Darek Gorecki Lab
Molecular medicine is now a well-established discipline where complementary molecular techniques are used to identify molecular defects behind human diseases and this knowledge is applied to develop molecular treatments.
The rapid development of molecular medicine techniques and their integration into everyday medical practice makes it difficult to imagine the 21st century biomedical scientists or physicians without, at least, a basic understanding of this subject. In our laboratory we attempt integration of molecular and clinical research to study the mechanisms of human diseases, providing research-led education.
Our laboratory is a member of the LARC Neuroscience Network and benefits from extensive European collaborations established through large European projects: The Advanced Microscopy Network (AdMiN), The Transchannel Neuroscience Network (TC2N) and The Peptide Research Network of Excellence (PeReNE). We also collaborate with the Nencki Institute, Warsaw and our research is supported by the Muscular Dystrophy Association USA.
Rare Diseases, 2016
Our current laboratory research
P2RX7 Purinoceptor as a therapeutic target for treatment of Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease, which leads to severe disability and death of young men. Currently, there is no treatment, which improves the long-term outcome. Therefore, new therapeutic modalities are urgently needed and abnormalities downstream from the absence of dystrophin are realistic targets.
We have discovered that DMD mutations alter extracellular ATP (eATP) signalling via P2RX7 purinoceptor up-regulation (Yeung et al., 2006; Young et al., 2012), which triggers a unique mechanism of autophagic death in dystrophic muscle cells (Young et al., 2015). Furthermore, ATP is a “danger signal” and P2RX7 its “danger receptor” activating inflammatory responses, which are prominent in DMD muscles. Therefore, the eATP-P2RX7 axis contributes to DMD pathology by stimulating harmful inflammatory responses. We have demonstrated, that genetic ablation or pharmacological inhibition of P2RX7 in the mdx mouse model of DMD, produced significant functional attenuation of both muscle and non-muscle symptoms (Sinadinos et al., 2015). Hence, the P2RX7 receptor is an attractive therapeutic target with clinical potential.
Crucially, the purinergic abnormality has been proven to affect dystrophic myoblasts, contrary to the previously established belief that muscle cells are not affected at this stage of differentiation. Our findings challenge the central hypothesis stating, that the DMD pathology results from sarcolemma fragility due to the absence of dystrophin in differentiated myofibres. Therefore, we are currently investigating the molecular mechanism leading from DMD mutations to the plethora of abnormalities such as in cell proliferation, differentiation, energy metabolism (Onopiuk et al., 2009) , Ca2+ homeostasis (Onopiuk et al., 2015) and death, contributing to loss of myogenic cells and thus impaired muscle regeneration. Understanding how DMD mutations cause such a range of abnormalities is vital for developing effective therapies.
Figure legend: Dying muscle releases large quantities of DAMPs, including ATP, which trigger chronic inflammation. P2RX7 activation on dystrophic myofibers exacerbates injury by promoting intracellular Ca2+ build-up and autophagic cell death. Infiltrating macrophages (Mφ), T-cells, and granulocytes (GrC) cause further myofiber damage, while chronically elevated levels of inflammatory mediators may disturb normal brain and bone functions. Chronic inflammation also reduces repair by altering satellite cell (SC) activation and muscle precursor cell differentiation, while high eATP levels combined with P2RX7 overexpression contribute to myogenic cell death and thus reduce muscle regeneration further still (Sinadinos et al., 2015).
- Sinadinos A, Young C, AlKhalidi R, Teti A, Kalinski P, Mohamad S, Floriot L, Henry T, Tozzi G, Jiang T, Wurtz O, Lefebvre A, Shugay M, Tong J, Vaudry D, Arkle S, doRego JC, Górecki DC. P2RX7 purinoceptor: a therapeutic target for ameliorating the symptoms of Duchenne muscular dystrophy. PLoS Med, 2015, 12(10): e1001888. doi:10.1371/journal.pmed.1001888
- Young C, Sinadinos A, Lefebvre A, Chan P, Arkle S, Vaudry D and Górecki DC. A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large pore formation and HSP90. Autophagy 2015; 11(1):113-30.
- Young C, Brutkowski W, Lien C-F, Arkle S, Lochmüller H, Zabłocki K and Górecki DC. P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: Relationship to pathology and potential target for treatment." J. Cell Mol Med. 16(5):1026-37, 2012
- Yeung D, Zablocki K, Lien C-F, Jiang T, Arkle S, Brutkowski W, Brown J, Lochmuller H, Simon J, Barnard E.A, Górecki D.C. Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells. FASEB J. 20(6): 610-20, 2006.
- Onopiuk M, Brutkowski W, Wierzbicka K, Wojciechowska S, Szczepanowska J, Fronk J, Lochmüller H, Górecki DC, Zabłocki K. Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts. Biochem. Biophys. Res. Com. 2009; 386(3): 463-466.
- Onopiuk M, Brutkowski W, Young CNJ, Krasowska E, Róg J, Ritso M, Wojciechowska S, Arkle S, Zabłocki K, Górecki DC. Store-operated calcium entry contributes to abnormal Ca2+ signalling in dystrophic mdx mouse myoblasts. Archives Biochem. Biophys 2015; 569:1-9.
Role of the dystrophin-associated protein (DAP) complex in the brain
Dystrophin functions as a molecular anchor for a number of proteins, which are collectively known as the dystrophin-associated protein (DAP) complex. These proteins have vital roles in various organs, for instance abnormalities of the DAP complex are implicated in brain formation and function. Our research programme aims to understand the role of DAPs in the brain.
We have recently shown that the absence of dystrophin results in a rearrangement of the precise spatio-temporal pattern of GABAergic synaptic transmission within the CA1 sub-field of the hippocampus (Krasowska et al., PLOS One 2014). These alterations in the molecular machinery, that underpins neuronal cell excitability, suggest a novel role of dystrophin in formation of specific subsets of synapses, in addition to its role in anchoring neurotransmitter receptors.
Moreover, a-dystrobrevin (alpha-DB), a protein contributing to the DAP complex in brain astrocytes, is essential for the formation and functioning of blood-brain barrier (BBB), which plays a key role in maintaining brain functionality. Although mammalian BBB is formed by endothelial cells, its function requires interactions between endotheliocytes and glia. Absence of glial alpha-DB causes abnormalities of BBB and progressive brain oedema (Lien et al, JBC, 2012). Furthermore, alpha-DB not dystrophin showed continuous expression throughout BBB development. Therefore, alpha-DB emerges as a central organiser of DAP in glial endfeet and is a rare example of a glial protein maintaining BBB functions. This finding may not only help to explain some of the cognitive deficits found in Duchenne muscular dystrophy but can also has implications for other neurological diseases where blood brain barrier function is impaired, such as stroke, dementia, epilepsy and secondary cancers.
- Lien C-F, Mohanta S,K, Frontczak-Baniewicz M, Swinny J, Zablocka B and Górecki DC. Absence of glial alpha-dystrobrevin causes abnormalities of the blood-brain barrier and progressive brain edema. J Biol Chem. 287, 49: 41374 –41385, 2012.
- Krasowska E, Zabłocki K, Górecki DC and Swinny JD. Aberrant location of inhibitory synaptic marker proteins in the hippocampus of dystrophin-deficient mice: implications for cognitive impairment in Duchenne muscular dystrophy. 2014 In : PLoS One. 9, 9, p. e108364.