Zebrafish model for an ultra-rare genetic disease identifies potential treatments

(Press-News.org) BIRMINGHAM, Ala. – Can a small fish help identify possible treatments for an ultra-rare inherited disease found in an Alabama boy? The genetic disease is XMEA, which progressively weakens the muscles and can affect the liver and heart. As of March 2024, only 33 cases had ever been seen worldwide.

After the DNA sequence of the boy’s genome showed a mutation in the VMA21 gene, one of the known causes of XMEA, University of Alabama at Birmingham and Children’s of Alabama pediatric neurologist Michael Lopez, M.D., Ph.D., referred the family to the UAB Center for Precision Animal Modeling, or C-PAM.

At C-PAM and in collaboration with a Canadian group, research led by Matthew Alexander, Ph.D., UAB Department of Pediatrics, Division of Pediatric Neurology, and Jim Dowling, M.D., Ph.D., Hospital for Sick Children, Toronto, Ontario, created a preclinical model of XMEA in zebrafish by mutating the fish gene that is analogous to VMA21. While this small, striped fish is commonly found in home aquariums, zebrafish also are a valuable animal model for human disease due to fast growth, large clutch sizes and easy genetic manipulation. They also are transparent as larvae.

In a study published in EMBO Molecular Medicine, Alexander and Dowling now show that their mutant zebrafish have weakened muscles and other symptoms that mirror human XMEA disease. With this simple model, they were able to test 30 clinically tested drugs and identify two that significantly improved XMEA symptoms in the zebrafish. They now are studying the VMA21 mutation in a mammalian model, the mouse, to further push research toward a possible clinical treatment.

“We have established the first preclinical animal model of XMEA, and we have determined that this model faithfully recapitulates most features of the human disease,” Alexander said. “It thus is ideally suited for establishing disease pathomechanisms and identifying therapies.”

Researchers used CRISPR-Cas9, often called molecular scissors for DNA, to create two mutants: a frameshift mutation caused by a one-base pair deletion, and a premature stop codon created during deletion of 14 base pairs and insertion of 21. Both loss-of-function mutations reduced VMA21 protein levels.

Both mutants showed changes consistent with altered muscle structure and function, such as shorter body length and non-inflated swim bladders. They had reduced ability to swim away from a stimulus, and they spent less time swimming and traveled less distance compared to wildtype zebrafish.

The key cellular change in human XMEA is impairment of autophagy, the cell’s recycling system. Autophagy takes place in cell organelles called lysosomes, and these need to be acidic to activate proteases that degrade proteins for recycling into new proteins. Like human XMEA, the mutant fish lysosomes showed a failure to acidify, and the muscle cells had characteristic vacuoles — fluid-filled enclosed structures. Like human XMEA patients, the fish also showed liver and heart pathologies.

Unlike human XMEA, which can vary from mild to moderate symptoms as a progressive disease, the mutant fish showed severe reductions in life span, presumably due to a more complete loss of VMA function compared to human patients.

Since the fish had impaired autophagy and since there are no therapies for XMEA patients, the researchers tested 30 clinically tested autophagy inhibitory compounds from the Selleckchem drug library on the XMEA fish.

Screening of clutches for changed muscle birefringence, a change in the refraction of polarized light that indicates reduced muscle organization, the team identified nine compounds that both reduced abnormal birefringence and prolonged fish survival. Long-term testing of the nine for improvements in survival and swimming showed that edaravone and LY294002 had the greatest therapeutic effects.

“Excitingly, we found that several autophagy antagonists could ameliorate aspects of the VMA21 zebrafish phenotype, and two compounds in particular improved the phenotype across multiple domains of birefringence, motor function and survival,” Alexander said. “The fact that multiple autophagy modulators ameliorated aspects of the phenotype supports an important role for autophagy in the disease process and lends confidence to the validity and potential translatability of the findings to patients.”

Co-authors with Alexander and Dowling in the study “X-linked myopathy with excessive autophagy: characterization and therapy testing in a zebrafish model,” are Lily Huang, Rebecca Simonian and Lacramioara Fabian, Hospital for Sick Children; and Michael A. Lopez, Muthukumar Karuppasamy, Veronica M. Sanders and Katherine G. English, UAB Department of Pediatrics, Division of Pediatric Neurology.

At UAB, Pediatrics is a department in the Marnix E. Heersink School of Medicine.

XMEA stands for X-linked myopathy with excessive autophagy.

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