Ham, Alexander. Studying the healthy, denervated and aged neuromuscular system using single nuclei RNA-seq. 2023, Doctoral Thesis, University of Basel, Faculty of Science.
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Abstract
The neuromuscular junction (NMJ) is the synapse formed between motor neurons and muscle fibers. In conditions like sarcopenia, the age-related loss of muscle mass and function, the NMJ destabilizes, ultimately leading to denervation and atrophy. Moreover, in a significant number of patients with neuromuscular disorders the etiology is unclear. Therefore, investigating how skeletal muscle supports NMJ maintenance is essential for advancing our understanding of neuromuscular disorders and developing potential therapies.
We conducted single nuclei RNA-seq (snRNA-seq) on mouse skeletal muscle to identify transcripts specific to or enriched in NMJ myonuclei. Transcriptomic analysis of NMJ myonuclei revealed a large number of genes that have not previously been described in the context of the NMJ. We selected five synaptic gene candidates and characterized their role in skeletal muscle by overexpression and knockout using adeno-associated viruses (AAVs) and muscle-specific Cas9-expressing mice. Taken together, we identify a transcription factor that promotes synaptic gene expression and a Golgi apparatus associated protein that interacts with Muscle-Specific Kinase (MuSK).
To study how sub-NMJ expressed genes are regulated, we investigated synaptic gene expression in muscles denervated by sciatic nerve transection and botulinum toxin injection, using snRNA-seq. Likewise, we perform snRNA-seq on muscles three weeks after knockout of Musk. Our data indicates that the downregulation of certain synaptic genes after denervation is due to the absence of electrical activity. Furthermore, we show that MuSK as well as other neurotrophic factors promote synaptic gene expression.
To study how the neuromuscular transcriptome changes in sarcopenia, we performed snRNA-seq on muscles and lumbar spinal cords of adult and aged mice. Sarcopenic muscles revealed denervated skeletal muscle populations, however interestingly, some muscle fibers showed markers of atrophy despite not expressing markers of denervation. This suggests that processes other than denervation contribute to atrophy in aged muscle. In the aged spinal cord, the most prominent feature was an increase in activated macrophages.
Taken together, we use snRNA-seq to identify genes specifically expressed by NMJ myonuclei and we analyze how these genes are regulated using different denervation models. We characterize five synaptic transcripts in mice and identify novel proteins involved in NMJ maintenance. Furthermore, using snRNA-seq we detect distinct age-associated nuclear populations in the muscle and spinal cord.
We conducted single nuclei RNA-seq (snRNA-seq) on mouse skeletal muscle to identify transcripts specific to or enriched in NMJ myonuclei. Transcriptomic analysis of NMJ myonuclei revealed a large number of genes that have not previously been described in the context of the NMJ. We selected five synaptic gene candidates and characterized their role in skeletal muscle by overexpression and knockout using adeno-associated viruses (AAVs) and muscle-specific Cas9-expressing mice. Taken together, we identify a transcription factor that promotes synaptic gene expression and a Golgi apparatus associated protein that interacts with Muscle-Specific Kinase (MuSK).
To study how sub-NMJ expressed genes are regulated, we investigated synaptic gene expression in muscles denervated by sciatic nerve transection and botulinum toxin injection, using snRNA-seq. Likewise, we perform snRNA-seq on muscles three weeks after knockout of Musk. Our data indicates that the downregulation of certain synaptic genes after denervation is due to the absence of electrical activity. Furthermore, we show that MuSK as well as other neurotrophic factors promote synaptic gene expression.
To study how the neuromuscular transcriptome changes in sarcopenia, we performed snRNA-seq on muscles and lumbar spinal cords of adult and aged mice. Sarcopenic muscles revealed denervated skeletal muscle populations, however interestingly, some muscle fibers showed markers of atrophy despite not expressing markers of denervation. This suggests that processes other than denervation contribute to atrophy in aged muscle. In the aged spinal cord, the most prominent feature was an increase in activated macrophages.
Taken together, we use snRNA-seq to identify genes specifically expressed by NMJ myonuclei and we analyze how these genes are regulated using different denervation models. We characterize five synaptic transcripts in mice and identify novel proteins involved in NMJ maintenance. Furthermore, using snRNA-seq we detect distinct age-associated nuclear populations in the muscle and spinal cord.
Advisors: | Rüegg, Markus A. |
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Committee Members: | Scheiffele , Peter and Mei, Lin |
Faculties and Departments: | 05 Faculty of Science > Departement Biozentrum > Neurobiology > Cell Biology (Scheiffele) 05 Faculty of Science > Departement Biozentrum > Neurobiology > Pharmacology/Neurobiology (Rüegg) |
UniBasel Contributors: | Ham, Alexander Sebastian and Rüegg, Markus A. and Scheiffele, Peter |
Item Type: | Thesis |
Thesis Subtype: | Doctoral Thesis |
Thesis no: | 15315 |
Thesis status: | Complete |
Number of Pages: | 120 |
Language: | English |
Identification Number: |
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edoc DOI: | |
Last Modified: | 03 Apr 2024 04:30 |
Deposited On: | 02 Apr 2024 07:52 |
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