Functional Analysis of Class II Phosphoinositide 3-Kinases PI3KC2α and PI3KC2β in Neurotransmission

Abstract

The Class II Phosphoinositide 3-kinases (PI3Ks) are a family of structurally similar enzymes that phosphorylate the 3-position of the inositol ring of phosphoinositides (PIPs), lipids that control membrane identity and cell signalling. Two of the members are ubiquitously expressed, including in the central nervous system (CNS): PI3KC2α and PI3KC2β. Studies from immortalised cells have demonstrated that these two enzymes have important roles in vesicle cycling and nutrient signalling, respectively. PI3KC2α generated phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] recruits and activates Sorting Nexin 9 to late stage Clathrin-coated pits at the cell surface, which provides the constriction necessary for Dynamin-mediated scission from the plasma membrane. PI3KC2β meanwhile plays important roles during low nutrient signalling, inhibiting cellular growth and promoting autophagy by generating PI(3,4)P2 at the lysosome leading to 14-3-3 protein mediated inhibition of mammalian target of rapamycin complex 1 (mTORC1), a key member of the PI3K-AKT signalling pathway. Here we wanted to investigate how these two proteins contribute to normal brain function. By generating a brain specific knockout of PI3KC2α in mice, we were able to show that these mice were viable and had normal development and behaviour. Synaptic cycling was found to be mostly unaffected as was synaptic transmission. However, we found accumulation of Synaptotagmin 1 in neurons lacking PI3KC2α, suggesting an underlying defect in maintaining homeostatic protein levels. Together these data suggest PI3KC2α may be largely dispensable in postmitotic neurons in the CNS. We then assessed the role of PI3KC2β in the CNS, using PI3KC2β null mice as a model, demonstrating that loss of the enzyme in mice resulted in elevated mTORC1 signalling within the hippocampus and cortex. Stereological analysis demonstrated no change in the density of excitatory and inhibitory neurons within the CA1 region of the hippocampus, and glial coverage was also unaffected. However, we found electrophysiological defects in acute CA1 hippocampal recordings, with network hyperexcitability reminiscent of epilepsy. This was confirmed by collaborators via identification of patient mutations in PI3KC2β associated with focal epilepsies and drug specific epileptogenesis in mice. The mTORC1 activation was shown to be reversable with acute rapamycin treatment, and our collaborators demonstrated epileptogenesis was acutely reversible in the mouse model. This data opens the possibility of a future pathway for treatment of human PI3KC2β type epilepsies. These data unveil distinct and important roles of the Class II PI3Ks in the CNS that have implications in human health and disease.