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Towards Better Understanding of Etiological Mechanisms at the Neuromuscular Junction
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Rostedt Punga: Clinical Neurophysiology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

The neuromuscular junction (NMJ) serves as a model for understanding the mechanisms that determine communication between neurons and their target cells. Disorders of the NMJ can be either autoimmune or genetic (hereditary). The autoimmune disorder myasthenia gravis (MG) is caused by antibodies against the presynaptic nerve terminal or the postsynaptic muscle membrane, which make up the NMJ. The most common antibodies are directed against the acetylcholine receptor (AChR) or muscle specific tyrosine kinase (MuSK). An alternative to expand on preclinical in-vivo methods for studying mechanisms underlying diseases of neuromuscular transmission is to apply physiologic in-vitro models that would allow tissue-tissue as well as cell-cell interactions. A system that would allow cell-cell interactions in a biological fashion is the micro-electrode array (MEA) chip that allows co-culturing of motor neurons and muscle cells.

The primary hypothesis is that the suggested MEA can be used in creating a reliable model for healthy and diseased NMJ, allowing for manipulations and treatment assays. The secondary hypothesis is that small non-coding RNA, so called microRNAs (miRNA) have a specific role in neuromuscular transmission and in MG.

Study I demonstrated a method of long-term muscle cell culture on the MEA chips, which allows us to trace the development of muscle cells through the observation of their electrical activity at subcellular resolution. The maturation of skeletal muscle tissue was accompanied by a gradual increase in the amplitude and frequency of extracellular individual electrical spikes. The mature muscle tissue demonstrated the steady electrical activity with synchronized spike propagation in different directions across the chip.

Study II showed a specific upregulated profile of miRNAs in the muscles of MuSK antibody seropositive MG mice. Transfection of these miRNAs, miR-1933 and miR-1930, promoted downregulation of several proteins and further confirmation with qPCR revealed a specific blocking of IMPA1 and MRPL27, which are involved in intracellular signal transduction and mitochondrial biogenesis in skeletal muscles.

Study III revealed no correlation between the morphology of skeletal muscle cells and their electrical activity at an early developmental stage. However, the application of recombinant rat agrin increased the number of AChRs clusters in the culture of skeletal muscle and promoted a higher degree of spontaneous activity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 55
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1607
Keywords [en]
Neuromuscular junction, Muscle cells, Microelectrode array, MicroRNA
National Category
Neurosciences
Research subject
Clinical Neurophysiology; Biomedical Laboratory Science
Identifiers
URN: urn:nbn:se:uu:diva-395581ISBN: 978-91-513-0791-6 (print)OAI: oai:DiVA.org:uu-395581DiVA, id: diva2:1364901
Public defence
2019-12-11, The library of the Department of Clinical Neurophysiology, Uppsala University Hospital, entrance 85, 3rd floor, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-20 Created: 2019-10-23 Last updated: 2019-11-20
List of papers
1. Long-Term High-Density Extracellular Recordings Enable Studies of Muscle Cell Physiology
Open this publication in new window or tab >>Long-Term High-Density Extracellular Recordings Enable Studies of Muscle Cell Physiology
2018 (English)In: Frontiers in Physiology, ISSN 1664-042X, E-ISSN 1664-042X, Vol. 9, article id 1424Article in journal (Refereed) Published
Abstract [en]

Skeletal (voluntary) muscle is the most abundant tissue in the body, thus making it an important biomedical research subject. Studies of neuromuscular transmission, including disorders of ion channels or receptors in autoimmune or genetic neuromuscular disorders, require high-spatial-resolution measurement techniques and an ability to acquire repeated recordings over time in order to track pharmacological interventions. Preclinical techniques for studying diseases of neuromuscular transmission can be enhanced by physiologic ex vivo models of tissue-tissue and cell-cell interactions. Here, we present a method, which allows tracking the development of primary skeletal muscle cells from myoblasts into mature contracting myotubes over more than 2 months. In contrast to most previous studies, the myotubes did not detach from the surface but instead formed functional networks between the myotubes, whose electrical signals were observed over the entire culturing period. Primary cultures of mouse myoblasts differentiated into contracting myotubes on a chip that contained an array of 26,400 platinum electrodes at a density of 3,265 electrodes per mm(2). Our ability to track extracellular action potentials at subcellular resolution enabled study of skeletal muscle development and kinetics, modes of spiking and spatio-temporal relationships between muscles. The developed system in turn enables creation of a novel electrophysiological platform for establishing ex vivo disease models.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2018
Keywords
muscle, spikes, spike analysis, physiology, microelectrode array (MEA) chip, skeletal
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-367028 (URN)10.3389/fphys.2018.01424 (DOI)000446852800001 ()30356837 (PubMedID)
Funder
Swedish Research Council, VR-523-2014-2048Swedish Research Council, VR-2016-2184Göran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2018-11-28 Created: 2018-11-28 Last updated: 2019-10-23Bibliographically approved
2. miR-1933-3p is upregulated in skeletal muscles of MuSK+ EAMG mice and affects Impa1 and Mrpl27.
Open this publication in new window or tab >>miR-1933-3p is upregulated in skeletal muscles of MuSK+ EAMG mice and affects Impa1 and Mrpl27.
2019 (English)In: Neuroscience research, ISSN 0168-0102, E-ISSN 1872-8111, article id S0168-0102(18)30649-7Article in journal, Editorial material (Refereed) Published
Abstract [en]

MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) typically affects skeletal muscles of the bulbar area, including the omohyoid muscle, causing focal fatigue, weakness and atrophy. The profile of circulating extracellular microRNA (miRNA) is changed in MuSK + MG, but the intracellular miRNA profile in skeletal muscles of MuSK + MG and MuSK + experimental autoimmune MG (EAMG) remains unknown. This study elucidated the intracellular miRNA profile in the omohyoid muscle of mice with MuSK + EAMG. The levels of eleven mouse miRNAs were elevated and two mouse miRNAs were reduced in muscles of MuSK + EAMG mice. Transient expression of miR-1933-3p and miR-1930-5p in mouse muscle (C2C12) cells revealed several downregulated genes, out of which five had predicted binding sites for miR-1933-3p. The mRNA expression of mitochondrial ribosomal protein L27 (Mrpl27) and Inositol monophosphatase I (Impa1) was reduced in miR-1933-3p transfected C2C12 cells compared to control cells (p = 0.032 versus p = 0.020). Further, transient expression of miR-1933-3p reduced Impa1 protein accumulation in C2C12 cells. These findings provide novel insights of dysregulated miRNAs and their intracellular pathways in muscle tissue afflicted with MuSK + EAMG, providing a possible link to mitochondrial dysfunction and muscle atrophy observed in MuSK + MG.

Keywords
EAMG, Experimental autoimmune myasthenia gravis, MuSK antibody, miR-1933-3p, microRNA
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-395566 (URN)10.1016/j.neures.2019.02.003 (DOI)30763589 (PubMedID)
Available from: 2019-10-21 Created: 2019-10-21 Last updated: 2019-10-23
3. Evaluation of muscle action potential parameters in relation to morphology of skeletal muscle cell culture on high-density microelectrode array chips
Open this publication in new window or tab >>Evaluation of muscle action potential parameters in relation to morphology of skeletal muscle cell culture on high-density microelectrode array chips
(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-395669 (URN)
Available from: 2019-10-22 Created: 2019-10-22 Last updated: 2019-10-23

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