Dane M Chetkovich
Northwestern University At Chicago
Project start date: 2008-02-01
Project end date: 2013-01-31
Sponsored Links Excellgen http://Excellgen.com
HCN CHANNEL TRAFFICKING IN EPILEPSY
Dane M Chetkovich, Assistant Professor
Northwestern University, 750 N. Lake Shore Drive, 7th, Chicago, Il 60611
Grant 5R01NS059934-03 from National Institute Of Neurological Disorders And Stroke
Abstract: Temporal lobe epilepsy (TLE) is a common cause of refractory seizures. Increased seizure propensity in TLE is caused by abnormal neuronal excitability. One candidate for manifesting excitability changes in TLE is the hyperpolarization-activated cyclic nucleotide-gated channel (h channel). H channels mediate the hyperpolarization-activated current, Ih, which is critical for membrane potential homeostasis and neuronal excitability. In hippocampal pyramidal neurons, h channel subunits HCN1 and HCN2 are dramatically enriched in distal compared to proximal dendrites, a phenomenon critical for neuronal excitability; upregulation of dendritic Ih reduces excitability, whereas reduction increases excitability. In a rat model of TLE, we found that excitability was reduced and Ih and distal enrichment of HCN1 was enhanced 24h after status epilepticus, a time point before onset of spontaneous seizures (latency). In contrast, after onset of spontaneous seizures, we found increased excitability coupled with reduced Ih and relocalization of HCN1 from distal dendrites to soma. Interestingly, interaction with an h channel binding protein was dramatically reduced after onset of spontaneous seizures. Furthermore, in slice cultures we found that HCN1 distal dendritic localization is controlled in an activity-dependent manner, requiring activation of N-methyl-D- aspartate receptors (NMDAR) and calmodulin-dependent protein kinase II (CaMKII) activity. We reason that early enhancement of h channel distal enrichment in TLE is a homeostatic response to increased activity that reduces excitability, whereas h channel relocalization from distal dendrites to soma during hippocampal epileptogenesis represents an aberrant process that contributes to increased excitability and the development of spontaneous seizures. We hypothesize that 1) control of h channel localization regulates excitability in TLE, 2) activity of inputs from entorhinal cortex to CA1 dendrites controls h channel localization through NMDAR-mediated activation of protein kinases and HCN subunit phosphorylation, and 3) Abnormal phosphorylation of HCN subunits disrupts h channel trafficking and overrides normal homeostatic, activity-dependent control of h channel localization, leading to onset of spontaneous seizures in TLE. We will utilize physiological, cell biological and biochemical techniques to address the following specific aims To determine whether 1) distal dendritic h channels are increased during latency and reduced after onset of spontaneous seizures in TLE, 2) Temporoammonic inputs from entorhinal cortex to CA1, NMDAR activation, CaMKII activity, and phosphorylation of HCN subunits control h channel localization, and 3) abnormal HCN subunit phosphorylation prevents interactions important for trafficking and targeting and mislocalizes h channels in chronic TLE. With respect to public health, despite numerous new medical and surgical treatments, refractory seizures remain a significant cause of disability in patients with temporal lobe epilepsy (TLE). The ultimate goal of this work is to gain a better understanding of molecular factors controlling the onset of spontaneous and refractory seizures in TLE, with the hope of identifying novel targets for the prevention and treatment of this common cause of epilepsy
Keywords: ATP-protein phosphotransferase; Address; Ammon Horn; Animal Care Assistants; Animal Care Technicians; Animal Technicians; Animals; Attenuated; Autoregulation; Binding Proteins; Biochemical; Biochemistry; Biological; CNG channel (rod); CaM KII; CaM PK II; CaM kinase II; CaMKII; Cell Body; Cells; Chemistry, Biological; Chronic; Comment; Comment (PT); Comment [Publication Type]; Commentary; Commentary (PT); Common Rat Strains; Cornu Ammonis; Coupled; Critiques; Data; Dendrites; Development; Distal; EEG; Editorial Comment; Editorial Comment (PT); Electrodes; Electroencephalography; Electrophysiology; Electrophysiology (science); Entorhinal Area; Entorhinal Cortex; Epilepsy; Epileptic Seizures; Epileptics; Epileptogenesis; Evaluation; Exhibits; Figs; Figs - dietary; Goals; Hippocampus; Hippocampus (Brain); Homeostasis; Implant; Institution; Left; Life; Ligand Binding Protein; Logistics; Mammals, Rats; Mediating; Medical; Membrane Potentials; Methods and Techniques; Methods, Other; Modeling; Molecular; Monitor; N-Methyl-D-Aspartate Receptors; NMDA Receptor-Ionophore Complex; NMDA Receptors; Neurophysiology / Electrophysiology; Neurosciences; Operation; Operative Procedures; Operative Surgical Procedures; Patients; Phosphorylation; Physiologic; Physiological; Physiological Homeostasis; Physiology; Prevention; Process; Protein Kinase; Protein Kinase Inhibitors; Protein Phosphorylation; Public Health; Published Comment; Pyramidal neuron; Rat; Rattus; Reading; Receptor Activation; Receptors, N-Methylaspartate; Refractory; Resting Potentials; Role; Seizure Disorder; Seizures; Slice; Status Epilepticus; Status Epilepticus, Generalized; Structure of entorhinal cortex; Surface; Surgical; Surgical Interventions; Surgical Procedure; Techniques; Temporal Lobe Epilepsy; Texas; Time; Transmembrane Potentials; Up-Regulation; Up-Regulation (Physiology); Upregulation; Veterinary Assistants; Veterinary Nurses; Veterinary Technicians; Viewpoint; Viewpoint (PT); Work; austin; calcium-dependent CaM kinase II; calmodulin-dependent protein kinase II; cationic channel protein (rod); cell body (neuron); common treatment; cyclic-nucleotide gated channel; cyclic-nucleotide gated ion channels; disability; entorhinal cortex; epilepsia; epileptiform; epileptogenic; experiment; experimental research; experimental study; glycogen synthase a kinase; hippocampal; hippocampal pyramidal neuron; hydroxyalkyl protein kinase; improved; neural cell body; neuronal cell body; neuronal excitability; novel; phosphorylase b kinase kinase; prevent; preventing; protein kinase inhibitor; public health medicine (field); public health relevance; research study; response; social role; soma; surgery; trafficking
Project start date: 2008-02-01
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
5R01NS059934-03 (2010): $334630
Grants awarded to Dane M Chetkovich
HCN CHANNEL TRAFFICKING IN EPILEPSY
Dane M Chetkovich, Assistant Professor
Northwestern University, 750 N. Lake Shore Drive, 7th, Chicago, Il 60611
Grant 3R01NS059934-02S1 from National Institute Of Neurological Disorders And Stroke
Abstract: This award is issued in response to Notice OD-09-060, Recovery Act Administrative Supplements Providing Summer Research Experiences for Students and Science Educators. Temporal lobe epilepsy (TLE) is a common cause of refractory seizures. Increased seizure propensity in TLE is caused by abnormal neuronal excitability. One candidate for manifesting excitability changes in TLE is the hyperpolarization-activated cyclic nucleotide-gated channel (h channel). H channels mediate the hyperpolarization-activated current, Ih, which is critical for membrane potential homeostasis and neuronal excitability. In hippocampal pyramidal neurons, h channel subunits HCN1 and HCN2 are dramatically enriched in distal compared to proximal dendrites, a phenomenon critical for neuronal excitability; upregulation of dendritic Ih reduces excitability, whereas reduction increases excitability. In a rat model of TLE, we found that excitability was reduced and Ih and distal enrichment of HCN1 was enhanced 24h after status epilepticus, a time point before onset of spontaneous seizures (latency). In contrast, after onset of spontaneous seizures, we found increased excitability coupled with reduced Ih and relocalization of HCN1 from distal dendrites to soma. Interestingly, interaction with an h channel binding protein was dramatically reduced after onset of spontaneous seizures. Furthermore, in slice cultures we found that HCN1 distal dendritic localization is controlled in an activity-dependent manner, requiring activation of N-methyl-D- aspartate receptors (NMDAR) and calmodulin-dependent protein kinase II (CaMKII) activity. We reason that early enhancement of h channel distal enrichment in TLE is a homeostatic response to increased activity that reduces excitability, whereas h channel relocalization from distal dendrites to soma during hippocampal epileptogenesis represents an aberrant process that contributes to increased excitability and the development of spontaneous seizures. We hypothesize that 1) control of h channel localization regulates excitability in TLE, 2) activity of inputs from entorhinal cortex to CA1 dendrites controls h channel localization through NMDAR-mediated activation of protein kinases and HCN subunit phosphorylation, and 3) Abnormal phosphorylation of HCN subunits disrupts h channel trafficking and overrides normal homeostatic, activity-dependent control of h channel localization, leading to onset of spontaneous seizures in TLE. We will utilize physiological, cell biological and biochemical techniques to address the following specific aims To determine whether 1) distal dendritic h channels are increased during latency and reduced after onset of spontaneous seizures in TLE, 2) Temporoammonic inputs from entorhinal cortex to CA1, NMDAR activation, CaMKII activity, and phosphorylation of HCN subunits control h channel localization, and 3) abnormal HCN subunit phosphorylation prevents interactions important for trafficking and targeting and mislocalizes h channels in chronic TLE. With respect to public health, despite numerous new medical and surgical treatments, refractory seizures remain a significant cause of disability in patients with temporal lobe epilepsy (TLE). The ultimate goal of this work is to gain a better understanding of molecular factors controlling the onset of spontaneous and refractory seizures in TLE, with the hope of identifying novel targets for the prevention and treatment of this common cause of epilepsy
Keywords: 21+ years old; 3-Pyrrolidineacetic acid, 2-carboxy-4-(1-methylethenyl)-, (2S-(2alpha, 3beta, 4beta))-; ATP-protein phosphotransferase; Address; Adult; Ammon Horn; Area; Autoregulation; Binding; Binding (Molecular Function); Binding Proteins; Biochemical; Biological; Brain; CNG channel (rod); CaM KII; CaM PK II; CaM kinase II; CaMKII; Cell Body; Cell membrane; Cells; Chronic; Common Rat Strains; Cornu Ammonis; Coupled; Cyclic Nucleotides; Cytoplasmic Membrane; Dendrites; Development; Digenic Acid; Distal; Electrons; Employee Strikes; Encephalon; Encephalons; Entorhinal Area; Entorhinal Cortex; Epilepsy; Epileptic Seizures; Epileptics; Epileptogenesis; Goals; Hippocampus; Hippocampus (Brain); Homeostasis; Human, Adult; Kainic Acid; Ligand Binding Protein; Long-Term Potentiation; Mammals, Rats; Mediating; Medical; Membrane Potentials; Methods and Techniques; Methods, Other; Microscopic; Modeling; Molecular; Molecular Interaction; N-Methyl-D-Aspartate Receptors; NMDA Receptor-Ionophore Complex; NMDA Receptors; Negative Beta Particle; Negatrons; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurons; Operation; Operative Procedures; Operative Surgical Procedures; Organelles; Pathway interactions; Patients; Pattern; Phosphorylation; Physiologic; Physiological; Physiological Homeostasis; Plasma Membrane; Play; Population; Prevention; Process; Property; Property, LOINC Axis 2; Protein Family; Protein Kinase; Protein Phosphorylation; Protein Subunits; Proteins; Public Health; Pyramidal neuron; Rat; Rattus; Receptor Activation; Receptors, N-Methylaspartate; Refractory; Resting Potentials; Role; Seizure Disorder; Seizures; Site; Slice; Status Epilepticus; Status Epilepticus, Generalized; Strikes; Strikes, Employee; Structure of entorhinal cortex; Surgical; Surgical Interventions; Surgical Procedure; Synapses; Synaptic; Synaptic plasticity; Techniques; Temporal Lobe Epilepsy; Time; Transmembrane Potentials; Up-Regulation; Up-Regulation (Physiology); Upregulation; Work; adult human (21+); animal tissue; calcium-dependent CaM kinase II; calmodulin-dependent protein kinase II; cationic channel protein (rod); cell body (neuron); common treatment; cyclic-nucleotide gated channel; cyclic-nucleotide gated ion channels; disability; entorhinal cortex; epilepsia; epileptiform; epileptogenic; fascinate; gene product; glycogen synthase a kinase; hippocampal; hippocampal pyramidal neuron; hydroxyalkyl protein kinase; immunoreactivity; neural cell body; neuronal; neuronal cell body; neuronal excitability; novel; pathway; phosphorylase b kinase kinase; plasmalemma; prevent; preventing; public health medicine (field); public health relevance; resistance to therapy; resistant to therapy; response; social role; soma; surgery; therapy resistant; trafficking
Project start date: 2008-02-01
Project end date: 2010-08-31
Budget start date: 27-JUL-2009
Budget end date: 31-AUG-2010
PFA/PA: PA-07-070
3R01NS059934-02S1 (2009): $14752
MECHANISMS OF GLUTAMATE RECEPTOR SYNAPTIC CLUSTERING
Dane M Chetkovich, Assistant Professor
Northwestern University, 750 N. Lake Shore Drive, 7th, Chicago, Il 60611
Grant 5K02NS055995-05 from National Institute Of Neurological Disorders And Stroke
Abstract: The goal of this Award is to support Dr. Dane Chetkovich in his career as an independent physician-scientist. The candidate will continue his studies on glutamate receptor (GluR) synaptic clustering, a project he initiated under the mentorship of Dr. David Bredt at the University of California, San Francisco and has continued in his own laboratory at Northwestern University. GluRs are proteins responsible for most of the excitatory communication in the brain. Changes in neuronal communication efficiency are mediated by changing the number or properties of GluRs at synapses, and underlie aspects of learning and memory. Additionally, GluRs are implicated in the pathophysiology of many diseases, including Alzheimer´s disease. Stargazin is a GluR-binding protein that is mutated in the epileptic and ataxic stargazer mice. In the stargazer mouse brain, GluR is made normally, but it does not target to synapses. Dr. Chetkovich´s prior work demonstrated that stargazin delivery of GluRs to synapses is dependent on several parts of stargazin that interact with other proteins, including a protein known as nPIST (neuronal Protein interacting Specifically with TC10). This binding occurs at a region of stargazin that is critical for proper GluR targeting to the synapse. To further explore the role of nPIST in GluR targeting to the synapse, Dr. Chetkovich will use cellular biological, biochemical and electrophysiological approaches to test the hypothesis that nPIST chaperones the targeting of GluR receptors to the synapse by delivering the nPIST/stargazin/GluR receptor complex to synaptic scaffolding proteins, and that phosphorylation of the nPIST domain regulates this process. The Specific Aims of the proposed project are 1) To understand the role of nPISTPDZ domain interactions in GluR synaptic clustering; 2) To explore the role of nPIST phosphorylation in GluR synaptic clustering; and 3) To evaluate stargazin family member interactions with nPIST and GluR subunits in GluR synaptic clustering. These experiments will enhance understanding of the basic mechanism of GluR targeting to synapses, and should provide new insight into mechanisms by which abnormal GluR targeting contributes to neurological disease. These studies will hopefully lead to discoveries that identify novel targets for the development of treatments to fight disabling neurological diseases such as Alzheimer´s disease. In addition to the project´s goal of scientific progress in an area important to human disease, the K02 will allow Dr. Chetkovich protected and structured research time to continue his work at the bench and mentor his trainees and interact with scientific colleagues as he develops his scientific career. Northwestern University offers an outstanding environment for these studies, and the Department of Neurology has committed the resources for Dr. Chetkovich to succeed as an independent physician-scientist. It is anticipated that Dr. Chetkovich´s progress, fostered by this award and the dynamic and supportive environment of Northwestern University, will enable successful competition for R01 mechanism and other funding to further sustain his career development and scientific efforts
Keywords: AMPA Receptors; ATP-protein phosphotransferase; Address; Affect; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer`s; Alzheimer`s Disease; Alzheimers Dementia; Alzheimers disease; Ammon Horn; Antimorphic mutation; Area; Award; BEGAIN; Binding; Binding (Molecular Function); Binding Proteins; Biochemical; Biological; Blood Coagulation Factor IV; Brain; Ca++ element; Calcium; California; Casein Kinase TS; Casein Kinase-2; Cell Function; Cell Process; Cell model; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Cellular model; Central Nervous System; Chaperone; Coagulation Factor IV; Commit; Communication; Complex; Cornu Ammonis; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Development; Disease; Disorder; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; Dysfunction; Encephalon; Encephalons; Environment; Epilepsy; Epileptic Seizures; Epileptics; Factor IV; Family member; Fostering; Frequencies (time pattern); Frequency; Functional disorder; Funding; Glutamate Receptor; Goals; Golgi; Golgi Apparatus; Golgi Complex; Hippocampus; Hippocampus (Brain); Individual; Isoforms; Isoxazoles; Laboratories; Lead; Learning; Ligand Binding Protein; Ligands; Long-Term Depression (Neurophysiology); Long-Term Depression (Physiology); Long-Term Potentiation; Long-Term Synaptic Depression; Mammals, Mice; Mediating; Membrane Protein Traffic; Membrane Traffic; Memory; Mentors; Mentorship; Mice; Mice, Mutant Strains; Modeling; Modification; Molecular; Molecular Chaperones; Molecular Interaction; Murine; Mus; Mutant Strains Mice; Mutate; N-Methyl-D-Aspartate Receptors; NMDA Receptor-Ionophore Complex; NMDA Receptors; Nerve Cells; Nerve Unit; Nervous System Diseases; Nervous System, Brain; Nervous System, CNS; Neural Cell; Neural Transmission; Neuraxis; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neurologic Disorders; Neurological Disorders; Neurology; Neuromediator Receptors; Neurons; Neuroregulator Receptors; Neurotransmitter Receptor; Pb element; Phosphorylation; Physicians; Physiopathology; Play; Postsynaptic Membrane; Primary Senile Degenerative Dementia; Process; Property; Property, LOINC Axis 2; Propionic Acids; Protein Family; Protein Isoforms; Protein Kinase; Protein Kinase CK2; Protein Kinase CKII; Protein Phosphorylation; Proteins; R01 Mechanism; R01 Program; RPG; Receptor Protein; Receptors, N-Methylaspartate; Receptors, Neurohumor; Regulation; Regulatory Protein; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Resources; Role; San Francisco; Scaffolding Protein; Scientist; Seizure Disorder; Structure; Subcellular Process; Synapses; Synaptic; Synaptic Transmission; Synaptic plasticity; Testing; Therapeutic Intervention; Time; Universities; Work; brain-enriched GKAP; career; career development; casein kinase II; dementia of the Alzheimer type; disease/disorder; epilepsia; epileptiform; epileptogenic; experiment; experimental research; experimental study; fighting; gene product; genetic regulatory protein; glycogen synthase a kinase; guanylate kinase-associated protein; heavy metal Pb; heavy metal lead; hippocampal; human disease; hydroxyalkyl protein kinase; improved; insight; intervention development; intervention therapy; long term depression; mouse mutant; nervous system disorder; neurodegenerative illness; neurological disease; neuronal; novel; pathophysiology; phosphorylase b kinase kinase; postsynaptic; primary degenerative dementia; receptor; receptor binding; receptor function; regulatory gene product; research study; scaffold; scaffolding; senile dementia of the Alzheimer type; social role; stargazin; therapy development; trafficking; treatment development
Project start date: 2006-09-05
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-00-020
5K02NS055995-05 (2010): $171719
5K02NS055995-04 (2009): $166782
5K02NS055995-02 (2007): $180485
1K02NS055995-01 (2006): $167621
Stargazin In Targeting Glutamate Receptors To Synapses
Dane M Chetkovich
Neurologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 1K08NS041956-01 from National Institute Of Neurological Disorders And Stroke IRG: NST
Abstract: Glutamate receptors (GluRs) mediate most of the excitatory neurotransmission in the mammalian brain. Changes in synaptic efficacy underlie aspects of learning and memory and help sculpt neural networks in development. Furthermore, GluRs have been implicated in the pathophysiology of many diseases including schizophrenia, stroke, epilepsy and neurodegenerative diseases such as Alzheimer disease. Understanding synaptic regulation of GluR will therefore provide valuable insight into the mechanisms for brain physiology and disease. The major ionotropic GluRs include the -amino-3hydroxyl-5methyl4-isoxazolepropionate receptors (AMPARs) and NmethylDaspartate receptors (NMDARs). AMPARs are loosely associated with the synapse, and their density at synapses is tightly controlled by neuronal activity. Although many studies have focused on the interactions of AMPARs with molecules involved in vesicle fusion, not much is known about the role of AMPARbinding proteins in synaptic plasticity. The protein stargazin is a GluRbinding protein that is mutated in stargazer, a strain of mice with epilepsy and ataxia. The primary deficit in this mouse is abnormal targeting of GluR to synapses. The goal of this research plan is to explore the role of the protein stargazin in the regulation of AMPAR synaptic targeting. The primary hypothesis of the proposed research is that stargazin mediates synaptic targeting of AMPARs, and phosphorylation of stargazin modulates AMPAR localization and function in synaptic plasticity. The Specific Aims of the proposed project are 1) To characterize the interaction between stargazin and GluR subunits, 2) to analyze the role of stargazin phosphorylation in AMPAR synaptic targeting, and 3) to identify binding partners of stargazin and phosphorylated stargazin. Experimental design and methods to accomplish the first Aim include standard molecular biology techniques to generate deletion constructs of stargazin and GluR and coinmunoprecipitation from transfected cells. The second Aim will involve in vitro phosphorylation of peptide substrates, immunohistochemistry in transfected heterologous cells and neurons, and in vitro hippocampal slice preparation to evaluate the role of stargazin phosphorylation in longterm potentiation (LTP). The third Aim will involve the yeast 2hybrid screening method, followed by cloning of binding partners and analysis by immunoprecipitation and immunohistochemistry of brain slices and cortical neurons. The experiments described in the research plan should allow a detailed account of the basic mechanism of GluR targeting to synapses, and posssibly provide insight into mechanisms by which GluR targeting is deranged in neurological disease
Keywords: glutamate receptor, hippocampus, receptor expression, synapse binding protein, complementary DNA, endoplasmic reticulum, enzyme activity, ligand, long term potentiation, phosphoprotein, protein kinase laboratory mouse, laboratory rat, molecular cloning, site directed mutagenesis, tissue /cell culture, western blotting, yeast two hybrid system
Project start date: 2001-08-15
Project end date: 2002-07-31
1K08NS041956-01 (2001): $121770
5K08NS041956-05 (2005): $123049
5K08NS041956-04 (2004): $123049
5K08NS041956-03 (2003): $123049
Role Of HCN Channels In Epilepsy
Dane M Chetkovich
Northwestern University 750 N. Lake Shore Drive, 7th Chicago, Il 60611
Grant 5R21NS052595-02 from National Institute Of Neurological Disorders And Stroke IRG: CNNT
Abstract: Epilepsy, the condition in which affected individuals suffer recurrent seizures, affects up to 2% of the population. Many familial epilepsy syndromes have been characterized, pointing to genetic factors that may predispose toward or underlie many cases of epilepsy. In addition to genetic approaches to identifying genes involved in human epilepsy syndromes, mouse models of epilepsy have further illuminated the important role of ion channels in epilepsy, and have demonstrated overlap between human and mouse epilepsy genes. One category of voltage-gated ion channels implicated in epilepsy is the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, which underlies the hyperpolarization-activated current (In). HCN channels mediate rhythmic oscillations in neuronal membrane potential and play an important role in membrane excitability and dendritic signal integration. A spontaneous mutant mouse with a 4-nucleotide insertion in the C-terminus of the hyperpolarization-activated cyclic nucleotide-gated channel subunit 2 (HCN2) gene has recently been identified. This mutant, named apathetic, is ataxic, and exhibits brief behavioral arrest spells consistent with absence seizures and paroxysmal jumping behavior followed by post-ictal immobility that is consistent with myoclonic seizures. The apathetic mutation is predicted to produce a protein with a truncation of a large portion of the C-terminal tail of HCN2. Protein-protein interactions between ion channel C-termini and scaffolding proteins often regulate ion channel localization, and abnormalities of HCN channel subcellular distribution may play a role in epilepsy. This proposal will utilize EEG recording, behavioral monitoring, cell biological and biochemical techniques to test the hypothesis that truncation of the HCN2 C-terminus in apathetic mice causes epilepsy by mislocalization of HCN channels in thalamic neurons, as well as in cortical and hippocampal pyramidal neurons, resulting in abnormal excitability and epilepsy. The immediate aims of this proposal are to characterize a novel animal model of spontaneous genetic epilepsy and to add insight into the basic molecular mechanisms that underlie some forms of epilepsy, with the ultimate goal of identifying targets for new diagnostic testing and treatments for epilepsy.
Keywords: epilepsy, role, behavior, brain, calcium, cell, conditioning, convulsant, culture, dendrite, diagnostic test, electrical potential, element, emotion, febrile seizure, gene, gene mutation, genetically modified animal, genetics, heart, human, in situ hybridization, insight, intracellular, ion, laboratory mouse, lead, ligand, membrane, membrane potential, model, mutant, myoclonus epilepsy, neuron, northern blotting, nucleotide, phenotype, play, positional cloning, protein, protein protein interaction, soma, spelling, syndrome, western blotting
Project start date: 2006-03-01
Project end date: 2008-02-28
5R21NS052595-02 (2007): $162218
Sponsored Links Excellgen http://Excellgen.com
1R21NS052595-01A1 (2006): $200475
GENE THERAPY FOR TREATMENT OF EPILEPSY
Dane M Chetkovich
Northwestern University, 750 N. Lake Shore Drive, 7th, Chicago, Il 60611
Grant 5R21NS065391-02 from National Institute Of Neurological Disorders And Stroke
Abstract: Despite numerous advances in epilepsy therapeutics, seizures refractory to medical intervention remain a significant cause of morbidity and disability in patients with epilepsy. Longitudinal studies involving patients with epilepsy have shown reduction of frequency and severity of seizures in many patients, but both surgically and medically treated patients often demonstrate a progressive decline in memory function despite treatment. As such novel approaches to controlling seizures in epilepsy are warranted. Increased seizure propensity in epilepsy is caused by abnormal neuronal excitability. Voltage gated ion channels play an important role in controlling intrinsic neuronal excitability, and many have been implicated in human and animal epilepsy syndromes. One such class of voltage-gated ion channels is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, which mediates the cationic current, Ih. Four closely related genes (Hcn1-4) coding for distinct channel subunit proteins (HCN1-4) have been identified; these channels have been implicated in both generalized and focal epilepsies. Global deletion of Hcn2 causes absence epilepsy in mice, likely by increasing calcium channel bursting in thalamocortical neurons. We have recently characterized a novel spontaneous mouse neurological mutant, apathetic, which has absence epilepsy owing to a four base pair insertion in the Hcn2 gene that disrupts HCN2 protein expression. Because loss of HCN2 in thalamocortical neurons produces calcium channel-mediated bursting that leads to absence seizures in mice, we hypothesize that viral overexpression of HCN2 in thalamocortical neurons will restore normal h channel function and reduce seizures in the epileptic, apathetic mice. We will utilize physiological, cell biological and biochemical techniques to address the following specific aims 1) To determine whether viral overexpression of HCN2 in thalamocortical neurons of apathetic mice can restore HCN2 protein expression and reverse the deficit in Ih, and 2) to determine whether viral overexpression of HCN2 in thalamocortical neurons reduce seizures in apathetic mice. With respect to public health, despite numerous new medical and surgical treatments, refractory seizures remain a significant cause of disability in patients with epilepsy. The purpose of this work is to explore whether gene therapy utilizing viral delivery of specific ion channel genes to neurons can effectively reverse ion channel deficits and prevent seizures in epileptic brain, with the ultimate goal of exploring this tool as a novel therapy for medically refractory epilepsy
Keywords: 1, 4-Butanolide; 21+ years old; 4-Butyrolactone; 4-Hydroxybutyric Acid Lactone; Abscission; Absence Epilepsy; Absence Seizures; Address; Adult; Ammon Horn; Animal Model; Animal Models and Related Studies; Animals; Base Pairing; Biochemical; Biological; Body Tissues; Brain; CNG channel (rod); Calcium Channel; Calcium Channel Antagonist Receptor; Cardiac; Cells; Childhood Absence Epilepsy; Code; Coding System; Collaborations; Cornu Ammonis; Cyclic Nucleotides; Data; Dihydro-2(3H)-fura; EEG; Electroencephalography; Encephalon; Encephalons; Epilepsies, Partial; Epilepsy; Epilepsy, Focal; Epilepsy, Generalized; Epilepsy, Localization-Related; Epilepsy, Minor; Epileptic Seizures; Epileptics; Excision; Exhibits; Extirpation; Focal Seizure Disorder; Frequencies (time pattern); Frequency; Fura, tetrahydro-2-; Gated Ion Channel; Gene Expression; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Generalized Epilepsy; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Intervention; Genetic Models; Genetic defect; Goals; HCN2 protein; Hand; Hereditary; Hippocampus; Hippocampus (Brain); Human; Human, Adult; Human, General; Inherited; Injection of therapeutic agent; Injections; Intervention; Intervention Strategies; Intervention, Genetic; Investigators; Ion Channel; Ion Channels, Calcium; Ionic Channels; Juvenile Absence Epilepsy; Knockout Mice; Longitudinal Studies; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Medical; Membrane Channels; Memory; Methods and Techniques; Methods, Other; Mice; Mice, Knock-out; Mice, Knockout; Mice, Neurologic Mutants; Modeling; Molecular Biology, Gene Therapy; Morbidity; Morbidity - disease rate; Murine; Mus; Mutation; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurologic; Neurologic Mutants Mice; Neurological; Neurological Mutant Mouse; Neurons; Null Mouse; Operation; Operative Procedures; Operative Surgical Procedures; Partial Epilepsies; Patients; Petit Mal Convulsion; Petit Mal Epilepsy; Pharmaceutical Agent; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Physiologic; Physiological; Play; Protein Subunits; Public Health; Pykno-Epilepsy; Pyknolepsy; Receptors, Calcium Channel Blocker; Refractory; Removal; Research Personnel; Researchers; Risk; Role; Seizure Disorder; Seizure Disorder, Absence; Seizure Disorder, Generalized; Seizure Disorder, Partial; Seizures; Severities; Surgical; Surgical Interventions; Surgical Procedure; Surgical Removal; Syndrome; Techniques; Technology; Temporal Lobe Epilepsy; Testing; Thalamic structure; Thalamus; Therapeutic; Therapy, DNA; Time; Tissues; V (voltage); VDCC; Viral; Viral Genes; Viral Vector; Virus; Viruses, General; Voltage-Dependent Calcium Channels; Work; adult human (21+); cationic channel protein (rod); cyclic-nucleotide gated channel; cyclic-nucleotide gated ion channels; disability; epilepsia; epileptiform; epileptogenic; gamma-Butyrolactone; gene therapy; genetic therapy; genome mutation; hippocampal; interventional strategy; knockout gene; long-term study; minimally invasive; model organism; neuronal; neuronal excitability; new approaches; novel; novel approaches; novel strategies; novel strategy; overexpression; petit mal seizure; prevent; preventing; protein expression; public health medicine (field); public health relevance; resection; social role; spatiotemporal; surgery; thalamic; tool; vector-induced; voltage
Relevance: With respect to public health, despite numerous new medical and surgical treatments, refractory seizures remain a significant cause of disability in patients with epilepsy. The purpose of this work is to explore whether gene therapy utilizing viral delivery of specific ion channel genes to neurons can effectively reverse ion channel deficits and prevent seizures in epileptic brain, with the ultimate goal of exploring this tool as a novel therapy for medically refractory epilepsy
Project start date: 2009-09-15
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-06-181
5R21NS065391-02 (2010): $190816
1R21NS065391-01A1 (2009): $257894