2 purinergic (nucleotide) receptor super-family comprises of two families of protein. The P2X, which are channel-forming ionotropic receptors and the P2Y metabotropic receptors activating G protein-mediated signalling pathways. Members of both groups have been identified in skeletal muscle cells at different stages of differentiation. It is well documented that sequential expression and down-regulation of particular P2 receptors on the surface of sarcolemma is closely associated with muscle maturation during embryogenesis and postnatal growth. P2 receptors are also involved in muscle regeneration following injury. Moreover, enhanced expression of specific purinergic receptors together with increased availability of extracellular ATP in dystrophic muscles are important elements of the dystrophic pathophysiology considerably increasing severity

Neuronal glycogen synthesis contributes to physiological aging

Glycogen is a branched polymer of glucose and the carbohydrate energy store for animal cells. In the brain, it is essentially found in glial cells, although it is also present in minute amounts in neurons. In humans, loss-of-function mutations in laforin and malin, proteins involved in suppressing glycogen synthesis, induce the presence of high numbers of insoluble polyglucosan bodies in neuronal cells. Known as Lafora bodies (LBs), these deposits result in the aggressive neurodegeneration seen in Lafora's disease. Polysaccharide-based aggregates, called corpora amylacea (CA), are also present in the neurons of aged human brains. Despite the similarity of CA to LBs, the mechanisms and functional consequences of CA formation are yet unknown. Here, we show that wild-type laboratory mice also accumulate glycogen-based aggregates in the brain as they age. These structures are immunopositive for an array of metabolic and stress-response proteins, some of which were previously shown to aggregate in correlation with age in the human brain and are also present in LBs. Remarkably, these structures and their associated protein aggregates are not present in the aged mouse brain upon genetic ablation of glycogen synthase. Similar genetic intervention in Drosophila prevents the accumulation of glycogen clusters in the neuronal processes of aged flies. Most interestingly, targeted reduction of Drosophila glycogen synthase in neurons improves neurological function with age and extends lifespan. These results demonstrate that neuronal glycogen accumulation contributes to physiological aging and may therefore constitute a key factor regulating age-related neurological decline in humans

Generation and characterization of <i>mdx</i>/P2X7^−/− mice.

<p>(A) Schematics of mouse breeding. (B) Representative Western blots showing increased expression of P2RX7 in 4-wk-old <i>mdx</i> compared to wild-type (WT) gastrocnemius and its absence in <i>mdx</i>/P2X7^−/−. Use of separate Western blots is indicated by solid black lines. (C) Micrographs of P2RX7 immunofluorescence localization (green signal) in 4-wk-old tibialis anterior (TA) muscle from WT, <i>mdx</i>, and Pf-<i>mdx</i>/P2X7^−/− mice showing expression in areas rich with infiltrating cells, and negative control using no primary antibody and with a blue signal denoting nuclear counterstaining.</p