Dravet Syndrome Anti-Epileptic Control by Targeting GIRK Channels

项目摘要 Dravet综合征（DS）是一种由NaV1.1功能丧失引起的破坏性癫痫形式（80-90%的病例）， 主要的电压门控Na+通道表达在抑制性（GABA能）中间神经元中， 海马体和前额叶皮层。这导致抑制性神经递质（GABA）的释放减少， 导致过度兴奋。这种疾病在生命的第一年内表现出来，通常由以下因素引发： 体温过高会导致频繁和长时间的癫痫发作，导致一系列健康问题，包括 发育迟缓、言语障碍、共济失调、张力减退和睡眠障碍。 FDA最近批准了两种小分子单一疗法：Epidiolex（大麻二酚或 CBD）在2018年和Fintepla（芬氟拉明或FA）在2020年为2岁及以上的患者。即使 它们减少癫痫发作的频率，这些药物在其有效剂量下会引起多种副作用。他们的 减少癫痫样活动的作用机制仍然未知。 G蛋白门控内向整流K+通道（GIRK）与癫痫密切相关。他们是 由G蛋白的Gβγ二聚体和[Na+]i以协同方式激活。协同作用的基础在于 它们各自以变构方式工作，主要控制两个通道门：Gβγ，膜门和 Na+，胞质门。在GABA能中间神经元的情况下，我们假设NaV1.1和GIRK 1/2 是偶联的，使得Na+通过NaV1.1进入促进GIRK 1/2活性以使细胞过度增殖， 确保去除NaV1.1灭活，以快速加标。在DS中，该机制受到损害， 细胞去极化和电压门控通道的失活普遍存在于GABA能神经元中， 补偿NaV1.1的损失。我们使用GAT 1508，一种GIRK 1/2的特异性激活剂来补偿 钠离子进入受损。由于GAT 1508打开了细胞溶质门，我们问它是否与CBD协同作用（通过 CB 1 R）和FA（通过5-HT 1DR）以更完全地打开膜门。在目标1中，实验旨在测试 该假说的目的是在异源表达和天然GABA能神经元中的细胞水平。 在目的2和3中，我们利用DS小鼠模型，其为编码NaV1.1的Scn 1a基因的杂合，并且在目的4中， 目标2，我们在脑切片水平上检验这一假设，其中突触和递质释放保持完整， 将DS模型与野生型动物模型进行比较。在目标3中，我们在整个动物水平上进行实验 (DS模型），使用同步EEG和双光子显微镜来监测所涉及的神经元回路。

PROJECT SUMMARY Dravet Syndrome (DS) is a devastating form of epilepsy caused by loss of function of NaV1.1 (80-90% of cases), the predominant voltage-gated Na+ channel expressed in inhibitory (GABAergic) interneurons in the hippocampus and prefrontal cortex. This causes a decrease in the release of inhibitory neurotransmitter (GABA), resulting in hyperexcitability. The disease manifests itself within the first year of life and is usually triggered by hyperthermia causing frequent and prolonged seizures that result in a host of health problems including developmental delay, speech impairment, ataxia, hypotonia and sleep disturbances. Two small molecule monotherapies have been approved recently by the FDA: Epidiolex (Cannabidiol or CBD) in 2018 and Fintepla (fenfluramine or FA) in 2020 for patients two years of age and older. Even though they reduce the frequency of seizures, these drugs at their effective dosages cause multiple side effects. Their mechanism(s) of action to reduce epileptiform activity remain(s) unknown. G protein-gated inwardly rectifying K+ (GIRK) channels have been strongly implicated in epilepsy. They are activated by the Gβγ dimer of G proteins and by [Na+]i in a synergistic manner. The basis of synergism lies in that they each work allosterically to control predominantly the two channel gates: Gβγ, the membrane gate and Na+, the cytosolic gate. In the case of the GABAergic interneurons, we hypothesize that NaV1.1 and GIRK1/2 are coupled, such that the Na+ entering through NaV1.1 promotes GIRK1/2 activity to hyperpolarize the cell and ensure removal of NaV1.1 inactivation for fast spiking. In DS this mechanism becomes compromised causing cell depolarization and inactivation of voltage-gated channels at large present in GABAergic neurons failing to compensate for the loss of NaV1.1. We use GAT1508, a specific activator of GIRK1/2 to compensate for the compromised Na+ entry. Since GAT1508 opens the cytosolic gate, we ask whether it synergizes with CBD (via CB1R) and FA (via 5-HT1DR) to open more fully the membrane gates. In Aim 1, experiments designed to test the hypothesis are aimed at the cellular level in both heterologous expression and in native GABAergic neurons. In Aims 2 and 3, we utilize a DS mouse model, heterozygous for the Scn1a gene that encodes NaV1.1, and in Aim 2, we test the hypothesis at the brain slice level, where synapses and transmitter release remain intact, and compare the DS model to a wild-type animal model. In Aim 3, we pursue experiments at the whole animal level (DS model), using simultaneous EEG and 2-photon microscopy to monitor the neuronal circuits involved.