Molecular Nature and Functional Role of Dendritic Voltage-Gated Ion Channels
- Dax Hoffman, PhD, Head, Molecular Neurophysiology and Biophysics Section
- Jiahua Hu, PhD, Staff Scientist
- Lin Lin, PhD, Microbiologist
- Ying Liu, MD, Biologist
- Cole Malloy, PhD, Postdoctoral Fellow
- Meghyn Welch, PhD, Postdoctoral Fellow
- Maisie Ahern, BS, Postbaccalaureate Fellow
- Shalika Padhi, BS, Postbaccalaureate Fellow
- Ashley Pratt, BS, Postbaccalaureate Fellow
The central nervous system (CNS) underlies all our experiences, actions, emotions, knowledge, and memories. With billions of neurons each firing hundreds of times per second, the complexity of the brain is stunning. To pare down the task of understanding something so complex, our research approach calls for studying the workings of a single central neuron: the pyramidal neuron from the CA1 region of the hippocampus. In humans, the hippocampus is essential for long-term memory and is among the first brain regions affected by epilepsy and Alzheimer’s disease. To understand how the hippocampus stores and processes information, we focus on the CA1 pyramidal neuron, one of its principal cell types. Each of these cells receives tens of thousands of inputs onto its dendrites, and it is commonly thought that information is stored by altering the strength of individual synapses (synaptic plasticity). Recent evidence suggests that the regulation of synaptic surface expression of glutamate receptors can, in part, determine synaptic strength. However, the dendrites contain an abundance of ion channels that are involved in receiving, transforming, and relaying information in the dendrites, adding an additional layer of complexity to neuronal information processing.
We found that the A-type potassium channel subunit Kv4.2 is highly expressed in the dendritic regions of CA1 neurons in the hippocampus and, as one of the primary regulators of dendritic excitability, plays a pivotal role in information processing. Kv4.2 is targeted for modulation during the types of plasticity thought to underlie learning and memory. Moreover, we found that the functional expression level of Kv4.2 regulates the subtype expression of NMDA–type glutamate receptors, the predominant molecular devices controlling synaptic plasticity and memory. We are currently following up on these findings with more detailed investigations into the mechanisms of activity-dependent Kv4.2 regulation. In addition, we have begun to investigate the role of dendritic voltage-gated potassium and calcium channels in neuronal development and developmental disorders.
Role of voltage-gated ion channels in synaptic development and disease
Kv4.2 complex regulation and its role in cognitive flexibility
We recently identified a novel molecular cascade that regulates the potassium channel Kv4.2's association with the auxiliary subunit DPP6 and membrane-surface expression in hippocampal neurons. The cascade is initiated by various activity patterns impinging on the neuron, triggering activation of p38 mitogen–activated protein kinase, which phosphorylates the C-terminal motif T607 in Kv4.2 in an activity-dependent manner. Phosphorylation by p38 initiates subsequent isomerization by a prolyl isomerase, Pin1, which selectively binds to and isomerizes phospho-Ser/Thr-Pro bonds. Pin1 is a ubiquitous isomerase that has been implicated in a growing number of nervous-system pathologies, including Alzheimer’s disease, where it may protect against age-dependent neurodegeneration.
To address the role of the p38-Pin1-Kv4.2 in neuronal and neural circuit function, we developed a mutant knock-in mouse model with a Thr607-to-Ala substitution at the activity-induced p38 phosphorylation site (T607-to-A607; Kv4.2TA). The mutation significantly reduces p38 phosphorylation and Pin1 isomerization of this motif, and we observed impaired Kv4.2-DPP6 dynamics and loss of activity-induced internalization of Kv4.2 in such mice. Furthermore, we identified a reduction in intrinsic excitability of hippocampal CA1 pyramidal neurons using whole-cell patch clamp recordings in Kv4.2TA mice compared with wild-type (WT). The reduction in excitability can be traced to an increase in the density of Kv4.2–mediated outward K+ current (A-current), supporting biochemical analysis that suggests loss of Kv4.2 internalization in the Kv4.2TA mice (increased surface Kv4.2). The hypo-excitability in individual neurons observed within the hippocampus of Kv4.2TA mice extends to the circuit/network level, as we also identified reduced kainic acid–induced seizure intensity and progression in these mice.
Hypoactivity within the hippocampal circuit in Kv4.2TA mice may impact cognition. Perhaps most intriguingly, we found that Kv4.2TA mice exhibit normal initial learning and memory in the Morris Water Maze and Lever Press, two tests of hippocampal-dependent learning and memory. However, they exhibited better ‘reversal’ learning in both tests than did WT mice. The improvement in reversal learning indicates an enhancement in cognitive flexibility. Such data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility, i.e., the ability to appropriately adjust one’s behavior to a changing environment, which is impaired in various neuro-developmental disorders such as the autism-spectrum disorder.
Considering the finding that Kv4.2TA mice demonstrate enhanced cognitive flexibility, which to our knowledge represents the first mouse model exhibiting this phenotype, Cole Malloy is pursuing the mechanisms underlying this phenotype. We are focusing on potential differences in synaptic properties between WT and Kv4.2TA mice. Results to date have revealed a novel meta-plasticity mechanism in a Kv4.2 mouse model, which may provide insights into cognitive flexibility and which would be of interest in therapeutic design for treating neuro-developmental disorders characterized by impairments in cognitive flexibility.
Kv4.2 K+ channels are a Ube3A substrate and contribute to cognition in Angelman syndrome (AS).
Angelman syndrome (AS) is a severe, debilitating neuro-developmental disorder with an estimated incidence of 1 in 20,000. It is caused by loss of function of imprinted genes on human chromosome 15q11–13 or by mutations in the Ube3A gene, which resides in this region. Imprinting of this gene results in the exclusive expression of the maternal allele in hippocampal neurons and cerebellar Purkinje cells. Deficits of Ube3A lead to accumulation of its target proteins, which thus dysregulate neuronal function. Using a TAP-MS screen of Kv4.2–interacting proteins that we had previously developed, we identified Ube3A as a Kv4.2–binding protein. Follow-up biochemistry and cell-biology studies, led by Jiahua Hu, confirmed the interaction and demonstrated that Kv4.2–Ube3A binding is activity-dependent. We show that Ube3A binds to Kv4.2 at its N-terminus, and, using an in vitro ubiquitination assay, ubiquinates residue K103. Ubiquitination of a substrate by Ube3a usually causes the substrate degradation. We therefore examined whether Kv4.2 K103 ubiquitination affects the Kv4.2 protein level. The result showed that mutation of K103 significantly delayed protein loss compared with un-mutated Kv4.2 in response to AMPA treatment in cultured hippocampal neurons, suggesting that K103 is required for activity-induced Kv4.2 protein loss. In addition, we showed that Ube3A is associated with internalized Kv4.2, which complexes with the Kv4 auxiliary subunit DPP6.
To further study Kv4.2’s role in AS, we imported a mouse model of AS in which Ube3A is deleted. We found that the Kv4.2 protein level and A-type K+ current are significantly elevated in the hippocampus of AS mice compared with WT littermates. Seizure or neuronal activity leads to Kv4.2 protein degradation. We examined whether Ube3A is required for Kv4.2 protein degradation. We found that seizure-induced Kv4.2 protein loss is abolished in AS, suggesting that seizure-induced Kv4.2 degradation requires Ube3A. Moreover, using patch-clamp electrophysiology, we found deficits in mEPSC frequency and spike-timing–dependent LTP (long-term potentiation) in AS mice. To further study the physiological function of Kv4.2 in AS, we generated CRE–dependent conditional Kv4.2 KO (knockout) mice and crossed them with Emx1-CRE mice to obtain conditional Kv4.2 KO mice (Kv4.2cKO). We then mated AS mice with Kv4.2cKO mice so that for Cole Malloy and Meghyn Welch could examine whether electrophysiological deficits in AS mice can be rescued. Interestingly, deficits in mEPSC frequency and spike-timing–dependent LTP in AS mice were normal in AS/Kv4.2cKO mice. A behavioral test battery for mouse models of AS has been developed to assess phenotypes in the domains of motor performance, repetitive behavior, anxiety and to test drugs and novel Ube3A mutants. We examined the battery in WT littermates, AS mice, Kv4.2cKO mice, and AS/Kv4.2cKO DKO (double knockout) mice and found that locomotion and nesting behaviors can be partially rescued in the DKO mice. In learning and memory tests, AS mice showed impairments in initial learning and reversal learning in an operant reversal test. However, the deficits in AS mice in reversal learning can be rescued by DKO mice. These findings reveal a novel Ube3A–downstream pathway regulating plasticity and cognitive behaviors, and they provide potential targets for the treatment of AS.
Figure 1.
Click image to view.
Working model of neuronal activity or seizure-induced Kv4.2-DPP6 complex remodeling that may contribute to abnormal phenotypes in Angelman syndrome (AS) mice
DPP6 impacts brain development, function, and Alzheimer’s disease/dementia.
In 2022, we reported [Reference 2] that DPP6–KO mice show enhanced neuro-degeneration associated with AD pathology. We also found that aging DPP6–KO mice display circadian dysfunction by home-cage tasks. To further study whether DPP6–KO mice have sleep disorders related to AD/dementia, we used an in vivo detection system by surgical implantation of HD-XO2 implantable telemetry, and recorded EEG/EMG/ activity from aging DPP6–KO mice brains. Electrophysiological data were collected for five days using Ponemah Physiology Platform software. We used NeuroScore software to analyze the sleep/wake time and perform power spectral analysis. From preliminary data, we found that 12-month-old DPP6–KO mice show less total sleep time, less slow-wave sleep duration, and more wake duration compared with WT.
To continue our examination of DPP6 function and its novel roles in preventing neuro-degeneration diseases such as AD/dementia, we are working on another in vivo assay of BioID (proximity-dependent biotin identification) by intra-cerebroventricular injection in neonatal mice with AAV(adeno-associated viral)-DPP6-BioID, to identify other proteins that can form dynamic DPP6–binding complexes, including those involved in transient interactions during cell trafficking as well as components of synaptic adhesion. Biotinylated proteins are isolated by affinity capture and identified by mass spectrometry. We found some interesting binding-partner candidates for further confirmation and functional study. These include, for example, the cell adhesion proteins that function in synapse maturation and enhancement and are involved in autism-spectrum disorders, schizophrenia, and neuro-degeneration diseases such as AD.
Publications
- Murphy JG, Gutzmann JJ, Lin L, Hu J, Petralia RS, Wang YX, Hoffman DA. R-type voltage-gated Ca2+ channels mediate A-type K+ current regulation of synaptic input in hippocampal dendrites. Cell Rep 2022 38(3):110264.
- Lin L, Petralia RS, Holtzclaw L, Wang YX, Abebe D, Hoffman DA. Alzheimer's disease/dementia-associated brain pathology in aging DPP6-KO mice. Neurobiol Dis 2022 174:105887.
- Malloy C, Ahern M, Lin L, Hoffman DA. Neuronal roles of the multifunctional protein dipeptidyl peptidase-like 6 (DPP6). Int J Mol Sci 2022 23(16):9184.
- Hu JH, Malloy C, Tabor GT, Gutzmann JJ, Liu Y, Abebe D, Karlsson RM, Durell S, Cameron HA, Hoffman DA. Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility. Nat Commun 2020 11(1):1567.
- Hu JH, Malloy C, Hoffman DA. P38 regulates kainic acid-induced seizure and neuronal firing via Kv4.2 phosphorylation. Int J Mol Sci 2020 21(16):5921.
- Lin L, Petralia RS, Lake R, Wang YX, Hoffman DA. A novel structure associated with aging is augmented in the DPP6-KO mouse brain. Acta Neuropathol Commun 2020 8(1):197.
Collaborators
- Heather Cameron, Section on Neuroplasticity, NIMH, Bethesda, MD
- Constantine A. Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD
Contact
For more information, email hoffmand@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/hoffman.
