Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

Isomerase regulation of potassium channel trafficking and function. The transient voltage-gated K+ current (IA) mediated by Kv4.2 in CA1 hippocampal pyramidal neurons regulates dendritic excitability, synaptic plasticity, and learning. Weve recently identified a novel molecular cascade initiated by the activation of p38 kinase and subsequent isomerization of a C-terminal motif (T607) in Kv4.2 that triggers dissociation from its auxiliary subunit DPP6, a reduction IA and increase of neuronal excitability. The phosphorylation of Kv4.2 T607 site is induced by novel environment exposure or seizure and is mediated by p38 MAPK. To investigate the consequences of this cascade on behavior and neuronal physiology, we used Crispr-Cas9 techniques to generate a knockin mouse in which the isomerase binding site is specifically abolished (Kv4.2TA). The mice are viable and appear normal although activity-dependent dissociation of the Kv4.2-DPP6 complex is impaired. Dr. Cole Malloy used patch clamp electrophysiology in pyramidal cells of hippocampal slices from Kv4.2TA and WT mice to decipher the role of p38-Pin1-mediated regulation of Kv4.2 on neuronal excitability. He found that Kv4.2TA cells displayed a reduction in AP firing relative to WT in response to somatic current injections. This reduced excitability is traced to increased Kv4.2-mediated current in Kv4.2TA cells in outside-out somatic patches. Pharmacological block of both p38 kinase and Pin1 in WT recapitulated the impact of the mutation on neuronal firing properties and IA, confirming the specificity of this cascade underlying these effects. To detect how these alterations in neuronal physiology may manifest in behavioral changes, Dr. Jiahua Hu performed a battery of tests probing seizure susceptibility and learning and memory capability. In response to IP kainic acid injection, Kv4.2TA mice exhibited reduced seizure intensity over an hour-long period relative to WT mice. The reduced seizure intensity also could be recapitulated in WT mice with pharmacological block of p38 kinase and Pin1. Therefore, we have identified a novel signaling cascade that can be a target for therapeutic intervention to mitigate seizure intensity in epilepsy by reducing Kv4.2 downregulation. Kv4.2TA mice exhibit normal initial learning and memory in the Morris Water Maze however they exhibited better 'reversal' learning in Morris Water Maze than did WT mice. The data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility. Cognitive flexibility is the ability to appropriately adjust ones behavior to a changing environment and is impaired in various neurodevelopmental disorders such as the autism spectrum disorder. Considering the finding that Kv4.2TA mice exhibit enhanced cognitive flexibility, ongoing experiments investigate potential differences in synaptic properties between WT and Kv4.2TA mice. Collectively, these experiments will reveal the cellular mechanisms underlying the reversal learning phenotype in Kv4.2TA mice and will provide further insight into mechanisms impacting cognitive flexibility. Ca2+ regulation of potassium channel function. Dr. Jonathan Murphy found that Ca2+ entry mediated by the voltage-gated Ca2+ channel subunit Cav2.3 regulates Kv4.2 function both in a heterologous expression system and endogenously in CA1 pyramidal neurons through Ca2+ binding auxiliary subunits known as K+ channel interacting proteins (KChIPs). KChIPs are calcium-sensing molecules containing four EF-hands which are dysregulated in several diseases and disorders including epilepsy, Huntingtons disease, and Alzheimers disease. He characterized a KChIP-independent interaction between Cav2.3 and Kv4.2 using immunofluorescence colocalization, coimmunoprecipitation, electron microscopy, FRAP, and FRET. We found that Ca2+-entry via Cav2.3 increases Kv4.2-mediated whole-cell current due in part to an increase in Kv4.2 surface expression. In hippocampal neurons, pharmacological block of Cav2.3 reduced whole-cell IA. We also found a reduction in whole-cell IA in Cav2.3 knockout (KO) mice mouse neurons with a loss of the characteristic dendritic IA gradient. Furthermore, the Cav2.3-Kv4.2 complex was found to regulate the size of synaptic currents and spine Ca2+ transients. These results reveal an intermolecular Cav2.3-Kv4.2 complex impacting synaptic integration in CA1 hippocampal neurons. To directly test if the binding interaction of Cav2.3 and Kv4.2 is required for Cav2.3 regulation of Kv4.2 function, we have worked toward developing tools to disrupt the Cav2.3-Kv4.2 interaction while sparing the expression of Cav2.3. KChIP protein, but not mRNA expression, has been shown to be reduced in Kv4.2 KO mouse brains, suggesting increased KChIP protein degradation in the absence of Kv4.2. We hypothesized that KChIP protein degradation is dependent on binding to Kv4.2 and that there is increased KChIP protein degradation in the absence of Kv4.2. We aimed to elucidate the undetermined molecular mechanism of KChIP protein degradation and its effect on Kv4.2 protein levels and function. Joe Krzeski has identified the pathway through which KChIP is degraded and a novel function for KChIP regulation of Kv4.2. Dr. Jiahua Hu generated a conditional Kv4.2 KO mouse using Crispr-Cas9 techniques. Joe Krzeski injected AAV-CRE-GFP virus into the CA1 in hippocampus of the conditional Kv4.2 KO mice. We found that Kv4.2 protein level is significantly reduced in the CRE-positive area. Interestingly, KChIP protein level is also significantly decreased in the same area. These data suggest KChIP protein can be dynamically regulated by Kv4.2 expression. Joe Krzeski identified a conserved lysine residue that can be ubiquitinated. Further studies will elucidate the mechanism of KChIP degradation and its regulation by Kv4.2. A mechanistic understanding of KChIP protein degradation is important, as it may lead to new therapeutic strategies to treat diseases in which KChIPs are dysregulated. DPP6 plays a role in Brain Development, Function and Behavior We have previously shown that the Kv4 auxiliary subunit DPP6 has a novel function in regulating dendritic filopodia formation and stability, affecting synaptic development and function (Lin et al. 2013). Recently, using immunofluorescence and electron microscopy, in a project lead by Dr. Lin Lin, we have discovered a novel structure in hippocampal area CA1 that was significantly more prevalent in DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appeared as clusters of large puncta that colocalized NeuN, synaptophysin, and chromogranin A. Electron microscopy revealed that these structures are abnormal, enlarged presynaptic swellings filled with mainly fibrous material with occasional peripheral, presynaptic active zones forming synapses. We found diagnostic biomarkers of Alzheimers disease present in abnormal levels in DPP6-KO mice including accumulation of amyloid and APP in the hippocampal CA1 area and a significant increase in expression of hyper-phosphorylated tau. The amyloid and phosphorylated tau pathologies were associated with neuroinflammation characterized by activation of microglia and astrocytes. We also found that activated astrocytes and microglia were significantly increased in DPP6-KO brain sections. We show that DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimer disease. Together these results indicate that DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of Alzheimers disease associated with a novel structure resulting from synapse loss and neuronal death. We continue to investigate DPP6 in neurodegeneration.