Dynamin 2 mechanoenzyme is a key regulator of membrane remodeling and gain-of-function mutations in its gene cause centronuclear myopathies. Here, we investigate the functions of dynamin 2 isoforms ...and their associated phenotypes and, specifically, the ubiquitous and muscle-specific dynamin 2 isoforms expressed in skeletal muscle. In cell-based assays, we show that a centronuclear myopathy-related mutation in the ubiquitous but not the muscle-specific dynamin 2 isoform causes increased membrane fission. In vivo, overexpressing the ubiquitous dynamin 2 isoform correlates with severe forms of centronuclear myopathy, while overexpressing the muscle-specific isoform leads to hallmarks seen in milder cases of the disease. Previous mouse studies suggested that reduction of the total dynamin 2 pool could be therapeutic for centronuclear myopathies. Here, dynamin 2 splice switching from muscle-specific to ubiquitous dynamin 2 aggravated the phenotype of a severe X-linked form of centronuclear myopathy caused by loss-of-function of the MTM1 phosphatase, supporting the importance of targeting the ubiquitous isoform for efficient therapy in muscle. Our results highlight that the ubiquitous and not the muscle-specific dynamin 2 isoform is the main modifier contributing to centronuclear myopathy pathology.
Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa with distinct endotypes including type 2 (T2) high eosinophilic CRS with nasal polyps (eCRSwNP), T2 low ...non-eosinophilic CRS with nasal polyps (neCRSwNP), and CRS without nasal polyps (CRSsNP).
Given the heterogeneity of disease, we hypothesized that assessment of single cell RNA sequencing (scRNA-seq) across this spectrum of disease would reveal connections between infiltrating and activated immune cells and the epithelial and stromal populations that reside in sinonasal tissue.
Here we find increased expression of genes encoding glycolytic enzymes in epithelial cells (EpCs), stromal cells, and memory T-cell subsets from patients with eCRSwNP, as compared to healthy controls. In basal EpCs, this is associated with a program of cell motility and Rho GTPase effector expression. Across both stromal and immune subsets, glycolytic programming was associated with extracellular matrix interactions, proteoglycan generation, and collagen formation. Furthermore, we report increased cell-cell interactions between EpCs and stromal/immune cells in eCRSwNP compared to healthy control tissue, and we nominate candidate receptor-ligand pairs that may drive tissue remodeling.
These findings support a role for glycolytic reprograming in T2-elicited tissue remodeling and implicate increased cellular crosstalk in eCRSwNP.
Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism regulating extracellular Ca2+ entry to control a multitude of Ca2+-dependent signaling pathways and cellular processes. SOCE relies on the ...concerted activity of the reticular Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel ORAI1, and dysfunctions of these key factors result in human pathologies. STIM1 and ORAI1 gain-of-function (GoF) mutations induce excessive Ca2+ influx through SOCE over-activation, and cause tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK), two overlapping disorders characterized by muscle weakness and additional multi-systemic signs affecting growth, platelets, spleen, skin, and intellectual abilities. In order to investigate the pathophysiological effect of overactive SOCE on muscle function and structure, we combined transcriptomics with morphological and functional studies on a TAM/STRMK mouse model. Muscles from Stim1R304W/+ mice displayed aberrant expression profiles of genes implicated in Ca2+ handling and excitation-contraction coupling (ECC), and in vivo investigations evidenced delayed muscle contraction and relaxation kinetics. We also identified signs of reticular stress and abnormal mitochondrial activity, and histological and respirometric analyses on muscle samples revealed enhanced myofiber degeneration associated with reduced mitochondrial respiration. Taken together, we uncovered a molecular disease signature and deciphered the pathomechanism underlying the functional and structural muscle anomalies characterizing TAM/STRMK.
Myotubular myopathy, also called X-linked centronuclear myopathy (XL-CNM), is a severe congenital disease targeted for therapeutic trials. To date, biomarkers to monitor disease progression and ...therapy efficacy are lacking. The Mtm1−/y mouse is a faithful model for XL-CNM, due to myotubularin 1 (MTM1) loss-of-function mutations. Using both an unbiased approach (RNA sequencing RNA-seq) and a directed approach (qRT-PCR and protein level), we identified decreased Mstn levels in Mtm1−/y muscle, leading to low levels of myostatin in muscle and plasma. Myostatin (Mstn or growth differentiation factor 8 Gdf8) is a protein released by myocytes and inhibiting muscle growth and differentiation. Decreasing Dnm2 by genetic cross with Dnm2+/− mice or by antisense oligonucleotides blocked or postponed disease progression and resulted in an increase in circulating myostatin. In addition, plasma myostatin levels inversely correlated with disease severity and with Dnm2 mRNA levels in muscles. Altered Mstn levels were associated with a generalized disruption of the myostatin pathway. Importantly, in two different forms of CNMs we identified reduced circulating myostatin levels in plasma from patients. This provides evidence of a blood-based biomarker that may be used to monitor disease state in XL-CNM mice and patients and supports monitoring circulating myostatin during clinical trials for myotubular myopathy.
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X-linked centronuclear myopathy (XL-CNM) is a severe congenital disease targeted for therapeutic trials. The authors identified decreased plasma myostatin levels in XL-CNM patients and mice. Decreasing Dnm2 in mice blocked or postponed disease progression and resulted in an increase in circulating myostatin, supporting monitoring circulating myostatin in XL-CNM clinical trials.
Omics analyses are powerful methods to obtain an integrated view of complex biological processes, disease progression, or therapy efficiency. However, few studies have compared different disease ...forms and different therapy strategies to define the common molecular signatures representing the most significant implicated pathways. In this study, we used RNA sequencing and mass spectrometry to profile the transcriptomes and proteomes of mouse models for three forms of centronuclear myopathies (CNMs), untreated or treated with either a drug (tamoxifen), antisense oligonucleotides reducing the level of dynamin 2 (DNM2), or following modulation of DNM2 or amphiphysin 2 (BIN1) through genetic crosses. Unsupervised analysis and differential gene and protein expression were performed to retrieve CNM molecular signatures. Longitudinal studies before, at, and after disease onset highlighted potential disease causes and consequences. Main pathways in the common CNM disease signature include muscle contraction, regeneration and inflammation. The common therapy signature revealed novel potential therapeutic targets, including the calcium regulator sarcolipin. We identified several novel biomarkers validated in muscle and/or plasma through RNA quantification, western blotting, and enzyme-linked immunosorbent assay (ELISA) assays, including ANXA2 and IGFBP2. This study validates the concept of using multi-omics approaches to identify molecular signatures common to different disease forms and therapeutic strategies.
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A multi-omics analysis is performed on transgenic mouse lines faithfully modeling different forms of centronuclear myopathies treated or not with several therapeutic approaches. The common disease and therapy signatures are described at the molecular level. Potential circulating biomarkers are identified and await confirmation in patients.
Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in ...human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (T
2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed T
2-multipotent progenitors (T
2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (T
) cells and follicular helper T (T
) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between T
2-MPP, T
2 effectors, T
cells and T
cells. T
2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify T
2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.
Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic ...signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.
Secondary lymphedema occurs in up to 20% of patients after lymphadenectomy performed for the surgical management of tumors involving the breast, prostate, uterus, and skin. Patients develop ...progressive edema of the affected extremity due to retention of protein-rich lymphatic fluid. Despite compression therapy, patients progress to chronic lymphedema in which noncompressible fibrosis and adipose tissue are deposited within the extremity. The presence of fibrosis led to our hypothesis that rosiglitazone, a PPARγ agonist that inhibits fibrosis, would reduce fibrosis in a mouse model of secondary lymphedema after hind limb lymphadenectomy. In vivo, rosiglitazone reduced fibrosis in the hind limb after lymphadenectomy. Our findings verified that rosiglitazone reestablished the adipogenic features of TGF-β1-treated mesenchymal cells in vitro. Despite this, rosiglitazone led to a reduction in adipose tissue deposition. Single-cell RNA-Seq data obtained from human tissues and flow cytometric and histological evaluation of mouse tissues demonstrated increased presence of PDGFRα+ cells in lymphedema; human tissue analysis verified these cells have the capacity for adipogenic and fibrogenic differentiation. Upon treatment with rosiglitazone, we noted a reduction in the overall quantity of PDGFRα+ cells and LipidTOX+ cells. Our findings provide a framework for treating secondary lymphedema as a condition of fibrosis and adipose tissue deposition, both of which, paradoxically, can be prevented with a pro-adipogenic agent.
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