Evaluation Of Novel Epilepsy Treatment Approaches

该项目的总体目标是研究治疗癫痫的新策略。研究继续评价神经类固醇在癫痫中的作用以及神经类固醇（及其合成类似物）在癫痫治疗中的可能用途。神经类固醇是内源性类固醇激素，其通过直接作用于膜离子通道（包括GABA-A和NMDA受体）而迅速改变神经元的兴奋性。在之前的报告期间，我们证实生殖激素孕酮具有强大的抗惊厥活性。孕激素受体敲除（PRKO）小鼠的研究表明，孕激素对癫痫易感性的影响不是由其同源核激素受体介导的。相反，我们发现孕酮是抗惊厥的，因为它转化为神经类固醇别孕烯醇酮。我们提出，月经期月经癫痫，癫痫发作频率的增加，许多妇女的经验，月经时间（当孕酮水平下降）可能与撤回allopregnanolone。目前，对于月经性癫痫还没有特效的治疗方法。然而，我们对月经性癫痫动物模型的研究表明，神经类固醇替代可能是有用的。此外，我们还研究了神经类固醇在应激诱导的癫痫发作易感性改变中的作用，特别关注脱氧皮质酮（DOC），一种肾上腺类固醇，其合成在应激过程中增强。我们的研究结果表明，DOC是急性应激的生理效应，可能有助于通过其转换为神经甾体与GABA-A受体，包括四氢脱氧皮质酮（THDOC）的调节作用，在癫痫发作易感性的应激诱导的变化的介质。我们的研究结果进一步表明神经活性类固醇作为应激相关癫痫发作的治疗方法的作用。最近的研究集中在雄激素相关的类固醇在癫痫易感性的调节中的作用。男性癫痫患者通常有性或生殖异常，这归因于雄激素水平的改变，包括低于正常的游离睾酮。睾酮的主要代谢物-雄甾酮（5 α-雄甾烷-3 α-醇-17-酮），一种神经类固醇，作为GABA-A受体的正变构调节剂，及其5 β-差向异构体etiocholanolone（5 β-雄甾烷-3 α-醇-17-酮）的水平也可能在癫痫中降低。我们以前报道，雄酮和etiocholanolone具有抗惊厥特性，我们提出，他们可能代表癫痫发作易感性的内源性调节剂。因此，在癫痫患者中这些代谢物的低水平可能导致癫痫发作易感性增强和癫痫发作控制不良。在月经性癫痫，神经活性类固醇替代是一个潜在的治疗策略。在本报告所述期间，我们通过研究雄激素相关类固醇雄烯醇继续研究雄激素相关类固醇在癫痫发作易感性调节中的作用，雄烯醇是一种属于有气味的16-雄甾烯组的甾体化合物，首先从公猪睾丸中分离，也在人类中发现。雄甾烯醇在动物和人类中都具有信息素样性质，但其信息素活性的分子靶点尚不清楚。认识到雄烯醇在结构上与内源性A环还原神经类固醇相似，我们假设它可以作为GABA-A受体阳性调节剂，并且具有抗惊厥特性。这一假设在小脑颗粒细胞的全细胞记录中得到证实，其中雄甾烯醇（而不是其3 β-差向异构体）引起GABA激活电流的浓度依赖性增强，并延长自发和微型抑制性突触后电流的持续时间。雄甾烯醇还增强了用重组α 1 β 2 γ 2和α 2 β 2 γ 2 GABA-A受体转染的人胚肾293细胞中GABA激活电流的幅度，并且在高浓度下，直接激活这些细胞中的电流。雄甾烯醇全身给药在旷场试验中引起小鼠抗焦虑样作用，在强迫游泳试验中引起零迷宫升高和抗抑郁样作用。雄甾烯醇，而不是其3 β-差向异构体，赋予癫痫发作保护在6赫兹电休克和戊四氮模型。雄甾烯醇在整个动物模型中的各种作用与其作为GABA-A受体调节剂的活性一致。与其他雄激素相关的神经类固醇一样，雄甾烯醇可能是癫痫易感性的内源性调节剂。在本报告所述期间，我们启动了一个新的神经类固醇研究方向，在该方向中，我们研究了神经类固醇可能在颞叶癫痫发生模型中调节癫痫发展的可能性。在这些研究中，经历匹鲁卡品诱导的癫痫持续状态的大鼠在潜伏期后表现出自发性复发性癫痫发作。我们发现，P450 SCC，类固醇合成的限速酶，在海马神经胶质细胞在潜伏期上调。更长时间的癫痫持续状态与更大的P450 SCC表达和更长的延迟癫痫发作的发展，这表明增强类固醇合成延缓癫痫发生。5 α-还原酶抑制剂非那普利可阻断神经类固醇的合成，缩短了潜伏期，表明神经类固醇可能具有抗癫痫作用。这些研究提出了神经类固醇可能在预防易感个体癫痫发展方面具有实用性的可能性。

The overall goal of this project is to investigate new strategies for the treatment of epilepsy. Research was continued evaluating the role of neurosteroids in epilepsy and the possible uses of neurosteroids (and their synthetic analogs) in epilepsy therapy. Neurosteroids are endogenous steroid hormones that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity. Studies with progesterone receptor knockout (PRKO) mice demonstrated that the effects of progesterone on seizure susceptibility are not mediated by its cognate nuclear hormone receptors. Instead, we found that progesterone is anticonvulsant as a result of its conversion to the neurosteroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. In addition, we have investigated the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on deoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. Our results demonstrated that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including tetrahydrodeoxycorticosterone (THDOC). Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. Recent studies have focused on the role of androgen-related steroids in the regulation of seizure susceptibility. Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone--androsterone (5alpha-androstan-3alpha-ol-17-one), a neurosteroid that acts as a positive allosteric modulator of GABA-A receptors, and its 5beta-epimer etiocholanolone (5beta-androstan-3alpha-ol-17-one)--also may be reduced in epilepsy. We previously reported that androsterone and etiocholanolone have anticonvulsant properties and we proposed that they could represent endogenous modulators of seizure susceptibility. Thus, low levels of these metabolites in men with epilepsy could lead to enhanced seizure susceptibility and poor seizure control. As in catamenial epilepsy, neuroactive steroid replacement is a potential therapeutic strategy. In the present reporting period, we continued to investigate the role of androgen-related steroids in the regulation of seizure susceptibility through a study of the androgen-related steroid androstenol, which is a steroidal compound belonging to the group of odorous 16-androstenes, first isolated from boar testes and also found in humans. Androstenol has pheromone-like properties in both animals and humans, but the molecular targets of its pheromonal activity are unknown. Recognizing that androstenol is structurally similar to endogenous A-ring reduced neurosteroids, we hypothesized that it could serve as a GABA-A receptor positive modulator and that it would have anticonvulsant properties. This hypothesis was confirmed in whole-cell recordings from cerebellar granule cells, where androstenol (but not its 3beta-epimer) caused a concentration-dependent enhancement of GABA-activated currents and prolonged the duration of spontaneous and miniature inhibitory postsynaptic currents. Androstenol also potentiated the amplitude of GABA-activated currents in human embryonic kidney 293 cells transfected with recombinant alpha1beta2gamma2 and alpha2beta2gamma2 GABA-A receptors and, at high concentrations, directly activated currents in these cells. Systemic administration of androstenol caused anxiolytic-like effects in mice in the open-field test and elevated zero-maze and antidepressant-like effects in the forced swim test. Androstenol, but not its 3beta-epimer, conferred seizure protection in the 6-Hz electroshock and pentylenetetrazol models. The various actions of androstenol in the whole-animal models are consistent with its activity as a GABA-A receptor modulator. Like other androgen-related neurosteroids, androstenol could represent an endogenous modulator of seizure susceptibility. During this reporting period we initiated a new direction of research on neurosteroids in which we examined the possibility that neurosteroids may modulate the development of epilepsy in a model of temporal lobe epileptogenesis. In these studies, rats experiencing pilocarpine-induce status epilepticus exhibited spontaneous recurrent seizures following a latent period. We found that P450scc, the rate-limiting enzyme in steroid synthesis, is upregulated in hippocampal glia during the latent period. More prolonged status epilepticus was associated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retards epileptogenesis. The 5alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that neurosteroids may be antiepileptogenic. These studies raise the possibility that neurosteroids may have utility in preventing the development of epilepsy in susceptible individuals.