Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis.

Misplaced Golgi Elements Produce Randomly Oriented Microtubules and Aberrant Cortical Arrays of Microtubules in Dystrophic Skeletal Muscle Fibers.

Differentiated mammalian cells and tissues, such as skeletal muscle fibers, acquire an organization of Golgi complex and microtubules profoundly different from that in proliferating cells and still poorly understood. In adult rodent skeletal muscle, the multinucleated muscle fibers have hundreds of Golgi elements (GE), small stacks of cisternae that serve as microtubule-organizing centers. We are interested in the role of the GE in organizing a peculiar grid of microtubules located in the fiber cortex, against the sarcolemma.

Persistent upregulation of the β-tubulin tubb6, linked to muscle regeneration, is a source of microtubule disorganization in dystrophic muscle.

In healthy adult skeletal muscle fibers microtubules form a three-dimensional grid-like network. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), microtubules are mostly disordered, without periodicity. These microtubule defects have been linked to the mdx mouse pathology.

mGluR5 mediates post-radiotherapy fatigue development in cancer patients.

Cancer-related fatigue (CRF) is a common burden in cancer patients and little is known about its underlying mechanism. The primary aim of this study was to identify gene signatures predictive of post-radiotherapy fatigue in prostate cancer patients. We employed Fisher Linear Discriminant Analysis (LDA) to identify predictive genes using whole genome microarray data from 36 men with prostate cancer.

β-Actin shows limited mobility and is required only for supraphysiological insulin-stimulated glucose transport in young adult soleus muscle.

Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the β-actin isoform, which in many cell types is the main actin isoform. However, it is not clear that β-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on β-actin nor actin reorganization upon glucose transport have been tested in mature muscle.

A High-Throughput Real-Time Imaging Technique To Quantify NETosis and Distinguish Mechanisms of Cell Death in Human Neutrophils.

Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by various mechanisms, including formation of neutrophil extracellular traps through a recently described distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in an unbiased, reproducible, and efficient way are lacking, considerably limiting the advancement of research in this field. We optimized and validated, a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using the IncuCyte ZOOM imaging platform and the membrane-permeability properties of two DNA dyes.

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease.

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood.

An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome.

Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1β and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion led to successful treatment with IL-1-blocking agents. Herein we report a de novo missense mutation (c.

Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements.

Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that muscle MTs are dynamic, growing at the typical speed of ∼9 µm/min, and forming small bundles that build a durable network.

Sirt1-deficient mice exhibit an altered cartilage phenotype.

Objective: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here we attempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice.

Method: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wild type (WT) or null Sirt1 gene.

Sirtuin 1 enzymatic activity is required for cartilage homeostasis in vivo in a mouse model.

Objective: We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage.

Methods: Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.

Increased apoptotic chondrocytes in articular cartilage from adult heterozygous SirT1 mice.

Objective: A growing body of evidence indicates that the protein deacetylase, SirT1, affects chondrocyte biology and survival. This report aims to evaluate in vivo attributes of SirT1 in cartilage biology of 129/J murine strains.

Methods: Heterozygous haploinsufficient (SirT1(+/-)) and wild-type (WT; SirT1(+/+)) 129/J mice aged 1 or 9 months were systematically compared for musculoskeletal features, scored for osteoarthritis (OA) severity, and monitored for chondrocyte apoptosis in articular cartilage.

Who needs microtubules? Myogenic reorganization of MTOC, Golgi complex and ER exit sites persists despite lack of normal microtubule tracks.

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2.

Differences in the predominance of lysosomal and autophagic pathologies between infants and adults with Pompe disease: implications for therapy.

Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase, the enzyme that degrades glycogen in the lysosomes. The disease manifests as a fatal cardiomyopathy and skeletal muscle myopathy in infants; in milder late-onset forms skeletal muscle is the major tissue affected. We have previously demonstrated that autophagic inclusions in muscle are prominent in adult patients and the mouse model.

A role for gamma/delta T cells in a mouse model of fracture healing.

Objective: Fractures can initiate an immune response that disturbs osteoblastic and osteoclastic cellular homeostasis through cytokine production and release. The aim of our study was to investigate gamma/delta T cells, innate lymphocytes known to be involved in tissue repair, as potential cellular components of the osteoimmune system's response to an in vivo model of bone injury. The absence of such cells or their effector cytokines influences the fate of other responder cells in proliferation, differentiation, matrix production, and ultimate callus formation.

Microtubule plus-end binding protein EB1 is necessary for muscle cell differentiation, elongation and fusion.

During muscle differentiation, microtubule stability, nucleation and orientation all undergo profound changes, which are simultaneous with and possibly necessary for the elongation and fusion of muscle cells. We do not yet understand these events, but they present similarities with the polarized migration of fibroblasts, in which EB1 is necessary for microtubule stabilization. However, it was recently reported that EB3, not EB1, is involved in muscle cell elongation and fusion, and that neither of these two proteins influences microtubule stabilization.

Myofibril assembly visualized by imaging N-RAP, alpha-actinin, and actin in living cardiomyocytes.

N-RAP is a striated muscle-specific scaffolding protein that organizes alpha-actinin and actin into symmetrical I-Z-I structures in developing myofibrils. Here we determined the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either alpha-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of alpha-actinin subsequently organizing along these structures.

Murine muscle cell models for Pompe disease and their use in studying therapeutic approaches.

Lysosomes filled with glycogen are a major pathologic feature of Pompe disease, a fatal myopathy and cardiomyopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase (GAA). To facilitate studies germane to this genetic disorder, we developed two in vitro Pompe models: myotubes derived from cultured primary myoblasts isolated from Pompe (GAA KO) mice, and myotubes derived from primary myoblasts of the same genotype that had been transduced with cyclin-dependent kinase 4 (CDK4). This latter model is endowed with extended proliferative capacity.

Autophagy and mistargeting of therapeutic enzyme in skeletal muscle in Pompe disease.

Enzyme replacement therapy (ERT) became a reality for patients with Pompe disease, a fatal cardiomyopathy and skeletal muscle myopathy caused by a deficiency of glycogen-degrading lysosomal enzyme acid alpha-glucosidase (GAA). The therapy, which relies on receptor-mediated endocytosis of recombinant human GAA (rhGAA), appears to be effective in cardiac muscle, but less so in skeletal muscle. We have previously shown a profound disturbance of the lysosomal degradative pathway (autophagy) in therapy-resistant muscle of GAA knockout mice (KO).

Autophagy and lysosomes in Pompe disease.

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, intralysosomal glycogen accumulates in multiple tissues, with skeletal and cardiac muscle most severely affected.(1) Complete enzyme deficiency results in rapidly progressive infantile cardiomyopathy and skeletal muscle myopathy that is fatal within the first two years of life. Patients with partial enzyme deficiency suffer from skeletal muscle myopathy and experience shortened lifespan due to respiratory failure.

Dysfunction of endocytic and autophagic pathways in a lysosomal storage disease.

Objective: To understand the mechanisms of skeletal muscle destruction and resistance to enzyme replacement therapy in Pompe disease, a deficiency of lysosomal acid alpha-glucosidase (GAA), in which glycogen accumulates in lysosomes primarily in cardiac and skeletal muscles.

Methods: We have analyzed compartments of the lysosomal degradative pathway in GAA-deficient myoblasts and single type I and type II muscle fibers isolated from wild-type, untreated, and enzyme replacement therapy-treated GAA knock-out mice.

Results: Studies in myoblasts from GAA knock-out mice showed a dramatic expansion of vesicles of the endocytic/autophagic pathways, decreased vesicular movement in overcrowded cells, and an acidification defect in a subset of late endosomes/lysosomes.

Reorganization of microtubule nucleation during muscle differentiation.

Skeletal muscle differentiation involves a complete reorganization of the microtubule network. Nearly 20 years ago, Tassin et al. [1985: J Cell Biol 100:35-46] suggested a mechanism for this reorganization by showing a redistribution of the microtubule organizing center from the centrosome to the nuclear membrane.

Role of Grb2 in EGF-stimulated EGFR internalization.

Grb2 is an adaptor molecule that couples membrane receptors such as the epidermal growth factor receptor (EGFR) to intracellular signaling pathways. To gain insight into the trafficking pathways followed by these molecules after activation by EGF, we visualized Grb2 and EGFR fused to GFP spectral variants in single live cells. In nonstimulated cells, Grb2-YFP was primarily localized diffusely in the cytoplasm, whereas EGFR-CFP was found on the plasma membrane and in endocytic structures localized in the perinuclear area.