32 research outputs found
Aberrant location of inhibitory synaptic marker proteins in the hippocampus of dystrophin-deficient mice
Duchenne muscular dystrophy (DMD) is a neuromuscular disease that arises from mutations in the dystrophin-encoding gene. Apart from muscle pathology, cognitive impairment, primarily of developmental origin, is also a significant component of the disorder. Convergent lines of evidence point to an important role for dystrophin in regulating the molecular machinery of central synapses. The clustering of neurotransmitter receptors at inhibitory synapses, thus impacting on synaptic transmission, is of particular significance. However, less is known about the role of dystrophin in influencing the precise expression patterns of proteins located within the pre- and postsynaptic elements of inhibitory synapses. To this end, we exploited molecular markers of inhibitory synapses, interneurons and dystrophin-deficient mouse models to explore the role of dystrophin in determining the stereotypical patterning of inhibitory connectivity within the cellular networks of the hippocampus CA1 region. In tissue from wild-type (WT) mice, immunoreactivity of neuroligin2 (NL2), an adhesion molecule expressed exclusively in postsynaptic elements of inhibitory synapses, and the vesicular GABA transporter (VGAT), a marker of GABAergic presynaptic elements, were predictably enriched in strata pyramidale and lacunosum moleculare. In acute contrast, NL2 and VGAT immunoreactivity was relatively evenly distributed across all CA1 layers in dystrophin-deficient mice. Similar changes were evident with the cannabinoid receptor 1, vesicular glutamate transporter 3, parvalbumin, somatostatin and the GABAA receptor alpha1 subunit. The data show that in the absence of dystrophin, there is a rearrangement of the molecular machinery, which underlies the precise spatio-temporal pattern of GABAergic synaptic transmission within the CA1 sub-field of the hippocampus
Store-operated calcium entry contributes to abnormal Ca<sup>2+</sup> signalling in dystrophic mdx mouse myoblasts
Sarcolemma damage and activation of various calcium channels are implicated in altered Ca2+ homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca2+ concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease
Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts
Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblastsâthe effector cells of muscle growth and regenerationâare affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmd(mdx) myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmd(mdx-ÎČgeo) myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease
P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: Relationship to pathology and potential target for treatment.
Open AccessDuchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca2 homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca2 and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca2 influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degenerationâregeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease
Recommendations of procedures to follow in the case of ovarian lesions in girls
This study presents current recommendations of the Polish Association of Pediatric Surgeons (PTChD) regarding diagnostics and treatment of ovarian lesions in girls. They are based on many years of the authorsâ clinical experience as well as a review of international literature and include practical clinical guidelines. The recommendations were formulated in cooperation with the Polish Association of Pediatric Oncology and Hematology (PTOHD), Polish Pediatric and Adolescent Gynecology Section of the Polish Society of Gynecologists and Obstetricians (PTG) and Polish Pediatric Section of the Polish Society of Radiology (PLTR). Only better understanding of prepubertal ovarian biology and natural history of its pathology may help to introduce efficient and safe diagnostic and therapeutic strategies for girls. The prepared document has been supplemented with treatment algorithms.
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 ATPâP2RX7 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
P2RX7 purinoceptor as a therapeutic target â the second coming?
.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
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