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Principal Investigator: Dax A Hoffman
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2023
Award: $2,031,130
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
Kv4.2 complex regulation and its role in cognitive flexibility
We have recently identified a novel molecular cascade that regulates the potassium channel Kv4.2 association with the auxiliary subunit DPP6 and membrane surface expression neurons. This cascade is initiated by various patterns of 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. This phosphorylation by p38 initiates subsequent isomerization by a prolyl isomerase, Pin1, that selectively binds to and isomerizes phosphor-Ser/Thr-Pro bonds. Pin1 is a ubiquitous isomerase that has been implicated in a growing number of nervous system pathologies, including Alzheimers 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 (Kv4.2TA). This 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 these mice. Furthermore, we identified a reduction in intrinsic excitability of hippocampal CA1 pyramidal neurons using whole-cell patch clamp recordings in Kv4.2TA mice relative to WT. This reduction in excitability is traced to an increase in the density of Kv4.2-mediated outward K+ current (A-current), supporting biochemical analysis suggesting loss of Kv4.2 internalization in the Kv4.2TA mice (increased surface Kv4.2). The hypoexcitability in individual neurons observed within the hippocampus of Kv4.2TA mice extends to the circuit/network level, as we identified reduced kainic acid-induced seizure intensity and progression in these mice as well.
We have 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. This improvement in reversal learning indicates an enhancement in cognitive flexibility. These data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility- 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 demonstrate enhanced cognitive flexibility Dr. 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 that may provide insights into cognitive flexibility and be of interest in therapeutic design in treating neurodevelopmental disorders characterized by impairments in cognitive flexibility.
Kv4.2 K+ channels are a Ube3A substrate and contributes to cognition in Angelman syndrome (AS)
AS is a severe debilitating neurodevelopmental disorder with an estimated incidence of 1 in 20,000. It is caused by loss of function of imprinted genes on human chromosome 15q1113 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 and thus dysregulate neuronal function. Using a TAP-MS screen of Kv4.2 interacting proteins that we developed previously, we identified Ube3A as a Kv4.2 binding protein. Follow up studies led by Dr. 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 ubiquitinates residue K103 using in vitro ubiquitination assay. Ubiquitination of a substrate by Ube3a usually causes the substrate degradation. We, therefore, examined if Kv4.2 K103 ubiquitination affects Kv4.2 protein level. The result showed that mutation of K103 significantly delayed protein loss compared to un-mutated Kv4.2 in response to AMPA treatment in cultured hippocampal neurons, suggesting K103 is required for activity induced Kv4.2 protein loss. In addition, we showed that Ube3A is associated with internalized Kv4.2.
To further study the Kv4.2s role in AS, we imported a mouse model of AS where Ube3A is deleted. We find that Kv4.2 protein level and A-type K+ current are significantly elevated in hippocampus of AS mice compared to WT littermates. Seizure or neuronal activity leads to Kv4.2 protein degradation. We examined if Ube3A is required for Kv4.2 protein degradation. We find 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 find deficits in mEPSC frequency and spike-timing-dependent LTP in AS mice. To further study the physiological function of Kv4.2 in AS, we generated CRE-dependent conditional Kv4.2 KO mice and crossed with Emx1-CRE mice to obtain conditional Kv4.2 KO mice (Kv4.2cKO). We then mated AS mice with Kv4.2cKO mice for Drs. Malloy and Welch to examine if electrophysiological deficits in AS mice can be rescued. A behavioral test battery for mouse models of Angelman syndrome 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 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. 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 provide potential targets for the treatment of AS.
DPP6 impacts brain development, function, and Alzheimers disease/dementia
In 2022, we reported that DPP6-KO mice show enhanced neurodegeneration associated with AD pathology. We also found that aging DPP6-KO mice display circadian dysfunction by home-cage tasks. To further study if 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 5 days. We used 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 to WT.
To continue our examination of DPP6 function and its novel roles in preventing neurodegeneration diseases like AD/dementia, we are working on another in vivo assay of proximity-dependent biotin identification by ICV injection in neonatal mice with AAV-DPP6-BioID, to identify other protein 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 have 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 are involved in autism spectrum disorders, schizophrenia, and neurodegeneration diseases such as AD.
Terms: <AD dementia><AD pathology><Address><Adhesion Molecule><Adhesions><Affect><Affinity><Aging><Alleles><Allelomorphs><Alzheimer Type Dementia><Alzheimer disease dementia><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimer's disease pathology><Alzheimer's pathology><Alzheimers Dementia><Amentia><Ammon Horn><Angelman Syndrome><Anxiety><Assay><Autism><Autistic Disorder><Behavior><Binding><Binding Proteins><Bioassay><Biochemical><Biologic Assays><Biological Assay><Biotin><Biotinylation><Brain><Brain Nervous System><Brain region><C-terminal><CNS Diseases><CNS Nervous System><CNS disorder><CSAID-Binding Protein 1><CSAID-Binding Protein 2><CSBP2><Cell Adhesion Molecule Gene><Cell Adhesion Molecules><Cell Body><Cells><Central Nervous System><Central Nervous System Diseases><Central Nervous System Disorders><Circadian Dysregulation><Cognition><Cognitive><Complex><Computer software><Cornu Ammonis><Cytokine-Suppressive Antiinflammatory Drug-Binding Protein 1><Cytokine-Suppressive Antiinflammatory Drug-Binding protein 2><Data><Delta Wave><Delta Wave sleep><Dementia><Dendrites><Development><Digenic Acid><Disease><Disorder><E6AP><EEG><Early Infantile Autism><Electroencephalogram><Electroencephalography><Electrophysiology><Electrophysiology (science)><Emotions><Encephalon><Environment><Epilepsy><Epileptic Seizures><Epileptics><Escalante syndrome><Exhibits><Follow-Up Studies><Followup Studies><Fragile X><Fragile X Syndrome><Frequencies><Genes><Genetic Alteration><Genetic Change><Genetic defect><Glutamate Receptor><Happy Puppet Syndrome><Hippocampus><Home><Human><Human Chromosomes><Impairment><Implant><In Vitro><Incidence><Individual><Infantile Autism><Injections><Ion Channel><Ionic Channels><Isomerase><Isomerase Gene><Isomerism><K channel><KCND2 channel><KI mice><KO mice><Kainic Acid><Kanner's Syndrome><Knock-in Mouse><Knock-out Mice><Knockout Mice><Knowledge><Kv4.2 channel><Lead><Learning><Ligand Binding Protein><Ligand Binding Protein Gene><Locomotion><MAPK14><MAPK14 Mitogen-Activated Protein Kinase><MAPK14 gene><Martin-Bell Syndrome><Martin-Bell-Renpenning syndrome><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Membrane><Membrane Channels><Memory><Metabolic Protein Degradation><Mice><Mice Mammals><Mitogen-Activated Protein Kinase 14><Modern Man><Molecular><Molecular Interaction><Motor><Murine><Mus><Mutation><Mxi2><Nerve Cells><Nerve Degeneration><Nerve Unit><Nervous System><Nesting Behavior><Neural Cell><Neuraxis><Neurocyte><Neurodevelopmental Disorder><Neurologic Body System><Neurologic Organ System><Neurological Development Disorder><Neuron Degeneration><Neurons><Neurophysiology / Electrophysiology><Null Mouse><Operative Procedures><Operative Surgical Procedures><PPIase><Partner in relationship><Pathology><Pathway interactions><Pattern><Pb element><Peptidyl-Prolyl cis-trans-Isomerase><Peptidylproline cis-trans-isomerase><Peptidylprolyl Isomerase><Performance><Phenotype><Phosphorylation><Phosphorylation Site><Physiologic><Physiological><Play><Potassium Channel><Potassium Ion Channels><Primary Senile Degenerative Dementia><Process><Proline Isomerase><Proline Rotamase><Prolyl Isomerase><Property><Protein Binding><Protein Phosphorylation><Protein Turnover><Proteins><Puppet Children><Purkinje Cells><Purkinje's Corpuscles><Pyramidal neuron><Regulation><Regulatory Protein Degradation><Renpenning syndrome 2><Reporting><Research><Reversal Learning><Role><SAPK2A><Schizophrenia><Schizophrenic Disorders><Seizure Disorder><Seizures><Sleep><Sleep Disorders><Slow-Wave Sleep><Software><Stress-Activated Protein Kinase 2A><Surface><Surgical><Surgical Interventions><Surgical Procedure><Synapses><Synaptic><Synaptic plasticity><Telemetries><Telemetry><Testing><Therapeutic><Time><UBE3A><UBE3A gene><Ubiquitilation><Ubiquitin-Protein Ligase E3A><Ubiquitination><Ubiquitinoylation><Vitamin H><X-linked mental deficiency-megalotestes syndrome><X-linked mental retardation with fragile X syndrome><X-linked mental retardation-fragile site 1 syndrome><age dependent><age related><autism spectrum disorder><autism-fragile X (AFRAX) syndrome><autistic spectrum disorder><behavior test><behavioral test><bound protein><cell adhesion protein><cell type><cerebellar Purkinje cell><circadian abnormality><circadian disruption><circadian disturbance><circadian dysfunction><circadian impairment><coenzyme R><cognitive enhancement><dementia praecox><density><design><designing><detection platform><detection system><developmental><drug detection><drug testing><electrophysiological><epilepsia><epileptogenic><experience><flexibility><flexible><fra(X) syndrome><fra(X)(28) syndrome><fra(X)(q27) syndrome><fra(X)(q27-28) syndrome><fragile X-mental retardation syndrome><fragile Xq syndrome><fragile site mental retardation 1><genome mutation><heavy metal Pb><heavy metal lead><hippocampal><hippocampal pyramidal neuron><homes><implantation><imprint><improved><in vivo><information processing><insight><interest><isomer><knockin mice><long term memory><longterm memory><loss of function><macro-orchidism-marker X (MOMX) syndrome><macro-orchidism-marker X syndrome><mar(X) syndrome><marker X syndrome><mate><membrane structure><mental retardation-macroorchidism syndrome><morris water maze><morris watermaze><mouse model><murine model><mutant><neonatal mice><neural circuit><neural circuitry><neural degeneration><neurocircuitry><neurodegeneration><neurodegenerative><neurodevelopmental disease><neurological degeneration><neuronal><neuronal circuit><neuronal circuitry><neuronal degeneration><novel><p38><p38 MAP Kinase><p38 MAPK Gene><p38 Mitogen Activated Protein Kinase><p38 Protein Kinase><p38 SAPK><p38-Alpha><p38Alpha><patch clamp><pathway><prevent><preventing><primary degenerative dementia><protein degradation><protein function><puppetlike syndrome><repetitive behavior><response><schizophrenic><senile dementia of the Alzheimer type><sleep diseases><sleep dysfunction><sleep illness><sleep problem><social role><surgery><synapse><synaptic circuit><synaptic circuitry><telemetric><trafficking><ubiquination><ubiquitin conjugation><voltage gated channel>