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Generation and Characterization of a Skeletal Muscle Cell-Based Model Carrying One Single Gne Allele: Implications in Actin Dynamics

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Abstract

UDP-N-Acetyl glucosamine-2 epimerase/N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE myopathy (GNEM) characterized by adult-onset muscle weakness and degeneration. However, recent studies propose alternate roles of GNE in other cellular processes beside sialic acid biosynthesis, particularly interaction of GNE with α-actinin 1 and 2. Lack of appropriate model system limits drug and treatment options for GNEM as GNE knockout was found to be embryonically lethal. In the present study, we have generated L6 rat skeletal muscle myoblast cell-based model system carrying one single Gne allele where GNE gene is knocked out at exon-3 using AAV mediated SEPT homology recombination (SKM-GNEHz). The cell line was heterozygous for GNE gene with one wild type and one truncated allele as confirmed by sequencing. The phenotype showed reduced GNE epimerase activity with little reduction in sialic acid content. In addition, the heterozygous GNE knockout cells revealed altered cytoskeletal organization with disrupted actin filament. Further, we observed increased levels of RhoA leading to reduced cofilin activity and causing reduced F-actin polymerization. The disturbed signaling cascade resulted in reduced migration of SKM-GNEHz cells. Our study indicates possible role of GNE in regulating actin dynamics and cell migration of skeletal muscle cell. The skeletal muscle cell-based system offers great potential in understanding pathomechanism and target identification for GNEM.

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Data Availability

All the data are enclosed in the manuscript and supplementary materials. Anything else required shall be provided.

Code Availability

NA

Abbreviations

GNE:

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

ER:

Endoplasmic reticulum

AAV:

Adeno-associated viruses

SEPT:

Synthetic exon promoter trap

ERK:

Extracellular signal-regulated kinase

FAK:

Focal adhesion kinase

Src:

Proto-oncogene tyrosine-protein kinase (sarcoma)

ManNac:

N-acetylmannosamine

NCAM:

Neural cell adhesion molecule

UPR:

Unfolded protein response

CRMP1:

Collapsin response mediator protein 1

ROCK:

Rho-associated protein kinase

LIMK:

LIM (Lin-11 Isl-1 Mec-3) kinase

Aβ 1–42:

Amyloid-β 1–42

loxP:

Locus of X-over P1

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Acknowledgements

We acknowledge Prof. Sudha Bhattacharya, School of Environmental Sciences, Jawaharlal Nehru University, India, and Prof. Alok Bhattacharya, Ashoka University, for their support and help during the project. We also acknowledge Dr. Sivaprakash Ramalingam, IGIB, for valuable suggestions. We acknowledge Advanced Instrumentation Research Facility, JNU, and its staff members (Ashok Sahu and Prabhat Yadav) for helping in acquiring confocal images.

Funding

This work was supported by:

1. Science and Engineering Research Board, Department of Science and Technology, Ministry of Science and Technology, Government of India, New Delhi; Grant Number-EMR/2015/001798.

2. Department of Atomic Energy, Government of India; Grant Number-37(1)/14/31/2018-BRNS/37253

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Authors and Affiliations

  1. School of Biotechnology, Jawaharlal Nehru University, 110067, New Delhi, India

    Shamulailatpam Shreedarshanee Devi, Rashmi Yadav, Fluencephila Mashangva, Priyanka Chaudhary, Shweta Sharma & Ranjana Arya

  2. Special Centre for Systems Medicine (Concurrent Faculty), Jawaharlal Nehru University, New Mehrauli Road, 110067, New Delhi, India

    Ranjana Arya

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  1. Shamulailatpam Shreedarshanee Devi
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Contributions

RA conceptualized, designed, brought financial support through grants, and analyzed the data. RA compiled and wrote the manuscript. SSD performed the experiments, generated the cell line and characterized the cell line, and helped in manuscript draft writing. RY validated the data by performing experiments, blind study and editing of the manuscript. FM confirmed the cell lines through sequencing and performed sialic acid content study. PC characterized the cell line by performing epimerase assay and helped in literature search. SS helped in editing of the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Ranjana Arya.

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The study was performed in the laboratory cell lines and does not involve human or animal subjects.

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All the authors have given consent for publication.

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The authors declare no conflict of interest.

Competing Interest Statement

The current work is related to a patent application number 202011019014 dated on 4 May 2020 in the Indian Patent Office, titled 'A skeletal Muscle cell-based model for GNE Myopathy'. This association does not alter the author adherence to all Molecular Neurobiology policies on sharing data as detailed online.

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Devi, S.S., Yadav, R., Mashangva, F. et al. Generation and Characterization of a Skeletal Muscle Cell-Based Model Carrying One Single Gne Allele: Implications in Actin Dynamics. Mol Neurobiol 58, 6316–6334 (2021). https://doi.org/10.1007/s12035-021-02549-w

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