Mutations in ANO5 have been linked to several human diseases including muscular dystrophy. Ano5 is an intracellular membrane protein, belonging to the anoctamin protein family. Many of the proteins in this family have been found to possess the Ca2+-activated phospholipid scrambling activity. Despite the clear genetic linkage between ANO5 and muscular dystrophy in patients, we found that complete KO of Ano5 in mice showed no overt muscle pathology during our last funding period. This was independently confirmed by other investigators using a different line of complete Ano5-KO mice. These findings indicate that a potential compensatory mechanism, likely through other anoctamin proteins, is involved in minimizing the impact of complete Ano5 deficiency. Intriguingly, an Ano5-KO mouse expressing putatively a truncated Ano5 peptide developed clinical signs of muscular dystrophy with intracellular aggregates and defective membrane repair. Many of the ANO5 mutations associated with human muscular dystrophy are premature termination mutations. These findings raise an interesting question about how ANO5 mutations cause muscle degeneration in human patients: does the expression of mutant amino-terminal Ano5 peptide lead to muscular dystrophy by promoting the formation of intracellular aggregates and compromising membrane repair machinery? Our continuing research in this proposal is centered on determining the fundamental role of the amino-terminus of Ano5 in regulating the intrinsic lipid scrambling function of anoctamins proteins, membrane repair and its contribution to the pathogenesis of muscular dystrophy caused by ANO5 mutations. Moreover, our studies will reveal the compensatory mechanism underlying the lack of muscular dystrophy phenotype in complete Ano5-KO mice. Through the use of in vivo CRISPR gene editing, biochemical, histopathological, and living cell imaging studies with animal models, our planned experiments shall advance our understanding of the physiological and pathological roles of amino-terminal Ano5 peptides in muscle and also shed critical insights into the development of novel therapeutic strategies for the treatment of Ano5-related muscular dystrophy.

Public Health Relevance

ANO5 has been identified as a causative gene for muscular dystrophy. However, it remains to be determined the role of Ano5 in muscle and how mutations in ANO5 cause muscle degeneration. This project is designed to investigate the mechanisms underlying defective membrane repair and muscle degeneration associated with ANO5 mutations. These studies will advance our understanding of the biology of Ano5 and the pathogenesis of Ano5-related muscular dystrophy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL116546-08
Application #
9859226
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Desvigne-Nickens, Patrice
Project Start
2019-04-01
Project End
2023-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
8
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Ohio State University
Department
Surgery
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Xu, Li; Gao, Yandi; Lau, Yeh Siang et al. (2018) Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice. J Vis Exp :
Sui, Tingting; Xu, Li; Lau, Yeh Siang et al. (2018) Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations. Cell Death Dis 9:609
Sui, Tingting; Lau, Yeh Siang; Liu, Di et al. (2018) A novel rabbit model of Duchenne muscular dystrophy generated by CRISPR/Cas9. Dis Model Mech 11:
Lau, Yeh Siang; Xu, Li; Gao, Yandi et al. (2018) Automated muscle histopathology analysis using CellProfiler. Skelet Muscle 8:32
El Refaey, Mona; Xu, Li; Gao, Yandi et al. (2017) In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice. Circ Res 121:923-929
Xu, Li; Zhao, Lixia; Gao, Yandi et al. (2017) Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA. Nucleic Acids Res 45:e28
Xu, Li; Park, Ki Ho; Zhao, Lixia et al. (2016) CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice. Mol Ther 24:564-9
Zhu, Hua; Han, Renzhi; Duan, Dayue Darrel (2016) Novel Biomarkers and Treatments of Cardiac Diseases. Biomed Res Int 2016:1315627
Xu, Jing; El Refaey, Mona; Xu, Li et al. (2015) Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy. Skelet Muscle 5:43
Zhao, Piming; Torcaso, Audrey; Mariano, Andrew et al. (2014) Anoctamin 6 regulates C2C12 myoblast proliferation. PLoS One 9:e92749

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