Identifying the causes of autism spectrum disorder has proven to be a significant challenge. Both genetic and environmental risk factors contribute to the development of autism. However, despite being estimated to account for 41% of autism risk, we know very little about the environmental risk factors of autism. Moreover, current systems that we are using to study how environmental factors influence autism risk, such as rodent models, are slow and laborious, and therefore not suitable for conducting large-scale systematic investigations. My long-term goal is to study the effect of environmental factors on the development of autism. Because social deficit is one of the 2 core symptoms of autism, I developed a high-throughput screening method to systematically search for chemicals that inhibit the development of social behavior in zebrafish. Using this system, I screened 1120 compounds, and identified a group of compounds - topoisomerase II (Top2) inhibitors - that effectively inhibited the normal development of social behavior in zebrafish. Prenatal exposure to the Top2 inhibitor ICRF-193 in mouse also induced behavioral defects related to the two core symptoms of autism. The main objective of this grant is to characterize the role of Top2 in the development of social behavior. Top2 is involved in gene regulation during brain development. Its two isoforms Top2a and Top2b are expressed in dividing and postmitotic tissues, respectively, during development, and modulate the expression of distinct sets of developmental genes. Top2 also regulates DNA methylation by binding to Uhrf1, a key adapter for Dnmt1. My preliminary results demonstrated that: 1) Top2a rather than Top2b is likely to be the biological target responsible for the social deficit phenotype; 2) chemical inhibition of Dnmts phenocopied Top2 inhibition and induced a similar social deficit phenotype in zebrafish; and 3) transient overexpression of Dnmt1 in zebrafish embryos rescued the social deficit phenotype induced by Top2 inhibitors. In addition, previous literatures have linked human mutations in TOP2A, TOP2B, and DNMT1 to increased autism risk. I thus propose the central hypothesis that Top2 plays a critical role in the development of social behavior through regulation of Dnmt1 and DNA methylation, and disruption of this pathway leads to deficits in sociality and other autism-related behaviors. To test this hypothesis, I propose to: 1) determine which Top2 isoform (Top2a or Top2b) is responsible for the social deficit phenotype induced by Top2 inhibition; 2) investigate the role of Dnmt1 and global DNA methylation in the development of social deficit following Top2 inhibition; and 3) To systematically discover environmental factors that affect the development of social behavior by screening the Tox21 library. Together, my proposed studies will have a broad impact on the field and may ultimately reveal novel therapeutic targets to prevent or treat autism.
Both genetic and environmental risk factors play a part in the development of autism, yet despite being estimated to account for 41% of autism risk, we know very little about what environmental factors contribute to autism risk. Because social deficit is one of the 3 core symptoms of autism, I developed a high-throughput screening approach to systematically search for chemicals that inhibit the development of social behavior. This screen revealed topoisomerase II as a key regulator of the development of social behavior in zebrafish and mouse, which is investigated in further detail in this proposal.
