Kv4.2 trafficking in CA1 pyramidal neuron dendrites. We previously reported that neuronal stimulation results in a redistribution of Kv4.2 channels away from dendritic spines to the dendritic shaft. This activity-dependent redistribution of Kv4.2 required activation of NMDA-type glutamate receptors and calcium influx, two requirements shared with synaptic plasticity, which is thought to underlie learning and memory. Given the nonuniform distribution of Kv4.2 channels in CA1 dendrites, Mike Nestor performed experiments to test the hypothesis that Kv4.2 channels are differentially trafficked at different regions along the apical dendrite during basal activity and upon stimulation in CA1 neurons. Proximal (50-150 um from the soma, primary and oblique) and distal (>200 um) apical dendrites were selected. The fluorescence recovery after photobleaching (FRAP) technique was used to measure basal cycling rates of EGFP-tagged Kv4.2 (Kv4.2g). We found that the cycling rate of Kv4.2 channels was one order of magnitude slower at both primary and oblique dendrites between 50-150 um from the soma. Kv4.2 channel cycling increased significantly at 200-250 um from the soma. Expression of a Kv4.2 mutant lacking a phosphorylation site for protein kinase-A (Kv4.2gS552A) abolished this distance-dependent change in channel cycling;demonstrating that phosphorylation by PKA underlies the increased mobility in distal dendrites. These results indicate that distance-dependent Kv4.2 mobility is regulated by activity-dependent phosphorylation of Kv4.2 by PKA. Postbac Josh Lee has followed up this project, aiming to uncover the pathway of internalized Kv4.2 as well as proteins that may be involved in directing its path. To date, he has shown that ubiquitin and ubiquitin ligase Nedd4-2 interact with Kv4.2 when overexpressed in a non-neuronal cell line. In the same cell line, he has shown that ubiquitin facilitates degradation of Kv4.2 compared to the basal rate of degradation. We are simultaneously conducting experiments to visually track the movement of Kv4.2. Colocalizing Kv4.2 with markers of well characterized endosomal compartments will reveal its pathway. In a collaboration with the Juan Bonifacino lab, Postbac Laura Long is examining the interaction of Kv4.2 with the clathrin-associated adaptor protein complex AP-1A. AP-1A is responsible for intracellular trafficking and has recently been implicated in determining receptor polarity in neurons. Molecular approaches have confirmed that Kv4.2 does indeed interact with AP-1A, and coimmunoprecipitation and yeast in situ hybridization assays suggest that this binding occurs on both the N terminal and C terminal tails of Kv4.2. She is currently investigating the functional implications of this interaction through imaging and shRNA knockouts. Functional role of the Kv4.2 auxiliary subunits: DPP6 Studies in heterologous expression systems have shown that Kv4.2 subunits interact with transmembrane DPP6 proteins to regulate channel trafficking and properties. The DPP6 auxiliary subunit protein, which is expressed in CA1 neurons, has recently been identified in large copy-number variants screens from some populations as an Autism Spectrum Disorder and ALS target gene. DPP6 enhances the opening probability of Kv4 channels and increases channel surface expression in heterologous systems. In dendritic recordings from DPP6 knock out mice, graduate student Wei Sun discovered that DPP6 is critical for generating the A-type K+ current gradient observed in CA1 dendrites. The loss this gradient led to hyper-excitable dendrites, with implications for information storage and coding. This year, researcher Lin Lin showed in a Nature Communications paper that knockdown and genetic deletion of DPP6 reveals its importance for the formation and stability of dendritic filopodia during early neuronal development. Additionally, hippocampal neurons lacking DPP6 showed a sparser dendritic branching pattern along with fewer spines throughout development and into adulthood. In electrophysiological and imaging experiments we showed that these deficits lead to fewer functional synapses and occur independently of the potassium channel subunit Kv4.2. We report that the extracellular domain of DPP6 interacts with a filopodia-associated myosin as well as with fibronectin in the extracellular matrix. DPP6 therefore plays an unexpected but important role in cell-adhesion and motility, impacting hippocampal synaptic development and function. Role of Kv4.2 channels in synaptic plasticity and development We have found that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced A-type K+ current (IA) showed a decrease in relative synaptic NR2B/NR2A subunit composition and do not exhibit a form of synaptic plasticity called long-term potentiation or LTP. Conversely, reducing IA by expression of a Kv4.2 dominant negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDA receptor activation to control synaptic NMDA receptor expression and plasticity. Additional advances from postdoc Eunyoung Kim included an investigation into the role of Kv4.2 in controlling the expression of synaptic NMDA receptors in vivo and during development. Synaptic NR2B fraction is developmentally regulated with implications for synaptic plasticity and learning and memory as well as diseases associated with learning impairments. Dr. Kim published a 2013 Journal of Neuroscience paper showing that in vivo injection of virus to alter Kv4.2 expression levels bidirectionally regulates NR2B subunit expression throughout development. Dendritic intrinsic excitability changes in disease Intrinsic excitability changes have been observed upon drug addiction. Cocaine is an addictive drug with psychostimulant effects that are attributed to inhibition of the dopamine transporter, which increases dopaminergic transmission. Chronic exposure to cocaine leads to neurodaptations in several voltage membrane conductances of neurons localized in the medial prefrontal cortex (mPCF) and nucleus accumbens. Postdoc Emily Campanac published a paper this year showing repeated cocaine exposure increases fast-spiking interneuron excitability in the rat mPFC. After cocaine withdrawal, interneurons showed an increase in action potential firing, increased input resistance, and decreased hyperpolarization-activated current. We also observed a reduction in miniature excitatory postsynaptic currents, whereas miniature inhibitory postsynaptic current activity was unaffected. In animals with cocaine history, dopamine receptor D(2) activation was less effective in increasing interneuron intrinsic excitability. Interestingly, these alterations are only observed 1 wk or more after the last cocaine exposure. This suggests that the dampening of D(2)-receptor-mediated response may be a compensatory mechanism to rein down the excitability of interneurons. Neuronal hyperexcitability is an early feature of Alzheimers disease. The underlying cellular mechanisms are unclear however. Postbac Ben Throesch directly examined dendritic excitability by patch-clamp recordings on dendrites of hippocampal neurons in mice expressing increased levels of amyloid-β. In a paper in preparation, he describes his results showing that neuronal dendrites were hyper-excitable due to a decrease in Kv4.2 expression, while the soma showed normal firing.

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Liu, Xiaozhuo; Campanac, Emilie; Cheung, Hoi-Hung et al. (2016) Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons. Mol Neurobiol :
Murase, Sachiko; Lantz, Crystal L; Kim, Eunyoung et al. (2016) Matrix Metalloproteinase-9 Regulates Neuronal Circuit Development and Excitability. Mol Neurobiol 53:3477-93
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Lin, Lin; Sun, Wei; Throesch, Ben et al. (2013) DPP6 regulation of dendritic morphogenesis impacts hippocampal synaptic development. Nat Commun 4:2270

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