Some of the trickier medical problems to deal with are those that are developmental in nature, as these disorders can arise through complicated menageries of genes and regulatory systems to create the negative outcome. And even if the culprit is just a single mutation on a specific gene, it often remains difficult to isolate, identify, and fundamentally prove the responsible genetic mutation for what it is. Doubly so for human disorders, as our options to prove developmental deficits may not translate into animal models effectively. But a group of researchers from a trio of universities, Portsmouth, Southampton and Copenhagen, decided to get together to tackle a newly noted child neurodevelopmental disorder and see if they could figure out the genes behind it. And the strange thing about this team is that it included not just the usual group of clinical geneticists and biochemists, but also frog geneticists.
Their target was a set of specific receptors in the central nervous system called α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors or AMPARs for short. These assist in transmitting the electrical signals required for our nervous system to function, specifically the neurotransmitter L-glutamate, and are also likely involved in the parts of our neurons responsible for memory and the ability to learn. In turn, the receptors are composed of four protein subunits collectively referred to by the gene names GRIA 1-4. Past research has discovered that GRIA2 through 4 are involved in causing other neurodevelopmental disorders, but no specific disorders have been shown for GRIA1 despite it being a high likelihood of having the same effect.
The researchers were able to use global genetic databases in order to find people who had developed such a disorder and that, through whole genome sequencing, were found to have a mutation in the GRIA1 gene. There were 7 people found in total, aged between 7 and 26, with none of them having been evaluated for GRIA1 being the potential cause of their disorders. Since the gene is not available through the common commercially sold gene panels, research on it has been stilted until this team decided to tackle the problem. The group were able to definitively show that GRIA1 had mutations in all the individuals, with several of them having the same exact mutation occurring and all resulting in speech and memory delays, hormonal imbalances, and seizures in some cases. They determined that the autosomal recessive inheritance models for the mutation resulted in the more minor effects on speaking and mental retainment, but it was the dominant inheritance version that caused severe seizures and strong impacts due to completely blocking the GRIA1 protein production pathway.
This is where the frogs come into the story or, more specifically, the tadpoles. Several of the researchers on the team have long been proponents of tadpoles as a model organism over mice, at least in regards to such medical research. To test their utility in this study, the group designed a trio of CRISPR insertions targeting different portions of the GRIA1 gene and added in a green fluorescent protein (GFP) marker to be able to identify where the CRISPR changes were being expressed in the body. The tadpoles used were of the western clawed frog (Xenopus tropicalis) variety available through the European Xenopus Resource Centre. Additionally, they expressed the CRISPR transgenes in neural tissue cells alongside testing it in tadpoles in order to have a comparison experimental group.
After infecting the frog egg cells and allowing the tadpoles to hatch and grow, they were examined for physical phenotype changes from the gene mutation. Then they were put through a series of maze tests to determine if they were statistically handicapped on retaining memory and on their physical capabilities as compared to a control group. The free movement pattern (FMP) Y-maze test is more often used on zebrafish in order to test spatial memory and cognitive flexibility in navigation. While physical abnormalities weren’t seen in the tadpoles, behavioral differences were noted, with several having “manic” bouts of movement consistent with how seizures function in tadpoles. The maze test had an additional layer beyond the control group where a group of non-CRISPRed tadpoles were given chemical agents known to impact memory retention in the species to see how they compared to the modified groups.
The maze tested whether the tadpoles followed consistent searching methods by alternating directions chosen or if they consistently took repetitious directions, indicating a lack of memory retention for prior decisions, referred to as working memory. The modified tadpoles exhibited a significant reduction in alternating direction decisions and also a lowered amount of decisions made in general as the maze testing progressed. While statistically significant results, further testing is required to shore up the specifics on how the tadpoles have been developmentally impacted and to what extent.
Neurodevelopment and Model Organisms
In total, the research team was able to showcase the effectiveness of using tadpoles as a model organism for testing genetic disorders. This is particularly so for how conserved genetic sequences are between tadpoles and humans for not only GRIA genes, but many others, and this offers an alternative and additional testing model beyond using mice models as a whole. The direct confirmation of GRIA1 as responsible for this emerging child neurodevelopmental disorder also opens the door to potential treatments, both pharmaceutically and genetically as our technology advances in the future. Hopefully further research such as this can benefit humanity in identifying and treating disorders that currently remain unknown and untested by the scientific community.
Photo CCs: Tadpoles (5886189230).png from Wikimedia Commons