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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., (© 2022 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function., (© 2022. The Author(s).)

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS., Competing Interests: Disclosures: A. D’Alessandro reported “other” from Omix Technologies Inc, personal fees from Hemanext Inc., and personal fees from Forma Therapeutics outside the submitted work. No other disclosures were reported., (© 2022 Giordano et al.)

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The neuromuscular junction (NMJ) has been the model of choice to understand the principles of communication at chemical synapses. Following groundbreaking experiments carried out over 60 years ago, many studies have focused on the molecular mechanisms underlying the development and physiology of these synapses. This Review summarizes the progress made to date towards obtaining faithful models of NMJs in vitro We provide a historical approach discussing initial experiments investigating NMJ development and function from Xenopus to mice, the creation of chimeric co-cultures, in vivo approaches and co-culture methods from ex vivo and in vitro derived cells, as well as the most recent developments to generate human NMJs. We discuss the benefits of these techniques and the challenges to be addressed in the future for promoting our understanding of development and human disease., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.