SU60 - MOLECULAR STUDIES OF THE ANKRYIN3 BIPOLAR DISORDER GWAS GENE IMPLICATE A ROLE IN MICROTUBULE DYNAMICS

Abstract

Background Ankyrin3 has been identified as a risk gene for Bipolar Disorder (BD) and autism spectrum disorders by multiple genome-wide and targeted association studies as well as sequencing studies. Carriers of the BD risk alleles have reduced ankyrin3 expression in the brain, suggesting that ankyrin3 suppression contributes to disease. However, the mechanism through which ankyrin3 confers risk is unknown. Ankyrin3 encodes the ankyrinG protein that tethers integral membrane proteins to the cytoskeleton. We previously reported that Ank3+/- heterozygous mice, which have ~50% reduced ankyrinG in the brain, exhibit behavioral changes reminiscent of bipolar mania (impulsivity, increased motivation for reward), and altered expression of proteins involved in neuron axonal transport. Both the behavioral and protein expression changes in Ank3+/- mice are normalized by lithium treatment, supporting the disease relevance of these findings. This study sought to further examine the molecular mechanism underlying the behavioral and neuronal changes induced by ankyrin3 suppression in brain. Methods We performed RNA sequencing of hippocampus from Ank3+/- heterozygous and Ank3+/+ wildtype mice, followed by identification of differentially expressed genes using the Tuxedo package. Biological pathways implicated by RNAseq analysis were verified by Western blot of hippocampal protein and live cell imaging of forebrain primary neurons. CRISPR/dCas9 technology was used to repress ankyrin3 transcription in a mouse neuronal system in order to conduct in-depth biochemical analysis. Neurons were treated with lithium and CHIR-99021, a selective inhibitor of the lithium target GSK3B, as well as an inhibitor of CRMP2, an axon-specific substrate of GSK3B. Results RNAseq identified 283 differentially expressed genes between Ank3+/- and Ank3+/+ mouse hippocampus (fold change>1.2, p 20% increased EB3, p 30%, p Discussion Ankyrin3 disruption in mice is associated with altered neuronal microtubule dynamics. Lithium reversal of both the microtubule and behavioral changes of Ank3+/- mice suggest that microtubule defects underlie manic-like behaviors observed in these mice. Our results support the investigation of risk genes using mouse and neuronal models to elucidate the neural mechanisms underlying psychiatric illness.