Functional Analysis of Pathogenic Human PTEN Variants in Early Neurodevelopment

Project Summary/Abstract PTEN is highly associated with autism, macrocephaly, and congenital hydrocephalus, which are increasingly prevalent neurodevelopmental disorders that present in early childhood. Currently, there are no treatments that address the cause of these conditions, despite mounting genetic evidence through large scale whole exome sequencing studies that mutations in specific genes, including PTEN, confer increased risk for these disorders. While these studies produce compelling targets for investigation, the role of PTEN in neurodevelopmental disorders remains poorly understood. We seek to understand how PTEN, through its role as a negative regulator of the greater mTOR pathway, contributes to early neurodevelopment using CRISPR-Cas9 loss-of-function models in zebrafish. We have generated zebrafish mutants with frameshift mutations in exon 5 of both pten alleles present in the teleost duplicated genome: ptena (Δ10) and ptenb (Δ2). We first aim to characterize neurodevelopmental abnormalities in these fish, which preliminary data indicates have significant differences in brain volume, brain activity, brain ventricle size, and startle response. We will further explore the function of PTEN in regulating cell proliferation, differentiation, and establishment of the excitatory and inhibitory circuits in the brain. Secondly, we aim to use this model to provide a new and accessible tool for screening the PTEN variants identified in children with neurodevelopmental disorders, by injecting embryos with human mRNA constructs at the 1-cell stage and evaluating neurodevelopmental changes. Lastly, we will perform a high-throughput drug screen of mTOR pathway inhibitors and additional compounds which oppose or match the behavioral changes observed in our mutant larvae. This will identify new potential pharmacological candidates. Our preliminary data already shows partial phenotype rescue upon treatment with mTORC1-inhibitor sirolimus. In summary, these loss of function lines demonstrate PTEN plays a critical role in early vertebrate neurodevelopment. Furthermore, these models provide a new and accessible tool for screening the PTEN variants identified in neurodevelopmental disorders as well as potential pharmacological candidates.