Granulomatous myositis: characteristics and outcome from a monocentric retrospective cohort study.

Granulomatous myositis is a clinical-pathological entity, which has been rarely reported, mostly described in sarcoidosis. Currently, no clear and simple prognostic factor has been identified to predict granulomatous myositis evolution. The clinical, anatomopathological, imaging, and biological characteristics of 26 patients with granulomatous myositis were retrospectively collected to describe clinical presentation and outcomes of this condition. Twenty-six patients with granulomatous myositis were included (14 males) with a median age of symptom onset of 65 years. 54 % of patients presented a severe form of the disease defined as a Rankin score ≥2 at last follow-up visit or a progressive form of the disease (no improvement under treatment). Etiology were sarcoidosis (n = 14), inclusion body myositis (n = 4), autoimmune disease (n = 1), hematological malignancy (n = 1), and idiopathic (n = 6). Distal deficit and amyotrophy were more frequent among those with a severe disease. Corticosteroids led to improvement in 75 % of cases, but 66 % of responders relapsed. Methotrexate appeared as a promising second line therapy with clinical improvement in 50 % of patients, and no relapse in responders. Granulomatous myositis is often a severe and difficult-to-treat disease in which patients frequently progress towards severe disability. The presence of muscle atrophy and distal weakness appears to be frequently associated with a severe form of the disease.

Spatial Transcriptomics Reveals Signatures of Histopathological Changes in Muscular Sarcoidosis.

Sarcoidosis is a multisystemic disease characterized by non-caseating granuloma infiltrating various organs. The form with symptomatic muscular involvement is called muscular sarcoidosis. The impact of immune cells composing the granuloma on the skeletal muscle is misunderstood. Here, we investigated the granuloma-skeletal muscle interactions through spatial transcriptomics on two patients affected by muscular sarcoidosis. Five major transcriptomic clusters corresponding to perigranuloma, granuloma, and three successive muscle tissue areas (proximal, intermediate, and distal) around the granuloma were identified. Analyses revealed upregulated pathways in the granuloma corresponding to the activation of T-lymphocytes and monocytes/macrophages cytokines, the upregulation of extracellular matrix signatures, and the induction of the TGF-ß signaling in the perigranuloma. A comparison between the proximal and distal muscles to the granuloma revealed an inverse correlation between the distance to the granuloma and the upregulation of cellular response to interferon-γ/α, TNF-α, IL-1,4,6, fibroblast proliferation, epithelial to mesenchymal cell transition, and the downregulation of muscle gene expression. These data shed light on the intercommunications between granulomas and the muscle tissue and provide pathophysiological mechanisms by showing that granuloma immune cells have a direct impact on proximal muscle tissue by promoting its progressive replacement by fibrosis via the expression of pro-inflammatory and profibrosing signatures. These data could possibly explain the evolution towards a state of disability for some patients.

Immune-Mediated Rippling Muscle Disease Associated With Thymoma and Anti-MURC/Cavin-4 Autoantibodies.

OBJECTIVES: Rippling muscle disease (RMD) is characterized by muscle stiffness, muscle hypertrophy, and rippling muscle induced by stretching or percussion. Hereditary RMD is due to sequence variants in the CAV3 and PTRF/CAVIN1 genes encoding Caveolin-3 or Cavin-1, respectively; a few series of patients with acquired autoimmune forms of RMD (iRMD) associated with AChR antibody-positive myasthenia gravis and/or thymoma have also been described. Recently, MURC/caveolae-associated protein 4 (Cavin-4) autoantibody was identified in 8 of 10 patients without thymoma, highlighting its potential both as a biomarker and as a triggering agent of this pathology. Here, we report the case of a patient with iRMD-AchR antibody negative associated with thymoma. METHODS: We suspected a paraneoplastic origin and investigated the presence of specific autoantibodies targeting muscle antigens through a combination of Western blotting and affinity purification coupled with mass spectrometry-based proteomic approaches. RESULTS: We identified circulating MURC/Cavin-4 autoantibodies and found strong similarities between histologic features of the patient's muscle and those commonly reported in caveolinopathies. Strikingly, MURC/Cavin-4 autoantibody titer strongly decreased after tumor resection and immunotherapy correlating with complete disappearance of the rippling phenotype and full patient remission. DISCUSSION: MURC/Cavin-4 autoantibodies may play a pathogenic role in paraneoplastic iRMD associated with thymoma.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Phosphorylated and aggregated TDP-43 with seeding properties are induced upon mutant Huntingtin (mHtt) polyglutamine expression in human cellular models.

The Tar DNA-Binding Protein 43 (TDP-43) and its phosphorylated isoform (pTDP-43) are the major components associated with ubiquitin positive/Tau-negative inclusions found in neurons and glial cells of patients suffering of amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration-TDP-43 (FTLD-TDP). Many studies have revealed that TDP-43 is also in the protein inclusions associated with neurodegenerative conditions other than ALS and FTLD-TDP, thus suggesting that this protein may be involved in the pathogenesis of a variety of neurological disorders. In brains of Huntington-affected patients, pTDP-43 aggregates were shown to co-localize with mutant Huntingtin (mHtt) inclusions. Here, we show that expression of mHtt carrying 80-97 polyglutamines repeats in human cell cultures induces the aggregation and the phosphorylation of endogenous TDP-43, whereas non-pathological Htt with 25 polyglutamines repeats has no effect. Mutant Htt aggregation precedes accumulation of pTDP-43 and pTDP-43 co-localizes with mHtt inclusions reminding what it was previously described in brains of Huntington-affected patients. Detergent-insoluble fractions from cells expressing mHtt and containing mHtt-pTDP-43 co-aggregates can function as seeds for further TDP-43 aggregation in human cell culture. The human cellular prion protein PrPC was previously identified as a negative modulator of mHtt aggregation; here, we show that PrPC-mediated reduction of mHtt aggregation is tightly correlated with a decrease of TDP-43 aggregation and phosphorylation, thus confirming the close relationships between TDP-43 and mHtt.

Development of an automated capillary nano-immunoassay-Simple Western assay-to quantify total TDP43 protein in human platelet samples.

Frontotemporal lobar degeneration syndrome is the second cause of young-onset dementia. Unfortunately, reliable biomarkers are currently lacking for the diagnosis of this disease. As TDP43 protein is one of the proteins pathologically involved in frontotemporal lobar degeneration, many studies have been performed to assess TDP43 protein diagnostic performances. Mixed results were obtained using cerebrospinal fluid and plasma samples so far. The aim of the study was to develop an automated capillary nano-immunoassay-Simple Western assay-to detect and quantify TDP43 protein simultaneously in human blood-based samples. Simple Western assay was developed with two different cell lysates used as positive controls and was compared to Western blot. TDP43 protein profiles in plasma samples were disappointing, as they were discordant to our positive controls. On the contrary, similar TDP43 patterns were obtained between platelet samples and cell lysates using both assays. Simple Western assay provided good quantitative performances in platelet samples: a linearity of signals could be observed (r2 = 0.994), associated to a within-run variability at 5.7%. Preliminary results based on a cohort of patients suffering from frontotemporal lobar degeneration showed large inter-individual variations superior to Simple Western's analytical variability. Simple Western assay seems to be suitable for detecting and quantifying TDP43 protein in platelet samples, providing a potential candidate biomarker in this disease. Further confirmation studies should now be performed on larger cohorts of patients to assess diagnostic performances of TDP43 protein in platelet samples.

Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages.

In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling.

Expression of Heterologous PrP and Prion Propagation in RK13 Cells.

Cultured cells are valuable models to study prion infections at the cellular level. Unfortunately, the vast majority of cell lines are resistant to the propagation of prion agents. The rabbit epithelial RK13 cell line is among the few cell lines permissive to prion infection. When genetically engineered to express heterologous PrP proteins, RK13 cells become permissive to several strains of prions from various animal species. Here, we describe the generation of stable RK13 cell clones expressing a heterologous PrP protein in an inducible manner, the establishment and maintenance of chronically infected cultures, and the selection of cell clones suitable for cell-based titration of prions.

[Endogenous retroviral sequences in the human genome can play a physiological or pathological role]. / Des séquences rétrovirales endogènes dans le génome humain peuvent jouer un rôle physiologique ou pathologique.

Human endogenous retroviruses (HERV) represent a large part of our genome and the few elements that have retained a potential of expression still remain "dormant" in physiological conditions. In some instances, they can be awakened by environmental factors activating their expression. The best studied conditions of HERV activation are infections caused by microorganisms such as viruses of the Herpesvirus family. This activation can thus lead to the expression of pathogenic proteins such as envelope proteins belonging to the HERV-W and HERV-K families, respectively involved in Multiple Sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Endogenous retroviral proteins can also acquire a physiological function beneficial for humans. This is the case of Syncytin-1 from the HERV-W family, that is involved in placenta formation.

Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway.

Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation.

Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells.

Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aß peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.

Co-infection with the friend retrovirus and mouse scrapie does not alter prion disease pathogenesis in susceptible mice.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Functional mechanisms of the cellular prion protein (PrP(C)) associated anti-HIV-1 properties.

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells.

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP(C)) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP(C) is converted into an abnormally folded isoform, called PrP(Sc), that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP(C) and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrP(Sc) production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.

Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles.

BACKGROUND INFORMATION: TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrP(Sc), a conformationally altered isoform of the normal prion protein (PrP(C)), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a). RESULTS: The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome-like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome-like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs. CONCLUSIONS: The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.

Retrovirus infection strongly enhances scrapie infectivity release in cell culture.

Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.

Analysis of the interactions between HIV-1 and the cellular prion protein in a human cell line.

The cellular prion protein (PrP(c)) is highly conserved in mammals and expressed widely in different tissues but its physiological role remains elusive. Recently, the human PrP(c) was shown to possess nucleic acid binding and chaperoning properties similar to human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, a key viral factor in virus structure and replication. These findings prompted us to determine if PrP(c) could influence HIV-1 replication. We used the human 293T cell line as a model system, since only a very low level of PrP(c) accumulates in these cells. Expression of PrP at a high level resulted in a specific decrease of HIV-1 Env and Vpr expression. Despite similar levels of intracellular Gag, virus production was reduced by eightfold and infectivity by three- to fourfold in the presence of PrP(c). A PrP(c) mutant lacking the glycosylphosphatidylinositol (GPI) anchor peptide did not impair HIV-1 production, suggesting that PrP(c) trafficking is critical for this inhibitory effect. Coexpressing HIV-1 and PrP(c) in these cells also caused a fraction of PrP(c) to become partially proteinase K-resistant (PrP(res)), further illustrating the interactions between HIV-1 and PrP(c).

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.