Functionalization of ASD Variants of PTEN in Rat and Xenopus
Objectives: We have developed a multiple-platform approach combining high-throughput and high-resolution assays to test large numbers of ASD-associated genes and their multiple variants in Saccharomyces, Drosophila, C. elegans, Xenopus, and mammalian hippocampal culture model systems. We focused on the ASD-associated gene PTEN (phosphatase and tensin homolog), a crucial negative regulator of the PI3K/mTOR pathway. In this presentation we discuss the impact of PTEN variants identified as altering PTEN function in high-throughput assays on the morphological growth and synaptogenesis of rat primary hippocampal neurons and newly differentiated neurons within the intact developing brain of Xenopus tadpoles.
Methods: We have expressed PTEN variants in primary neuron cultures with a PTEN knockdown background and describe effects on neuron morphology, synapse number and balance. Using in vivo time-lapse two-photon imaging in awake, transparent Xenopus laevis tadpoles, we report the effects of PTEN variants expressed by single-cell electroportation on the dendritic arbor growth of developing brain neurons.
Results: Rat hippocampal neurons overexpressing human WT PTEN demonstrate a decrease in spines and PSD95 puncta compared to controls, which is not observed in neurons overexpressing the loss-of-function variants. RNAi knockdown of PTEN increased spine number, PSD95 puncta and soma size. These phenotypes were not rescued by expressing loss-of-function human PTEN variants associated with ASD. In Xenopus, expression of ASD-associated variants induced abnormal dendritic arbor growth phenotypes in vivo.
Conclusions: Our high-resolution assays of neuronal morphogenesis and synaptogenesis provide insight into the underlying pathophysiology and neural circuit development abnormalities that may give rise to ASD.