Mechanisms of cellular, synaptic and circuit dysfunction in Kcnc1-related epileptic encephalopathy

Kcnc1相关癫痫性脑病的细胞、突触和回路功能障碍的机制

• 批准号: 10424981
• 负责人: Eric Ryan Wengert
• 金额: $ 6.72万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10424981
• 关键词: Action Potentials; Address; Ataxia; Axon; Behavior; Behavioral; Calcium; Cells; Cerebral cortex; Characteristics; Complex; Dendrites; Disease; Electrophysiology (science); Epilepsy; Event; Exhibits; Fellowship; Frequencies; Functional disorder; Generations; Genes; Goals; Image; Impaired cognition; Impairment; In Vitro; Individual; Intellectual functioning disability; Interneurons; Ion Channel; Knowledge; Lead; Light; Link; Measures; Mediating; Mentors; Methodology; Mus; National Research Service Awards; Neurodevelopmental Disorder; Neurologic; Neurons; Neurosciences; Optics; Other Genetics; Parvalbumins; Pathogenesis; Pathogenicity; Patients; Phenotype; Physiological; Physiology; Potassium; Potassium Channel; Presynaptic Terminals; Progressive Myoclonic Epilepsies; Property; Recovery; Recurrence; Research; Research Personnel; Role; Seizures; Sensory; Sodium Channel; Symptoms; Synapses; Synaptic Transmission; Technical Expertise; Techniques; Testing; Training; Variant; Vibrissae; Voltage-Gated Potassium Channel; Wild Type Mouse; awake; base; biophysical properties; calcium indicator; career; density; early onset; epileptic encephalopathies; excitatory neuron; experimental study; hippocampal pyramidal neuron; in vivo; insight; loss of function; mouse model; mutant; nervous system disorder; neural circuit; neuronal cell body; neuronal circuitry; neuronal excitability; neurotransmission; novel; patch clamp; response; synaptic inhibition; therapy development; two-photon; voltage

PROJECT SUMMARY I am applying for this NRSA postdoctoral fellowship with the long-term career goal of becoming an independent investigator capable of leading a highly productive research lab in the field of neuroscience. Under the guidance of Dr. Ethan Goldberg and Dr. Doug Coulter, this mentored fellowship will provide me the conceptual and hands-on training required to achieve my goal and thrive in the scientific enterprise. Pathogenic variants in the gene KCNC1, which encodes the voltage-gated potassium channel subunit Kv3.1, lead to severe neurological disease including epilepsy. While most patient-derived variants are loss-of- function, the precise mechanisms by which impaired Kv3.1 function alters individual neuron physiology and neural circuit function to result in spontaneous seizures are unclear. Kv3.1 is prominently expressed in parvalbumin-positive fast-spiking inhibitory interneurons (PV-IN) in various subcellular regions including the dendrites, soma, axon, and synaptic terminal where it critically contributes to reliable high-frequency action potential generation and propagation. Because PV-INs critically contribute to network dynamics and constrain excitability of nearby excitatory pyramidal neurons in cortical circuits, we hypothesize that Kv3.1 dysfunction impairs PV-IN high-frequency action potential generation and propagation, disinhibits pyramidal neurons, and contributes to aberrant network excitability to drive abnormal behavior and seizures. Therefore, the overarching objective of this study is to determine the effect of mutant Kv3.1 on PV-IN physiology as a potential major contributor to pathogenesis of KCNC1-related epilepsy. In aim 1, I will collect patch-clamp electrophysiology recordings of somatic and axonal potassium channel function and neuronal excitability in PV-INs from a novel mouse model of KCNC1 epilepsy which harbors the epileptic encephalopathy patient-derived p.A421V variant. Given the substantially reduced channel activity observed in the p.A421V variant, and that Kv3 currents are necessary for fast-spiking physiology, I anticipate that PV-INs from the mouse model of KCNC1 epileptic encephalopathy will exhibit impaired action potential generation at the soma, and unreliable propagation through the axon. In aim 2, I will interrogate deficits in inhibitory synaptic transmission in response to mutant Kv3.1 expression using multiple (6-8) simultaneous recordings of synaptically-connected PV-INs and pyramidal neurons in cortical microcircuits. Lastly, in aim 3 I will corroborate my in vitro findings in the in vivo context through calcium-imaging of neuronal activity in awake, behaving mice. To relate neuronal activity to relevant behavior, I will examine the excitability of both cortical pyramidal neurons and PV-INs in response to sensory stimulation by whisker deflection and test the hypothesis that altered Kv3.1 function in PV-INs compromises network inhibition in vivo. Overall, completion of the aims described in this mentored fellowship will provide significant insight into the mechanisms of epilepsy in KCNC1-related disorders and deeper understanding of the contribution of Kv3 channels to PV-IN physiology in general.

项目摘要 我正在申请这个NRSA博士后奖学金，其长期职业目标是成为 能够在神经科学领域领导高产研究实验室的独立研究者。在下面 Ethan Goldberg博士和Doug Coulter博士的指导，这项指导的奖学金将为我提供 在科学企业中实现我的目标并蓬勃发展需要概念和动手培训。 基因KCNC1中的致病变异，该变异编码电压门控通道亚基 Kv3.1，导致包括癫痫在内的严重神经系统疾病。虽然大多数患者衍生的变体是 - 功能，损害KV3.1功能的确切机制改变了单个神经元的生理和 导致自发癫痫发作的神经回路功能尚不清楚。 Kv3.1在 白蛋白阳性阳性快速加快抑制性中间神经元（PV-in），包括各种亚细胞区域 树突，躯体，轴突和突触终端，在其中有助于可靠的高频动作 潜在的产生和传播。因为PV-INS严重有助于网络动态并约束 在皮质回路中附近的兴奋性锥体神经元的兴奋性，我们假设KV3.1功能障碍 损害PV-IN高频动作电势产生和繁殖，Disbib抑制了金字塔神经元，并且 有助于促进异常行为和癫痫发作的异常网络兴奋性。因此，总体 这项研究的目的是确定突变体Kv3.1对PV-IN生理学的影响 KCNC1相关癫痫发病机理的贡献者。在AIM 1中，我将收集贴片钳电生理学 新颖的PV-IN中的体细胞和轴突钾通道功能和神经元兴奋性的记录 KCNC1癫痫的小鼠模型，该模型具有癫痫性脑病患者衍生的P.A421V变体。 鉴于在P.A421V变体中观察到的通道活性大大降低，并且KV3电流是 对于快速加速生理学所需的必要 脑病将在SOMA表现出动作潜在产生受损，并通过 轴突。在AIM 2中，我将响应突变体KV3.1询问抑制性突触传播的缺陷。 使用多个（6-8）同时记录突触连接的PV-INS和锥体的表达 皮质微电路中的神经元。最后，在AIM 3中，我将在体内的情况下证实我的体外发现 通过钙的钙成像在清醒中，行为小鼠。将神经元活性与相关性联系起来 行为，我将检查响应感觉的皮质金字塔神经元和PV-IN的兴奋性 晶须挠度刺激并测试了PV-INS妥协中KV3.1功能改变功能的假设 网络抑制体内。总体而言，该指导奖学金中描述的目标的完成将提供 对KCNC1相关性疾病中癫痫机制的重要洞察力，并更深入地了解 一般而言，KV3通道对PV-IN生理学的贡献。