Progesterone Receptors and Seizure Susceptibility

描述（由申请人提供）：预后（P）在“月经性癫痫”的病理生理学中起关键作用，月经周期相关的癫痫发作障碍影响许多女性癫痫患者。虽然P的内分泌作用是由孕激素受体（PR）介导的，但P在癫痫发作活动中的分子作用机制尚不清楚。P调节癫痫发作活动部分通过转化为别孕烯醇酮，这增强了GABAA受体功能。最近，我们有了新的发现，PRs在癫痫易感性中起着关键作用。我们的初步结果强调，PR基因敲除（PRKO）小鼠表现出癫痫发作阈值升高和抵抗点燃癫痫发生，这表明PR介导癫痫发作的易感性。在这个应用中，我们建议阐明的分子机制，从而PRs增加癫痫发作的易感性在海马，利用遗传，分子和电生理学的方法。我们推测孕酮对癫痫发作活动的作用部分是由于PR调节海马GABA^受体亚单位的表达和功能，导致抑制作用降低，从而降低癫痫发作的易感性。PR介导的“亚基转换”导致GABAA受体亚基组成和功能特性的改变是P控制癫痫易感性的分子机制。我们建议利用我们实验室中已建立的3种模型系统来严格检验这一假设：1）PRKO转基因小鼠模型; 2）野生型小鼠中的反义PR抑制; 3）RU-486阻断野生型小鼠中的PR功能。本提案的具体目的是：1）使用癫痫点燃模型确定PR是否增加癫痫发作易感性; 2）确定PR是否参与P诱导的GABAA受体亚单位表达和通道功能的改变。目的1将检查WT和PRKO小鼠中的海马点燃的速率，用和不用P和/或非那肽治疗，作为癫痫发生的指标。目的2将评估用P、P+非那肽处理的WT和PRKO小鼠以及神经类固醇停药后24小时海马子区域中GABAA受体亚单位的mRNA（实时PCR）和蛋白质水平（蛋白质印迹）。我们将通过使用膜片钳电生理学记录急性分离的海马神经元中GABA门控的CI”电流来验证P诱导的GABAA受体亚单位表达变化的功能重要性。意义这项研究将提供新的信息，P机制控制癫痫发作的易感性，这可能会导致更好地了解月经性癫痫。本案无关月经性癫痫的女性在其月经周期周围聚集发作。然而，目前这种情况的病理生理学尚未完全了解。本申请中提出的研究将有助于更好地了解疾病机制，从而允许开发针对月经性癫痫和与孕酮水平波动相关的其他病症的特定疗法。

DESCRIPTION (provided by applicant): Progesterone (P) plays a key role in the pathophysiology of "catamenial epilepsy", a menstrual cyclerelated seizure disorder that affects many women with epilepsy. While P's endocrine actions are mediated by the progesterone receptor (PR), P's molecular mechanism of action in seizure activity is not clearly understood. P modulates seizure activity partly through conversion to allopregnanolone, which enhances GABAA receptor function. Recently, we have made the novel discovery that PRs play a key role in seizure susceptibility. Our preliminary results underscore that PR knockout (PRKO) mice exhibit elevated seizure threshold and resistance to kindling epileptogenesis, suggesting that PRs mediate seizure susceptibility. In this application, we propose to elucidate the molecular mechanisms whereby PRs increase seizure susceptibility in the hippocampus, utilizing genetic, molecular and electrophysiological approaches. We hypothesize that progesterone's actions on seizure activity are due in part to PR regulation of hippocampal GABA^ receptor subunit expression and function that results in reduced inhibition and thereby seizure susceptibility. PR-mediated "subunit switching" leading to alterations of GABAA receptor subunit composition and functional properties are the molecular mechanism by which P controls seizure susceptibility. We propose to test this hypothesis critically utilizing 3 model systems that are well established in our lab: 1) PRKO transgenic mouse model; 2) Antisense PR inhibition in wild-type mice; and 3) RU-486 blockade of PR function in wild-type mice. The Specific Aims of this proposal are: 1) to determine whether PRs increase seizure susceptibility using the kindling model of epilepsy; and 2) to determine whether PRs are involved in P induced alterations in GABAA receptor subunit expression and channel function. Aim 1 will examine the rate of hippocampus kindling in WT and PRKO mice, with and without P and/or finasteride treatment, as an indicator of epileptogenesis. Aim 2 will assess mRNA (real-time PCR) and protein levels (Westerns blots) of GABAA receptor subunits in hippocampal subfields in WT and PRKO mice treated with P, P+finasteride, and 24 hrs after neurosteroid withdrawal. We will verify the functional importance of P induced changes in GABAA receptor subunit expression by recording GABA-gated CI" currents in acutely isolated hippocampal neurons using patch-clamp electrophysiology. Significance. The proposed study will provide novel information on the P mechanisms governing regulation of seizure susceptibility, which could lead to improved understanding of catamenial epilepsy. Relevance. Women with catamenial epilepsy have seizures clustered around their monthly cycle. However, currently the pathophysiology of this condition is not fully understood. The studies proposed in this application will help better understand the disease mechanism and thereby allow development of specific therapies for catamenial epilepsy and other conditions associated with fluctuations in progesterone levels.