Aims
Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the ...involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1.
Methods
By using micropatterned 96‐well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock‐out human‐induced pluripotent stem cells‐derived MNs and human healthy skeletal muscle cells.
Results
This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA‐binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal‐related processes regulated by these proteins that are commonly misregulated in DM1.
Conclusions
Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.
Myotonic dystrophy type 1 (DM1) is one of the most common hereditary muscular dystrophies in adult. In this study, we assessed the anterograde contribution of spinal motoneurons in the neuromuscular defects observed in DM1. By using micropatterned cocultures between human skeletal muscle cells and human‐induced pluripotent stem cells (hiPSC)‐spinal motoneurons derived from DM1 patients, our analysis revealed presynaptic defects including abnormal neuritogenesis, which affect the formation or stability of the neuromuscular junction. These defects can be reproduced by depleting muscleblind (MBNL) proteins whose loss‐of‐function is a key event in DM1 pathogenesis. Thus, our study highlighted the importance of considering the anterograde pathological contribution of spinal motoneurons in discovery future therapy for DM1.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to ...elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.
1 Laboratoire de Rétrovirologie, Institut Pasteur de la Guyane, French Guiana
2 Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France
3 Centre de ...Primatologie, Institut Pasteur de la Guyane, French Guiana
4 CNRS UPR 9051 (UMR 7151), Saint Louis Hospital, Paris, France
5 Viral Immunology Section, NINDS, National Institutes of Health, Bethesda, MD, USA
Correspondence Mirdad Kazanji m.kazanji{at}cirmf.org
A squirrel monkey model of human T-cell leukemia virus type 1 (HTLV-1) infection was used to evaluate the immunogenicity and protective efficacy of a chimeric peptide vaccine composed of a B-cell epitope from the envelope region (aa 175218) and three HLA-A*0201-restricted cytotoxic T-lymphocyte epitopes derived from Tax protein (Tri-Tax). These selected Tax peptides induced secretion of gamma interferon (IFN- ) in peripheral blood mononuclear cells obtained from monkeys chronically infected with HTLV-1. After immunization, a high titre of antibodies and a high frequency of IFN- -producing cells were detected against the Env and the Tri-Tax immunogens, but not against the individual Tax peptides. This might indicate that epitope(s) distinct from those recognized by humans are recognized by responder monkeys. After challenge, it was shown by competitive PCR that partial protection against HTLV-1 infection could be raised in immunized animals. Further studies should be developed to determine the duration of this protection.
Present address: Unité de Rétrovirologie, Centre International de Recherches Médicales de Franceville (CIRMF), BP 769, Franceville, Gabon.
Present address: National Institutes of Health, NCI/AMRVS, 9000 Rockville Pike, Bldg 41, Room C303, Bethesda, MD 20892-5055, USA.
Human T-cell lymphotropic virus type 1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATLL) has a poor prognosis owing to its intrinsic resistance to chemotherapy. Although zidovudine (AZT) and ...alpha interferon (IFN-α) give rise to some response and improve the prognosis of ATLL, alternative therapies are needed. Arsenic trioxide (As
2O
3) has been shown to synergize with IFN-α in arresting cell growth and inducing apoptosis of ATLL cells in vitro. In this study, we evaluated the toxicity and the efficacy of this combined treatment in HTLV-1-infected squirrel monkeys (
Saimiri sciureus) and HTLV-1 infected cell lines derived therefrom. We first show that treatment with As
2O
3 and IFN-α can induce growth arrest in HTLV-1-transformed monkey T-cell lines in vitro. We then show that treatment of squirrel monkeys with As
2O
3 in vivo is highly toxic at 0.9 or 0.3
mg/day but not at 0.14
mg/day for up to 2 weeks. Although the combination of As
2O
3 and IFN-α did not affect significantly the HTLV-1 proviral load in infected monkeys, it reduced the absolute numbers of CD3
+, CD4
+ and CD8
+ cells during treatment, with a significant reduction in the total number of circulating HTLV-1 flower cells in the infected monkeys with chronic ATLL-like disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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