A multidisciplinary approach identifying PDZ-RhoGEF (ARHGEF11) as a critical regulator of synapses and cognition

PROJECT SUMMARY Cognitive abilities have a profound impact on life outcomes. As such, there has been much scientific interest in identifying genes that are critical for regulating cognitive phenotypes. The miR-132 microRNA is among the few studied microRNAs that impacts learning and memory in rodents, is dysregulated in multiple human disorders typified by cognitive impairment, and regulates synaptic structure and function. Human genome wide association studies (GWAS) have identified numerous genes of potential relevance to general cognitive function, yet a challenge is identifying which genes, among the many, are likely to have direct effects on learning and memory. Genes that are targets of miR-132, and that have also been identified in GWAS of human cognitive function, could have a high likelihood of directly impacting learning and memory. The overlay of empirically identified high probability miR-132 gene targets with a list of genes that exhibit genome-wide significant association with human cognitive abilities lead to the identification of two genes, one of which is ARHGEF11. ARHGEF11 encodes PDZ- RhoGEF, a guanine nucleotide exchange factor (GEF) for the RhoA small GTPase. Within the brain, PDZ- RhoGEF is most highly detected in the cerebral cortex, including the prefrontal cortex (PFC). PDZ-RhoGEF is enriched in the dendrites and dendritic spines of pyramidal neurons, with minimal detectable expression in glia. Despite being among the most potent known activators of RhoA, no studies have investigated the functions of PDZ-RhoGEF in the brain. One overarching theory guiding this proposal is that PDZ-RhoGEF is a critical regulator of dendrite and dendritic spine stability, and that it also regulates synaptic function, neuronal engagement and cognition. A second overarching theory guiding this proposal is that PDZ-RhoGEF is a previously unrecognized component of multiple signaling pathways of known importance for controlling synaptic and cognitive phenotypes. Aim 1 will use viral-mediated gene transfer to determine if increased levels of PDZ- RhoGEF in the prefrontal cortex affects synaptic structure, synaptic function, neuronal engagement, and cognition in a manner that requires RhoA activation. Aim 2 will determine if the effects of altered miR-132 expression on dendritic spine stability and cognition are due, at least in part, to miR-132's ability to control levels of PDZ-RhoGEF. As part of the experimental design of Aim 2, we will also simultaneously determine the effects of PDZ-RhoGEF knockdown in the mouse prefrontal cortex on baseline dendritic spine and cognitive phenotypes. Aim 3 will use enzyme activity assessments, super resolution synaptic imaging, and viral gene transfer to characterize the physical and functional interaction between PDZ-RhoGEF and the scaffolding-like protein DISC1. As part of Aim 3, we will determine if the effects of DISC1 loss on synapse destabilization and cognitive impairment are due to excessive PDZ-RhoGEF activity. If our hypotheses are correct, these studies could identify a previously unrecognized role for PDZ-RhoGEF in controlling synaptic and cognitive phenotypes via multiple signaling pathways, and illuminate RhoA signaling as a target for future therapeutics.