Determining a Role for Protein Kinase A in Dendrite Development using a FRET-based Sensor

PROJECT SUMMARY Dendrite morphology determines many aspects of neuronal function, including action potential propagation and information processing. Although emerging evidence suggests that dendrite growth and branching is regulated locally, the lack of optimal measurements at local sites exists. Brain-derived neurotrophic factor (BDNF) is one of the most studied regulators of dendrite development, and we reported that BDNF exerts distinct local effects on the dendritic arbor depending on where on the arbor it is applied. BDNF triggers PKA activation to regulate dendrite branching, yet not much is known about how BDNF activates PKA to promote local dendrite branching. The proposed work aims to utilize cAMP-dependent protein kinase (PKA) activation sensors as part of a F?rster resonance energy transfer (FRET)-based imaging platform to study the spatiotemporal regulation of the dendritic arbor by PKA activation. First, we will locally apply PKA activator or inhibitor to the dendrite and show a functional relationship between PKA activity and dendrite branching. A microtubule targeted A-kinase activity reporter (tAKAR4?) that shows high sensitivity and dynamic range and is activated by neuromodulators will be expressed in cultured embryonic rat hippocampal neurons of both sexes. We will measure the time-dependent dynamic distribution of PKA in neurons as dendrites develop and branch over time. Second, it has been reported that nuclear signaling of activated PKA occurs and is highest after stimulation of secondary versus other order dendrites, regardless of distance from the soma. As such, we will construct a new PKA activation sensor, tAKAR4?, that will be targeted to the nucleus and use this new FRET sensor to determine whether nuclear PKA activity increases when secondary, but not other order, dendrites are stimulated with PKA activator. Third, we will use the tAKAR4? FRET sensor and nuclear- targeted AKAR4 probe to determine the mechanism by which BDNF locally regulates the arbor. These studies will shed light on mechanisms that shape neuronal morphology that can then be targeted for therapeutics to restore neuronal connectivity and circuitry after injury due to stroke or TBI or as a result of neurodegenerative diseases.