Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE267913 | GEO | 2025/02/10
REPOSITORIES: GEO
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Project description:The neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous undescribed NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected and Musk knockout muscles. These data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling, but is also affected by electrical activity and trophic factors other than agrin. By selecting three previously undescribed NMJ genes Etv4, Lrtm1 and Pdzrn4, we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression and AAV-CRISPR/Cas9-mediated knockout show that Etv4 is sufficient to upregulate expression of ~50% of the NMJ genes in non-synaptic myonuclei, while muscle-specific knockout of Pdzrn4 induces NMJ fragmentation. Further investigation of Pdzrn4 revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ and suggest a novel pathway for NMJ post-translational modifications.
2025-02-10 | GSE267910 | GEO
Project description:The neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous undescribed NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected and Musk knockout muscles. These data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling, but is also affected by electrical activity and trophic factors other than agrin. By selecting three previously undescribed NMJ genes Etv4, Lrtm1 and Pdzrn4, we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression and AAV-CRISPR/Cas9-mediated knockout show that Etv4 is sufficient to upregulate expression of ~50% of the NMJ genes in non-synaptic myonuclei, while muscle-specific knockout of Pdzrn4 induces NMJ fragmentation. Further investigation of Pdzrn4 revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ and suggest a novel pathway for NMJ post-translational modifications.
2025-02-10 | GSE267909 | GEO
Project description:Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance
| PRJNA1113617 | ENA
Project description:Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance (snRNA-Seq)
| PRJNA1113625 | ENA
Project description:Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance (bulk RNA-Seq)
| PRJNA1113626 | ENA
Project description:Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 μm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.
| S-EPMC3656228 | biostudies-literature
Project description:Vertebrate skeletal muscle fibers contain hundreds of nuclei, of which three to six are functionally specialized and stably anchored beneath the postsynaptic membrane at the neuromuscular junction (NMJ). The mechanisms that localize synaptic nuclei and the roles they play in neuromuscular development are unknown. Syne-1 is concentrated at the nuclear envelope of synaptic nuclei; its Caenorhabditis elegans orthologue ANC-1 functions to tether nuclei to the cytoskeleton. To test the involvement of Syne proteins in nuclear anchoring, we generated transgenic mice overexpressing the conserved C-terminal Klarsicht/ANC-1/Syne homology domain of Syne-1. The transgene acted in a dominant interfering fashion, displacing endogenous Syne-1 from the nuclear envelope. Muscle nuclei failed to aggregate at the NMJ in transgenic mice, demonstrating that localization and positioning of synaptic nuclei require Syne proteins. We then exploited this phenotype to show that synaptic nuclear aggregates are dispensable for maturation of the NMJ.
| S-EPMC555524 | biostudies-literature
Project description:BackgroundSkeletal muscle is a plastic tissue that can adapt to different stimuli. It is well established that Mammalian Target of Rapamycin Complex 1 (mTORC1) signalling is a key modulator in mediating increases in skeletal muscle mass and function. However, the role of mTORC1 signalling in adult skeletal muscle homeostasis is still not well defined.MethodsInducible, muscle-specific Raptor and mTOR k.o. mice were generated. Muscles at 1 and 7 months after deletion were analysed to assess muscle histology and muscle force.ResultsWe found no change in muscle size or contractile properties 1 month after deletion. Prolonging deletion of Raptor to 7 months, however, leads to a very marked phenotype characterized by weakness, muscle regeneration, mitochondrial dysfunction, and autophagy impairment. Unexpectedly, reduced mTOR signalling in muscle fibres is accompanied by the appearance of markers of fibre denervation, like the increased expression of the neural cell adhesion molecule (NCAM). Both muscle-specific deletion of mTOR or Raptor, or the use of rapamycin, was sufficient to induce 3-8% of NCAM-positive fibres (P < 0.01), muscle fibrillation, and neuromuscular junction (NMJ) fragmentation in 24% of examined fibres (P < 0.001). Mechanistically, reactivation of autophagy with the small peptide Tat-beclin1 is sufficient to prevent mitochondrial dysfunction and the appearance of NCAM-positive fibres in Raptor k.o. muscles.ConclusionsOur study shows that mTOR signalling in skeletal muscle fibres is critical for maintaining proper fibre innervation, preserving the NMJ structure in both the muscle fibre and the motor neuron. In addition, considering the beneficial effects of exercise in most pathologies affecting the NMJ, our findings suggest that part of these beneficial effects of exercise are through the well-established activation of mTORC1 in skeletal muscle during and after exercise.
| S-EPMC7015238 | biostudies-literature
Project description:Formation, maturation, stabilization, and functional efficacy of the neuromuscular junction (NMJ) are orchestrated by transsynaptic and autocrine signals embedded within the synaptic cleft. Here, we demonstrate that collagen XIII, a nonfibrillar transmembrane collagen, is another such signal. We show that collagen XIII is expressed by muscle and its ectodomain can be proteolytically shed into the extracellular matrix. The collagen XIII protein was found present in the postsynaptic membrane and synaptic basement membrane. To identify a role for collagen XIII at the NMJ, mice were generated lacking this collagen. Morphological and ultrastructural analysis of the NMJ revealed incomplete adhesion of presynaptic and postsynaptic specializations in collagen XIII-deficient mice of both genders. Strikingly, Schwann cells erroneously enwrapped nerve terminals and invaginated into the synaptic cleft, resulting in a decreased contact surface for neurotransmission. Consistent with morphological findings, electrophysiological studies indicated both postsynaptic and presynaptic defects in Col13a1(-/-) mice, such as decreased amplitude of postsynaptic potentials, diminished probabilities of spontaneous release and reduced readily releasable neurotransmitter pool. To identify the role of collagen XIII at the NMJ, shed ectodomain of collagen XIII was applied to cultured myotubes, and it was found to advance acetylcholine receptor (AChR) cluster maturation. Together with the delay in AChR cluster development observed in collagen XIII-deficient mutants in vivo, these results suggest that collagen XIII plays an autocrine role in postsynaptic maturation of the NMJ. Altogether, the results presented here reveal that collagen XIII is a novel muscle-derived cue necessary for the maturation and function of the vertebrate NMJ.
| S-EPMC2955218 | biostudies-literature
Project description:The RNA from NMJ and non-NMJs samples were examined using microarray analysis with of identifying novel regulatory genes that are preferentially expressed in the synaptic regions of the isolated muscle fibers.
2019-12-12 | GSE92400 | GEO