Friday, May 9, 2025
5/9/2025
In Vivo Analyses of Kinase Signaling in Learning/Memory Circuitry - Project Summary Circuit-localized kinase signaling in the brain modulates local circuit activity and consequent behavioral output. An enormous roadblock to understanding this core mechanism has been the inability to image kinase signaling in vivo, but this barrier has finally been overcome with separation of phases-based activity reporter of kinase (SPARK) biosensors, which can visualize rapid, reversible kinase enzymatic function in targeted brain circuits. This proposal images 3 key pathways – protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and calcium/calmodulin-dependent protein kinase II (CaMKII) – both independently and in combination. We will test roles of circuit activity, mapped neuromodulatory input, and kinase-kinase interaction in normal animals, as well as behavioral learning/memory dysfunction and epileptic seizure disease models. The exquisitely mapped Drosophila central brain mushroom body (MB) learning/memory center provides individually-identified MB inputs, core Kenyon cells and MB output neurons for circuit-level dissection of kinase signaling in vivo, within an expansive connectivity network. Learning acquisition and memory consolidation depends on Kenyon cells, which have well-defined input/output connectivity nodes ideal for informed, targeted SPARK imaging studies. In Aim 1, we image bidirectional activity-dependent kinase signaling with PKA- and ERK-SPARK biosensors. To test localized circuit signaling, we use neuron-targeted optogenetics, channel manipulations (e.g. TRPA1), and neurotransmission blockade (e.g. transgenic tetanus toxin) to dissect activity-dependent kinase signaling. We then assay the roles of this local kinase signaling on circuit function (employing GCaMP imaging) and on learning/memory behavioral output. In Aim 2, we image behavioral learning/memory dysfunction and epileptic seizure disease models for changes in circuit-localized kinase signaling. We then test the effects of genetic and pharmaceutical correction of kinase signaling on circuit function, learning/memory and seizure behavior. We assay the roles of defined neuromodulatory neuropeptide, serotonergic and dopaminergic synaptic inputs, using neuron-targeted ligand/receptor RNAi to dissect regulatory mechanisms. In Aim 3, we generate and test an essential new transgenic CaMKII-SPARK biosensor for in vivo circuit signaling studies. We image CamKII- SPARK in activity interaction (as in Aim 1) and behavioral mutant model (as in Aim 2) analyses. We then test kinase signaling, circuit function and behavioral output dependent on PKA/ERK/CaMKII signaling interactions. We image all three SPARK biosensors in combination with loss-of-function and gain-of-function of the other kinase signaling pathways, including Meng-Po kinase (human SBK1), which we propose balances/coordinates local circuit function. In multiply mutant kinase combinations, we will test local circuit activity with targeted GCaMP imaging, and consequent behavioral output in both learning/memory and seizure models. The overall goal of this proposal is the genetic dissection of circuit-localized PKA, ERK and CaMKII interactive signaling as an activity control in brain learning/memory circuitry, to improve both circuit function and behavior performance.
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