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 cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

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. In this study we have evaluated the contribution of the autophagic pathology in infants before and 6 months after enzyme replacement therapy. Single muscle fibers, isolated from muscle biopsies, were stained for autophagosomal and lysosomal markers and analyzed by confocal microscopy. In addition, unstained bundles of fixed muscles were analyzed by second harmonic imaging. Unexpectedly, the autophagic component which is so prominent in juvenile and adult patients was negligible in infants; instead, the overwhelming characteristic was the presence of hugely expanded lysosomes. After 6 months on therapy, however, the autophagic buildup becomes visible as if unmasked by the clearance of glycogen. In most fibers, the two pathologies did not seem to coexist. These data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults.

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. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The alpha-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the alpha-actinin dynamically exchanged during all stages of myofibril assembly. In contrast, less than 20% of the N-RAP in premyofibrils was exchanged during 10-20 min after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before alpha-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.

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. Modifications of this grid in the mdx mouse model of Duchenne muscular dystrophy have led to identifying dystrophin, the protein missing in both human disease and mouse model, as a microtubule guide. Compared to wild-type (WT), mdx microtubules are disordered and more dense and they have been linked to the dystrophic pathology. GE themselves are disordered in mdx. Here, to identify the causes of GE and microtubule alterations in the mdx muscle, we follow GFP-tagged microtubule markers in live mdx fibers and investigate the recovery of GE and microtubules after treatment with nocodazole. We find that mdx microtubules grow 10% faster but in 30% shorter bouts and that they begin to form a tangled network, rather than an orthogonal grid, right after nucleation from GE. Strikingly, a large fraction of microtubules in mdx muscle fibers seem to dissociate from GE after nucleation. Moreover, we report that mdx GE are mispositioned and increased in number and size. These results were replicated in WT fibers overexpressing the beta-tubulin tubb6, which is elevated in Duchenne muscular dystrophy, in mdx and in regenerating muscle. Finally, we examine the association of GE with ER exit sites and ER-to-Golgi intermediate compartment, which starts during muscle differentiation, and find it persisting in mdx and tubb6 overexpressing fibers. We conclude that GE are full, small, Golgi complexes anchored, and positioned through ER Exit Sites. We propose a model in which GE mispositioning, together with the absence of microtubule guidance due to the lack of dystrophin, determines the differences in GE and microtubule organization between WT and mdx muscle fibers.

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. Ingenuity Pathway Analysis was used to determine functional networks of the predictive genes. Functional validation was performed using a T lymphocyte cell line, Jurkat E6.1. Cells were pretreated with metabotropic glutamate receptor 5 (mGluR5) agonist (DHPG), antagonist (MPEP), or control (PBS) for 20 min before irradiation at 8 Gy in a Mark-1 γ-irradiator. NF-κB activation was assessed using a NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line. LDA achieved 83.3% accuracy in predicting post-radiotherapy fatigue. "Glutamate receptor signaling" was the most significant (p = 0.0002) pathway among the predictive genes. Functional validation using Jurkat cells revealed clustering of mGluR5 receptors as well as increased regulated on activation, normal T cell expressed and secreted (RANTES) production post irradiation in cells pretreated with DHPG, whereas inhibition of mGluR5 activity with MPEP decreased RANTES concentration after irradiation. DHPG pretreatment amplified irradiation-induced NF-κB activation suggesting a role of mGluR5 in modulating T cell activation after irradiation. These results suggest that mGluR5 signaling in T cells may play a key role in the development of chronic inflammation resulting in fatigue and contribute to individual differences in immune responses to radiation. Moreover, modulating mGluR5 provides a novel therapeutic option to treat CRF.

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)-1ß 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-1ß oversecretion led to successful treatment with IL-1-blocking agents. Herein we report a de novo missense mutation (c.1009A > T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1ß and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4-transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.

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-ß inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis.

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. METHODS: Tibia fractures were created in 60 gamma/delta T cell-deficient mice (also called delta T cell receptor [TCR]-knockout mice) and 60 control C57BL/6 mice. Analysis included radiographs, basic histology, mechanical testing, flow cytometry, and immunohistochemical localization of gamma/delta TCR-positive subsets from control animals and of CD44 expression from both groups, as well as enzyme-linked immunosorbent assay for the effector cytokines interleukin-2 (IL-2), interferon-gamma (IFNgamma), and IL-6. RESULTS: Animals deficient in gamma/delta T cells demonstrated more mature histologic elements and quantitative increases in the expression of major bone (bone sialoprotein) and cartilage (type II collagen) matrix proteins and in the expression of bone morphogenetic protein 2 at a critical reparative phase. Moreover, only gamma/delta T cell-deficient animals had a decrease in the osteoprogenitor antiproliferative cytokines IL-6 and IFNgamma at the reparative phase. The result was improved stability at the repair site and an overall superior biomechanical strength in gamma/delta T cell-deficient mice compared with controls. CONCLUSION: The evidence for a role of gamma/delta T cells in the context of skeletal injury demonstrates the importance of the immune system's effect on bone biology, which is relevant to the field of osteoimmunology, and offers a potential molecular platform from which to develop essential therapeutic strategies.

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. To re-examine the role of EB1, we have generated C2 cell lines permanently expressing EB1-targeted shRNAs. In these lines, EB1 is specifically knocked down by more than 90% before any differentiation-related changes can take place. We find that differentiation (assessed by myogenin expression), elongation and fusion are prevented. In addition, two early events that normally precede differentiation - microtubule stabilization and the accumulation of cadherin and beta-catenin on the plasma membrane - are inhibited. Re-expression of EB1 as EB1-GFP restores all aspects of normal differentiation, whereas overexpression of EB3-GFP restores elongation but not fusion. We conclude that EB1 is necessary for the early stages of muscle differentiation.

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. Little progress has been made since. It is still not clear whether centrosomal proteins are redistributed together, whether microtubules are nucleated at the nuclear membrane or transported there post-nucleation, and whether gamma-tubulin (gammatub) remains necessary for nucleation in myotubes. To investigate these questions, we have examined the redistribution of the centrosomal proteins pericentrin (PC), gammatub, and ninein in the C2 muscle cell line. Immunofluorescence of differentiated myotubes shows PC along the nuclear membrane whereas gammatub is only detected there after pre-fixation detergent extraction. After expression of a GFP-tagged gammatub, we observe a weak fluorescence along the nuclear membrane, confirming the presence of gammatub at a low concentration relative to PC. Microinjection of anti-gammatub antibodies into myotubes blocks microtubule growth from both nuclear membranes and centrosomal sites. The centrosomal microtubule-anchoring protein, ninein, is found at the nuclear membrane as well and its distribution appears independent of microtubule integrity. We conclude that centrosomal proteins are redistributed independently during muscle differentiation, to sites that nucleate microtubules both along the nuclear membranes and through the cytoplasm.