Biology subjects

Gorecki, D. C.

Publications and source records attributed to Gorecki, D. C..

6 recordsLinked to original sources

bioRxiv2025

Dystrophin Dp71 is essential for the development and function of macrophages

Mutations in the DMD gene, encoding dystrophins, cause progressive muscle degeneration with severe sterile inflammation. While macrophages predominate amongst muscle-infiltrating cells, being central to both damage and regeneration, they were not known to express dystrophin. Yet, we recently demonstrated Dp71 dystrophin expression correlating with tumour infiltrating macrophages. Here we report physiological, developmentally regulated expression of Dp71 in human and mouse hematopoietic stem cells, which decreases with cell maturation into bone marrow macrophages (BMM). Proteomics with molecular and functional analyses in mouse dystrophin-null BMM and peritoneal macrophages reveal that absence of dystrophin disturbs their development. Alterations in over 300 proteins mapped to pathways and networks relating to reduced migration and phagocytosis and increased NLRP3 inflammasome functions. These defects are Dp71-dependent and not caused by the dystrophic environment, since Dmdmdx mouse macrophages, which express Dp71, are not affected. Thus, we identify an important new role for the DMD gene. Altered Dp71 expression in tumour microenvironment cells and in dystrophin-null patients should be investigated to understand the commonalities between DMD and tumours, and potentially identify new treatments.

cell biology↗

bioRxiv2023

P2X7-dependent exchange of extracellular microparticles and mitochondria by mouse microglia

Microparticles (MPs) are ubiquitously secreted by all cells and play a fundamental role in numerous biological processes such as cell-to-cell communication, cell differentiation, inflammation, and cell energy transfer. Ligation of the P2X7 receptor (P2X7R) by extracellular ATP (eATP) is the well-known stimulus for MP release, which affects their contents in a cell-specific fashion. We investigated MP release and functional impact in mouse microglial cell lines characterized for high (N13-P2X7RHigh) or low (N13-P2X7RLow) expression of the P2X7R. Stimulation with extracellular ATP triggered a P2X7R-dependent release of a MP population enriched with naked mitochondria. Released mitochondria were taken up and incorporated into the mitochondrial network of the recipient cells in a P2X7R-dependent fashion. Other constituents of the MP cargo, e.g. NLRP3 and the P2X7R itself, were also delivered to the recipient cells. Transfer of mitochondria, NLRP3 and P2X7R increased the energy level of the recipient cells and conferred a pro-inflammatory phenotype. These data show that P2X7R-dependent exchange of MPs and mitochondria modulates energy metabolism and inflammatory responses, pointing to the P2X7R as a master regulator of intercellular organelle and MP trafficking in mouse microglia.

cell biology↗

bioRxiv2022

Full-length dystrophin is expressed across human tissues and DMD downregulation commonly occurring in tumours coincides with Duchenne-like molecular alterations

Mutations of the DMD gene, encoding dystrophins, cause Duchenne muscular dystrophy (DMD). Some tumors also display altered dystrophin expression and recent studies identified a developmental onset of DMD. Given that embryogenesis and carcinogenesis share many mechanisms, we analyzed a broad spectrum of tumors to establish whether dystrophin loss evokes related outcomes. Transcriptomic, proteomic, and mutation datasets from fifty tumor tissues and matching controls (10,894 samples) and 140 corresponding tumor cell lines were analyzed. Interestingly, DMD expression was widespread across healthy tissues at levels comparable to housekeeping genes. In 80% of tumors, DMD expression was reduced due to transcriptional downregulation and not somatic mutations. The full-length transcript encoding Dp427 was decreased in 68% of tumors, while Dp71 variants showed variability of expression. Hierarchical clustering analysis of DMD transcripts distinguished malignant from control tissues. Transcriptomes of primary tumors and tumor cell lines with low DMD expression showed enrichment of specific pathways in the differentially expressed genes. Pathways consistently identified: ECM-receptor interaction, calcium signaling and PI3K-Akt, are also altered in DMD muscle. Notably, low DMD expression was associated with a more advanced stage, older age of onset, and reduced survival across different tumors. Thus, DMD transcription occurs throughout a spectrum of normal tissues. The molecular signature associated with its frequent downregulation in malignancies is concordant with changes found in Duchenne muscles, even though these malignancies originate from tissues never previously associated with dystrophin expression or function. Therefore, the importance of this largest known gene extends beyond its roles identified in DMD, certainly into oncology.

genetics↗

bioRxiv2022

Metabotropic purinergic receptor profiles and calcium signalling in primary mice myoblasts differ depending on their muscle origin and are altered in cells with mutated dystrophin gene (mdx mice)

Mortality of Duchenne Muscular Dystrophy (DMD) is a direct consequence of progressive wasting of muscle fibres leading to skeletal muscle deterioration and cardiomyopathy. However pathophysiological effects of mutations in the dystrophin encoding gene, which result in improper muscle maturation are detectable in muscle precursor cells which do not express dystrophin gene at the protein level because of early stage of differentiation thus irrespectively of changes in dystrophin-encoding gene. Among these abnormalities elevated activity of P2X7 receptors and increased store-operated calcium entry, have been identified in mdx mouse myoblasts. Moreover the increased response of immortalized mdx myoblasts to agonists activating metabotropic purinergic receptors was characterised. Experiments on immortalized myoblasts do not allow indicating potentially specific effects of mdx mutation on cells derived from particular muscles. Moreover an immortalization of cells itself may influence their metabolism in poorly defined way. Therefore here the metabotropic response of primary myoblasts derived from various muscles of normal and mdx mice to nucleotide stimulation has been investigated. Transcript and protein level of P2Y receptors, sensitivity to antagonist, and cellular localization clearly indicate P2RY2 as the most affected in mdx myoblasts. This meets our previous conclusion drawn from experiments with immortalized cells. However a pattern of expression and activity of P2Y receptors among myoblasts derived from four muscles differ. Also cellular levels of some other proteins belonging to the "calcium signalling toolkit" differ in myoblasts from various muscle and are differently changed due to mdx mutation. Finally, these results complement and strongly support previously formulated conclusion that phenotypic effects of DMD emerge as early as in undifferentiated muscle and therefore traditional understanding of DMD pathogenesis needs re-evaluation.

biochemistry↗

bioRxiv2021

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 (SC), causing progressive muscle degeneration and repair defects. It was not known whether dystrophic myoblasts--the effector cells of muscle growth and regeneration--are affected. Using a combination of transcriptomic, molecular, functional analyses, and genome-scale metabolic modelling, we demonstrate, for the first time, convergent cell-autonomous abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx mouse myoblasts lacking full-length dystrophin transcripts, the expression of 170 other genes was significantly altered. Myod1 (p=2.9e-21) and key muscle genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology enrichment analysis indicated significant alterations in genes involved in muscle development and function. These transcriptomic abnormalities translated into functional alterations such as increased proliferation (p=3.0e-3), reduced chemotaxis towards both sera-rich (p=3.8e-2) and cytokine-containing medium (p=1.0e-2), and significantly accelerated differentiation in 3D organotypic cultures. These altered myoblast functions are essential for muscle regeneration. The defects were caused by the loss of expression of full-length dystrophin, as strikingly similar and not exacerbated alterations were also observed in dystrophin-null Dmdmdx-{beta}geo myoblasts. Corresponding abnormalities were identified in an established dystrophic mouse muscle (SC5) cell line and human DMD primary myoblasts, confirming universal, cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts indicated significant alteration in the rate of glycolysis/gluconeogenesis (log2FC = 4.8), leukotriene metabolism (log2FC = 4.754), mitochondrial beta-oxidation of branched-chain, odd-chain, and di-unsaturated fatty acids (n-6) (log2FC = -1.187, log2FC = -0.8295 and log2FC = -0.655). These results demonstrate the disease continuum: DMD defects in satellite cells cause myoblast dysfunctions affecting muscle regeneration, which is essential to counteract myofiber loss. Contrary to the established belief, our data demonstrate that typical DMD alterations occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.

pathology↗

bioRxiv2019

Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise - even asymptomatically - is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis, and compared their differentiation dynamics to that of healthy control cells by a comprehensive multi-omic analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time points demonstrated that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation endpoint; and 2) homogeneously and robustly recapitulates key developmental steps - mesoderm, somite, skeletal muscle - which offers the possibility to explore dystrophin functions and find earlier disease biomarkers. Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin functions during muscle development.

developmental biology↗