Functionalization of ASD Variants of PTEN in Rat and Xenopus

Thursday, May 11, 2017: 12:00 PM-1:40 PM
Golden Gate Ballroom (Marriott Marquis Hotel)
R. Dingwall1, M. Edwards1, K. Post2, P. Pavlidis3, T. O’Connor1, C. Rankin1, D. Allan1, C. Loewen1, S. Bamji1 and K. Haas1, (1)University of British Columbia, Vancouver, BC, Canada, (2)University of British Columbia, Vancouver, BC, CANADA, (3)MSL and Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada
Background: This study is part of a multi-model platform to functionalize Autism Spectrum Disorder (ASD) gene variants. One of the major challenges to understanding the mechanistic causes of ASD is the high, and ever growing, number of associated genes. To date the research community’s approach of focusing on one gene at a time using a limited set of very low-throughput assays has yielded potential cellular phenotypes for only a handful of genes. The complexity of this problem is confounded by the identification of multiple mutations of the same ASD-associated genes, culminating in many thousands of gene variants currently without functional phenotyping. A radically different approach is required in order to make significant headway in the near future.

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 SaccharomycesDrosophilaC. 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.

See more of: Animal Models
See more of: Animal Models