Progesterone Receptors and Seizure Susceptibility

描述（由申请人提供）：黄体酮(P)在“羊膜性癫痫”的病理生理学中起关键作用，羊膜性癫痫是一种影响许多女性癫痫患者的月经周期相关的癫痫发作障碍。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部分通过转化为异孕酮来调节癫痫发作活动，增强GABAA受体功能。最近，我们有了新的发现，pr在癫痫易感性中起关键作用。我们的初步结果强调，PR敲除（PRKO）小鼠表现出更高的癫痫阈值和对点燃癫痫发生的抗性，表明PR介导癫痫易感性。在这项应用中，我们建议利用遗传、分子和电生理方法来阐明PRs增加海马癫痫易感性的分子机制。我们假设黄体酮对癫痫活动的作用部分是由于PR对海马GABA受体亚基表达和功能的调节，从而导致抑制降低，从而降低癫痫易感性。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已经建立的3个模型系统来严格检验这一假设：1)PRKO转基因小鼠模型；2)野生型小鼠反义PR抑制；3) RU-486阻断野生型小鼠PR功能。本提案的具体目的是：1)利用癫痫点燃模型确定pr是否增加癫痫易感性；2)确定pr是否参与P诱导的GABAA受体亚基表达和通道功能的改变。目的1将检测WT和PRKO小鼠海马点燃率，并将P和/或非那雄胺治疗和不治疗作为癫痫发生的指标。目的2将评估经P、P+非那雄胺治疗的WT和PRKO小鼠海马亚区GABAA受体亚基的mRNA （real-time PCR）和蛋白水平（western blots）。我们将通过膜片钳电生理学记录急性分离海马神经元gaba门控CI电流，验证P诱导GABAA受体亚基表达变化的功能重要性。的意义。本研究将为P调控癫痫易感性的机制提供新的信息，有助于提高对癫痫的认识。的相关性。患有双侧癫痫的女性在月经周期前后发作。然而，目前对这种情况的病理生理尚不完全了解。本应用程序中提出的研究将有助于更好地了解疾病机制，从而允许开发针对晚眠性癫痫和其他与孕酮水平波动相关的疾病的特定治疗方法。

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.