Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA.
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA.
Epilepsia. 2020 Oct;61(10):2301-2312. doi: 10.1111/epi.16670. Epub 2020 Sep 17.
Neuroinflammation is a major theme in epilepsy, which has been characterized in acquired epilepsy but is poorly understood in genetic epilepsy. γ-Aminobutyric acid type A receptor subunit gene mutations are significant causes of epilepsy, and we have studied the pathophysiology directly resulting from defective receptor channels. Here, we determined the proinflammatory factors in a genetic mouse model, the Gabrg2 knockin (KI). We have identified increased cytokines in multiple brain regions of the KI mouse throughout different developmental stages and propose that accumulation of the trafficking-deficient mutant protein may increase neuroinflammation, which would be a novel mechanism for genetic epilepsy.
We used enzyme-linked immunosorbent assay, immunoprecipitation, nuclei purification, immunoblot, immunohistochemistry, and confocal microscopy to characterize increased neuroinflammation and its potential causes in a Gabrg2 KI mouse and a Gabrg2 knockout (KO) mouse, each associated with a different epilepsy syndrome with different severities.
We found that proinflammatory cytokines such as tumor necrosis factor alpha, interleukin 1-beta (IL-1β), and IL-6 were increased in the KI mice but not in the KO mice. A major underlying basis for the discrepancy in cytokine expression between the two mouse models is likely chronic mutant protein accumulation and endoplasmic reticulum (ER) stress. The presence of mutant protein dampened cytokine induction upon further cellular stimulation or external stress such as elevated temperature. Pharmacological induction of ER stress upregulated cytokine expression in the wild-type and KO but not in the KI mice. The increased cytokine expression was independent of seizure occurrence, because it was upregulated in both mice and cultured neurons.
Together, these data demonstrate a novel pathophysiology for genetic epilepsy, increased neuroinflammation, which is a common mechanism for acquired epilepsy. The findings thus provide the first link of neuroinflammation between genetic epilepsy associated with an ion channel gene mutation and acquired epilepsy.
神经炎症是癫痫的一个主要主题，在获得性癫痫中已有特征描述，但在遗传性癫痫中了解甚少。γ-氨基丁酸 A 型受体亚基基因突变是癫痫的重要原因，我们已经研究了直接由缺陷受体通道引起的病理生理学。在这里，我们在一个遗传小鼠模型中确定了促炎因子，即 Gabrg2 敲入（KI）。我们已经在 KI 小鼠的多个脑区确定了细胞因子的增加，并且在不同的发育阶段提出了积累转运缺陷的突变蛋白可能会增加神经炎症的假说，这将是遗传性癫痫的一种新机制。
我们使用酶联免疫吸附测定、免疫沉淀、核纯化、免疫印迹、免疫组织化学和共聚焦显微镜来描述 Gabrg2 KI 小鼠和 Gabrg2 敲除（KO）小鼠中神经炎症的增加及其潜在原因，每个模型都与不同严重程度的不同癫痫综合征有关。
我们发现促炎细胞因子，如肿瘤坏死因子-α、白细胞介素 1-β（IL-1β）和 IL-6，在 KI 小鼠中增加，但在 KO 小鼠中没有增加。两种小鼠模型中细胞因子表达差异的一个主要潜在基础可能是慢性突变蛋白积累和内质网（ER）应激。突变蛋白的存在抑制了进一步细胞刺激或外部应激（如高温）时细胞因子的诱导。ER 应激的药物诱导上调了野生型和 KO 小鼠但不是 KI 小鼠的细胞因子表达。细胞因子的表达增加与癫痫发作无关，因为它在两种小鼠和培养神经元中都上调。
这些数据共同证明了遗传性癫痫的一种新的病理生理学机制，即神经炎症增加，这是获得性癫痫的共同机制。这些发现为与离子通道基因突变相关的遗传性癫痫和获得性癫痫之间的神经炎症提供了第一个联系。
