A team of researchers from the University of Utah have discovered that inhibition of the enzyme topoisomerase IIα (Top2a) during early development leads to a reduction in social behaviours in zebrafish and in mice, leading to a phenotype similar to that of autism in humans. Furthermore, Top2a defects lead to the downregulation of genes known to be associated with autism. The work, published in Science, describes how environmental and genetic factors influence our behaviour at the earliest stages of life.
A sense of community
The vast majority of animals exhibit common social behaviours. These act to provide a sense of protection and community, and in humans can be seen almost immediately after birth. Sociality is thought to develop prenatally, yet little is known as to how.
Despite the pervasiveness of these behaviours, some individuals display reduced sociality; one of the common symptoms of autism spectrum disorder (ASD). ASD has been studied at length, but questions still remain as to how genetic or environmental factors influence the development of the autism phenotype.
To further investigate embryonic development of social behaviours, Randall Peterson and his Utah-based team chose to conduct experiments in zebrafish. Zebrafish are a useful model organism for a variety of reasons; their transparent embryos are easy to manipulate, and there is a significant amount of conservation between zebrafish and humans. Most importantly for this study, there are vast similarities in sociality between the species.
The researchers exposed zebrafish embryos to over 1000 drugs to assess if exposure led to defects in social behaviour. This was analysed through the use of “Fishbook” – a 3D-printed arena where fish could choose to swim towards a social stimulus. It was observed that fish who had been exposed to fluroquinolone antibiotics were less likely to do so, displaying reduced sociality.
Fluroquinolones act by inhibiting type II topoisomerases. These are enzymes that play a role in DNA replication and recombination, making them ideal for combatting bacterial infection. However, at high doses the drugs can influence processes in eukaryotic cells. To determine which topoisomerase was impacting the fish’ behaviour, the team used splice-blocking mechanisms to inhibit different isoforms. It was observed that a lack of Top2a lead to the lowered social interactions seen in the zebrafish.
From fish to mice
The next stages of the study involved inhibiting the expression of topoisomerases in mice. Pregnant mothers were treated with Top2a inhibitors, and their offspring raised to adulthood. Those who were treated engaged in less social interaction than untreated counterparts, confirming that the link between Top2a and sociality is conserved across different species. These mice also displayed a similar phenotype to that of ASD in humans, such as communication difficulties and engaging in repetitive behaviours.
RNA-sequencing of Top2a mutants revealed an association with a multitude of genes previously associated with ASD risk. Moreover, analysis showed that these genes, like others affected by Top2a inhibition, showed increased binding to PCR2 – a complex involved in H3K27me3 trimethylation – and were enriched for this biomarker.
A reversible phenotype
The use of PRC2 inhibitory drugs was shown to rescue the phenotype in Top2a depleted zebrafish and increase social behaviours once more. This result implies that Top2a plays an important role in an epigenetic pathway that impacts social behaviour. The ability to rescue the phenotype shocked Peterson, who stated that he “would’ve thought disrupting brain development when you’re an embryo would be irreversible.”
The results of the study further our understanding of how both genetic mechanisms and environmental factors such as drug use during pregnancy can influence our lives. It also provides a valuable insight as to how these two factors are intertwined. The results must however be validated in humans and the team were keen to stress that there is no evidence that the use of antibiotics causes autism, and that the results simply elucidate some underlying molecular mechanisms. Peterson confirmed “there is no reason to stop taking antibiotics.”