Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Isomerase regulation of potassium channel trafficking and function. We recently identified a novel molecular cascade initiated by the activation of p38 kinase and subsequent Pin1dependent isomerization of a C-terminal motif (T607) in Kv4.2 that triggers dissociation from its auxiliary subunit DPP6, a reduction IA, and an increase in neuronal excitability. Pin1 is a prolyl isomerase that selectively binds to and isomerizes phospho-Ser/Thr-Pro (pSer/Thr-Pro) bonds. Mis-regulation of Pin1 plays an important role in a growing number of pathological conditions including Alzheimer's disease, where it may protect against age-dependent neurodegeneration. Using biochemical and electrophysiological techniques, we showed that Pin1 activity is required for the dissociation of the Kv4.2DPP6 complex and that this action alters neuronal excitability. 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 the activity-dependent dissociation of the Kv4.2DPP6 complex is impaired. Seizure or neuronal activity leads to Kv4.2 protein degradation. We found that Kv4.2 degradation is dependent on the above Pin1 mechanism as Kv4.2 trafficking, and degradation are abolished in Kv4.2TA mice. Seizure or neuronal activity triggers Kv4.2 phosphorylation and subsequently isomerization by Pin1 that results in Kv4.2 dissociation with DPP6 and internalization. Internalized Kv4.2 underwent ubiquitination and degradation. In order to examine if DPP6 is required for this process, we employed DPP6 KO mice to test kainic acid-induced Kv4.2 protein loss. We found that seizure-induced Kv4.2 degradation is abolished in DPP6 KO mice, suggesting that seizure-induced Kv4.2 protein loss occurred in DPP6 containing Kv4.2 complex. Interestingly, we found that seizure-induced Kv4.2 phosphorylation at Pin1 isomerization site is also abolished in DPP6 KO mice while induction of p38 activity is normal in DPP6 KO, suggesting that seizure-induced Kv4.2 phosphorylation occurred in DPP6 containing Kv4.2 complex. These data are consistent and support the notion that seizure of neuronal activity induces phosphorylation of DPP6 containing Kv4.2 complex, which results in DPP6-Kv4.2 dissociation and internalization and subsequent ubiquitination and degradation. Kv4.2TA mice exhibit normal initial learning and memory in spatial memory tasks however they exhibited better 'reversal' learning 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. To determine the cellular/molecular correlate of this cognitive phenotype, Dr. Malloy used patch clamp electrophysiology in hippocampal CA1 pyramidal cells from Kv4.2TA and WT mice to record multiple forms of synaptic plasticity. Kv4.2TA mice exhibit similar basal synaptic transmission compared to WT mice. Additionally, no change was found in measures of spike-timing dependent long-term potentiation (STD-LTP) and long-term depression (LTD) in CA1 pyramidal neurons in acute hippocampal slices. However, intriguingly, a significant enhancement in the reversal of STD-LTP (depotentiation) magnitude in Kv4.2TA mice, which appears to be driven by differences in NMDA-mediated transmission. This is suggestive of a synapse state-dependent difference in synaptic plasticity in CA1 stratum-radiatum of the hippocampus facilitated by loss of dynamic regulation of the Kv4.2 complex. We have, therefore, revealed a novel metaplasticity mechanism in a Kv4.2 mouse model. 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. We identified Cav2.3 as a Kv4.2-interacting protein in a proteomic screen and we confirmed Cav2.3-Kv4.2 complex association using multiple techniques. Dr. Murphy 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 loss of Cav2.3 function leads to the enhancement of AMPA receptor-mediated synaptic currents and NMDA receptor-mediated spine Ca2+ influx. These results reveal an intermolecular Cav2.3-Kv4.2 complex impacting synaptic integration in CA1 hippocampal neurons. DPP6 impacts brain development, function and Alzheimer's disease/dementia In 2020 Lin et al. reported the novel structures in hippocampal area CA1 are significantly more prevalent in DPP6-KO aging mice compared to WT mice, also they are observed earlier during development in DPP6-KO mice. These novel structures apparently derived from degenerating presynaptic terminals, as clusters of large puncta that colocalize NeuN, synaptophysin, and chromogranin A, and also partially label for MAP2, amyloid , APP, a-synuclein, and phosphorylated tau, with synapsin-1 and VGluT1 labeling on their periphery. Following these finding, Dr. Lin recently found that DPP6-KO mice show enhanced neurodegeneration associated with AD pathology. By using immunofluorescence and electron microscopy, we confirm that both APP and amyloid are prevalent in these novel structures; and we show with immunofluorescence the presence of similar novel structures are found in human hippocampal CA1 of Alzheimers disease donors. In aged mice, we used in vivo MRI to show reduced size in DPP6-KO brain and hippocampus. Aging DPP6-KO hippocampi contained fewer total neurons and greater neuron death and had diagnostic biomarkers of Alzheimers disease present including accumulation of amyloid and APP and increase in expression of hyper-phosphorylated tau. The amyloid and phosphorylated tau pathologies were associated with neuroinflammation characterized by increases in microglia and astrocytes. Levels of proinflammatory or anti-inflammatory cytokines increased in aging DPP6-KO mice. We also show that aging DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimers disease. Together these results indicate that aging DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of dementia associated with a novel structure resulting from synapse loss and neuronal death.