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Principal Investigator: Dax A Hoffman
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2022
Award: $2,257,955
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
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.
Terms: <AD dementia><AD pathology><AMPA Receptors><Acute><Affect><Aging><Alzheimer><Alzheimer Type Dementia><Alzheimer disease><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimer's biomarker><Alzheimer's disease biological marker><Alzheimer's disease dementia><Alzheimer's disease pathology><Alzheimer's pathology><Alzheimers Dementia><Alzheimers disease><Alzheimer’s biological marker><Alzheimer’s disease biomarker><Amentia><Ammon Horn><Amyloid><Amyloid Substance><Anti-Inflammatories><Anti-Inflammatory Agents><Anti-inflammatory><Antiinflammatories><Antiinflammatory Agents><Area><Astrocytes><Astrocytus><Astroglia><Autism><Autistic Disorder><Axon Terminals><Behavior><Binding><Binding Sites><Biochemical><Brain><Brain Nervous System><Brain region><C-terminal><CNS Diseases><CNS Nervous System><CNS disorder><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><CSAID-Binding Protein 1><CSAID-Binding Protein 2><CSBP2><Calcium><Cas nuclease technology><Cell Body><Cells><Central Nervous System><Central Nervous System Diseases><Central Nervous System Disorders><Characteristics><Chromogranin A><Circadian Dysregulation><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Cognitive><Combining Site><Complex><Cornu Ammonis><Cytokine-Suppressive Antiinflammatory Drug-Binding Protein 1><Cytokine-Suppressive Antiinflammatory Drug-Binding protein 2><Data><Dementia><Dendrites><Depotentiation><Development><Digenic Acid><Disease><Disorder><Dissociation><EF Hand Motifs><EF Hands><Early Infantile Autism><Electron Microscopy><Electrophysiology><Electrophysiology (science)><Emotions><Encephalon><Environment><Epilepsy><Epileptic Seizures><Epileptics><Escalante syndrome><Exhibits><FRET><Fluorescence Resonance Energy Transfer><Fragile X><Fragile X Syndrome><Förster Resonance Energy Transfer><Glutamate Receptor><Hippocampus><Hippocampus (Brain)><Homosynaptic Depression><Hortega cell><Human><Huntington Chorea><Huntington Disease><Huntington's><Huntington's Disease><Huntington's Disease Pathway><Huntingtons Disease><Immunofluorescence><Immunofluorescence Immunologic><Immunofluorescence Microscopy><Impairment><Individual><Infantile Autism><Ion Channel><Ionic Channels><Isomerase><Isomerase Gene><K channel><KI mice><KO mice><Kainic Acid><Kanner's Syndrome><Kinases><Knock-in Mouse><Knock-out><Knock-out Mice><Knockout><Knockout Mice><Knowledge><Kv4 channel><Label><Learning><Long-Term Depression><Long-Term Potentiation><Long-Term Synaptic Depression><Longterm Potentiation><MAPK14><MAPK14 Mitogen-Activated Protein Kinase><MAPK14 gene><MR Imaging><MR Tomography><MRI><MRIs><Magnetic Resonance Imaging><Martin-Bell Syndrome><Martin-Bell-Renpenning syndrome><Measures><Mediating><Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance><Membrane Channels><Memory><Metabolic Protein Degradation><Metaplasia><Metaplastic Change><Mice><Mice Mammals><Microglia><Mitogen-Activated Protein Kinase 14><Modern Man><Molecular><Molecular Interaction><Murine><Mus><Mxi2><N Methyl D aspartic Acid><N methyl D aspartate><N-Methyl-D-Aspartate Receptors><N-Methyl-D-aspartate><N-Methylaspartate><N-Methylaspartate Receptors><NAC precursor><NMDA><NMDA Receptor-Ionophore Complex><NMDA Receptors><NMR Imaging><NMR Tomography><Nerve Cells><Nerve Degeneration><Nerve Unit><Neural Cell><Neural Transmission><Neuraxis><Neurocyte><Neurodevelopmental Disorder><Neurological Development Disorder><Neuron Degeneration><Neurons><Neurophysiology / Electrophysiology><Nuclear Magnetic Resonance Imaging><Null Mouse><PARK1 protein><PARK4 protein><PPIase><Parathyroid Secretory Protein 1><Pathologic><Pathology><Peptidyl-Prolyl cis-trans-Isomerase><Peptidylproline cis-trans-isomerase><Peptidylprolyl Isomerase><Pharmacology><Phenotype><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Physiology><Pituitary Secretory Protein I><Play><Potassium Channel><Potassium Ion Channels><Presynaptic Nerve Endings><Presynaptic Terminals><Primary Senile Degenerative Dementia><Process><Proline Isomerase><Proline Rotamase><Prolyl Isomerase><Protein Phosphorylation><Protein Turnover><Proteins><Proteomics><Pyramidal Cells><Pyramidal neuron><Reactive Site><Regulation><Regulatory Protein Degradation><Renpenning syndrome 2><Reporting><Research><Reversal Learning><Role><SAPK2A><SNCA><SNCA protein><Seizure Disorder><Seizures><Site><Slice><Spinal Column><Spine><Stress-Activated Protein Kinase 2A><Structure><Suggestion><Surface><Symptoms><Synapses><Synapsins><Synaptic><Synaptic Boutons><Synaptic Terminals><Synaptic Transmission><Synaptic Vesicle P38 Membrane Protein><Synaptic Vesicle Protein P38><Synaptic plasticity><Synaptophysin><System><Techniques><Testing><Time><Transmission><Transphosphorylases><Ubiquitilation><Ubiquitination><Ubiquitinoylation><Vertebral column><X-linked mental deficiency-megalotestes syndrome><X-linked mental retardation with fragile X syndrome><X-linked mental retardation-fragile site 1 syndrome><Zeugmatography><a-syn><a-synuclein><age dependent><age related><aged><alpha synuclein><alphaSP22><antiinflammatory><astrocytic glia><asyn><autism spectrum disorder><autism-fragile X (AFRAX) syndrome><autism-fragile X syndrome><autistic spectrum disorder><backbone><cell type><circadian abnormality><circadian disruption><circadian disturbance><circadian dysfunction><circadian impairment><common symptom><cytokine><dementia of the Alzheimer type><developmental><diagnostic biomarker><diagnostic marker><electrophysiological><epilepsia><epileptiform><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><gitter cell><hippocampal><hippocampal pyramidal neuron><hyper-phosphorylated tau><hyperphosphorylated tau><in vivo><information processing><knockin mice><long term memory><longterm depression><longterm memory><longterm synaptic depression><macro-orchidism-marker X (MOMX) syndrome><macro-orchidism-marker X syndrome><mar(X) syndrome><marker X syndrome><mental retardation-macroorchidism syndrome><mesoglia><microglial cell><microgliocyte><mouse model><murine model><nerve cell death><nerve cell loss><neural degeneration><neurodegeneration><neurodegenerative><neuroinflammation><neuroinflammatory><neurological degeneration><neuron cell death><neuron cell loss><neuron death><neuron loss><neuronal><neuronal cell death><neuronal cell loss><neuronal death><neuronal degeneration><neuronal excitability><neuronal loss><non A-beta component of AD amyloid><non A4 component of amyloid precursor><novel><p-tau><p-τ><p38><p38 MAP Kinase><p38 MAPK Gene><p38 Mitogen Activated Protein Kinase><p38 Protein Kinase><p38 SAPK><p38-Alpha><p38Alpha><patch clamp><perivascular glial cell><phospho-tau><phospho-τ><phosphorylated tau><primary degenerative dementia><protein degradation><senile dementia of the Alzheimer type><social role><spatial memory><synapse><tau-1><trafficking><transmission process><ubiquination><ubiquitin conjugation><voltage><voltage gated channel><α-syn><α-synuclein>