Role of VRK2 Kinase in Brain Development and Function

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PROJECT SUMMARY/ABSTRACT The goal of this project is to investigate the roles of Vrk2 kinase in zebrafish. Genomic studies have linked VRK2 to schizophrenia, bipolar disorder, sleep, major depression, and more. These numerous associations indicate that it may be a key factor underlying predisposition to neuropsychiatric disorders. Yet it remains unclear how VRK2 is functioning in the vertebrate brain. Preliminary studies led to the creation and initial characterization of vrk2 zebrafish mutants. These mutants had altered sleep behavior and reduced brain activity, particularly in areas of the hypothalamus that are implicated in sleep. For Aim 1, we will expand the characterization of the cellular, developmental, and behavioral differences in these mutants. We will further explore their sleep abnormalities, such as assessing homeostasis after deprivation and circadian response to challenges such as dark-only conditions. Identifying the timing and location of vrk2 action that drive these behavioral outcomes is the second goal of Aim 1. We hypothesize that vrk2 mutant phenotypes result from absent phosphorylation of its direct targets. Therefore, in Aim 2 we propose to pilot several strategies for discovering these targets in zebrafish. The choice of method will depend on our investigation of where and when vrk2 function is required for normal behavior and brain activity (Aim 1) and includes multiple backup strategies, as phosphoproteomics can be challenging. Proposed approaches include developing an analog- sensitive variant of Vrk2 to explicitly label only direct substrates of Vrk2 for subsequent immunoprecipitation. The variant and ATP analog will either be injected into single-cell embryos or exposed to purified zebrafish brain lysate, if vrk2 has a role in early embryos that influences later brain development or if we conclude that it likely induces direct phosphorylation of proteins involved in sleep signaling. These aims will provide insight into the molecular, cellular, developmental and behavioral processes regulated by vrk2. Defining the function of genes that increase risk for neuropsychiatric disorders will provide the foundation to understand their causes and develop new diagnostics and therapies.