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.

项目概要 我正在申请 NRSA 博士后奖学金，其长期职业目标是成为一名 独立研究员，有能力领导神经科学领域高效的研究实验室。在下面 在 Ethan Goldberg 博士和 Doug Coulter 博士的指导下，这个指导奖学金将为我提供 实现我的目标并在科学事业中蓬勃发展所需的概念和实践培训。 KCNC1 基因的致病性变异，该基因编码电压门控钾通道亚基 Kv3.1，导致严重的神经系统疾病，包括癫痫。虽然大多数源自患者的变异都丢失了 功能，受损的 Kv3.1 功能改变个体神经元生理学的精确机制以及 导致自发性癫痫发作的神经回路功能尚不清楚。 Kv3.1 突出地表达于 小清蛋白阳性快速尖峰抑制性中间神经元（PV-IN）位于各个亚细胞区域，包括 树突、体细胞、轴突和突触末梢，它对可靠的高频动作做出了至关重要的贡献 潜在的产生和传播。因为 PV-IN 对网络动态做出了至关重要的贡献并限制了 皮质回路中附近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 高频动作电位的产生和传播，抑制锥体神经元，以及 导致异常的网络兴奋性，从而导致异常行为和癫痫发作。因此，总体 本研究的目的是确定突变体 Kv3.1 对 PV-IN 生理学的影响作为潜在的主要 KCNC1 相关癫痫发病机制的贡献者。在目标 1 中，我将收集膜片钳电生理学信息 记录体细胞和轴突钾通道功能以及 PV-IN 中的神经元兴奋性 KCNC1 癫痫小鼠模型，含有癫痫性脑病患者衍生的 p.A421V 变体。 鉴于 p.A421V 变体中观察到的通道活动大幅减少，并且 Kv3 电流为 对于快速峰值生理学来说是必要的，我预计来自 KCNC1 癫痫小鼠模型的 PV-IN 脑病将表现出体细胞动作电位产生受损，并且通过不可靠的传播 轴突。在目标 2 中，我将探讨针对突变体 Kv3.1 的抑制性突触传递缺陷 使用突触连接的 PV-IN 和锥体的多个 (6-8) 同时记录进行表达 皮质微电路中的神经元。最后，在目标 3 中，我将在体内证实我的体外发现 通过对清醒、行为正常的小鼠的神经元活动进行钙成像。将神经元活动与相关的 行为，我将检查皮质锥体神经元和 PV-IN 对感觉的反应的兴奋性 通过晶须偏转进行刺激并测试改变 PV-IN 中 Kv3.1 功能的假设 体内网络抑制。总体而言，完成本指导奖学金中描述的目标将提供 对 KCNC1 相关疾病的癫痫机制有深入的了解，并对 KCNC1 相关疾病的癫痫机制有更深入的了解 Kv3 通道对 PV-IN 生理学的总体贡献。