A Delta GABA Receptor as a Target for Essential Tremor Therapy

描述（由申请人提供）： 原发性震颤影响0.5-0.9%的一般人群和约4.5%的65岁以上人群。治疗震颤的药物是根据经验而不是通过实验室研究发现的重新用途的药物。因此，它们缺乏效力和特异性，并且通常不能很好地耐受。在我们的项目中，我们寻求确定抗震颤药物可以设计的分子靶点，提供更大的靶向特异性和效力。一个线索是，患者经常报告说，只要1-2杯葡萄酒就可以抑制震颤。特发性震颤的脑映射研究表明，震颤的基础是小脑回路内的超振荡活动。 当患者服用低剂量的酒精时，不仅震颤减少，而且小脑皮层的过度活动也减少了。我们推测，低剂量酒精通过激活GABAA受体来治疗震颤，GABAA受体含有α 6，β 3和δ亚基，仅在小脑皮质中发现。如果我们能证明这种受体可以介导震颤抑制，它可能是抗震颤治疗的重要靶点。GABAA受体是由α、β和γ或δ亚基组成的五聚体。δ GABAA受体发挥紧张性抑制作用，并且对GABA更加敏感。对细胞中表达的受体和脑切片的研究表明，THIP、蝇蕈醇和神经类固醇如加奈索酮选择性地激活δ GABAA受体，而不是γ GABAA受体。此外，1-2杯葡萄酒中的乙醇被发现可以激活那些使用β 3亚基的δ GABAA受体。我们假设THIP、蝇蕈醇、加奈索酮和低剂量酒精对δ GABAA受体的激活将抑制野生型但非同窝δ基因敲除小鼠的特发性震颤发作模型中的震颤。此外，酒精在野生型小鼠中的震颤抑制作用应该被酒精拮抗剂Ro 15 -4513逆转，这进一步证明了酒精通过GABAA受体抑制震颤。我们使用的是与原发性震颤的震颤有许多相似之处的震颤模型。对运动功率进行数字分析，以提供震颤带宽中的运动的测量值作为总体运动功率的比率。我们的试验数据表明，THIP，蝇蕈醇，加奈索酮，和低剂量酒精都抑制野生型，但不是在δ基因敲除小鼠的震颤。低剂量酒精的作用被Ro 15 -4513阻断。我们建议重复这些发现（目标1）。δ亚基与α 4或α 4相关， 6在GABAA受体中。α 4-δ受体激活时介导镇静和运动障碍，而以前没有发现与α 6-δ受体激活相关的行为。我们的初步实验表明，THIP和低剂量酒精抑制野生型但不是在α 6基因敲除小鼠的震颤，表明α 6-δ受体介导震颤抑制。我们建议复制这一发现，并测试加奈索酮以及（目的2）。含有α 6-δ的GABAA受体仅在小脑皮质中发现，在那里它们与β 3，β 2或β 1相关。下一步是研究小脑β 3是否对这些δ受体作用药物的震颤抑制很重要，方法是繁殖在表达α 6的小脑部分缺失β 3的小鼠（目的3）。预计β 3对低剂量酒精的抗震颤作用至关重要，但对THIP或加奈索酮可能不重要。如果合适的基因敲除小鼠品系可用，我们将测试β 2或β 1是否对介导THIP和加奈索酮的抗震颤作用很重要。这些实验预期导致GABAA受体亚型α 6-β 2/3-δ的鉴定，其仅在小脑皮质中发现，将构成抗震颤疗法可被设计为激活的分子靶点。这种疗法，通过其高选择性，预计是有效的和良好的耐受性。作为第一 如果从实验室研究中开发出专门针对特发性震颤的药物，预计它将比现有药物具有更大的疗效，预防患有特发性震颤的退伍军人的残疾和失业。

DESCRIPTION (provided by applicant): Essential tremor affects 0.5-0.9% of the general population and about 4.5% of those above age 65. Medications for tremor are re-purposed drugs found empirically rather than through laboratory research. As such they lack potency and specificity, and are often not well tolerated. In our program we seek to identify molecular targets to which anti-tremor drugs could be designed, offering greater on-target specificity and potency. A clue is that patients often report that as little as 1-2 glasses of wine can suppress tremor. Brain mapping studies in essential tremor indicate that hyper-oscillatory activity within circuits of the cerebellum underlies tremor. When patients are given low doses of alcohol, not only does the tremor diminish, but excessive activity in the cerebellar cortex also diminishes. We postulate that low-dose alcohol works on tremor by activating GABAA receptors that contain alpha6, beta3 and delta subunits, found only in the cerebellar cortex. If we can show that this receptor can mediate tremor suppression, it could represent an important target for anti-tremor therapy. The GABAA receptor is a pentamer made of alpha, beta, and either gamma or delta subunits. Delta GABAA receptors exert tonic inhibition, and are much more sensitive to GABA. Studies with receptors expressed in cells, and with brain slices, have shown that THIP, muscimol and neurosteroids such as ganaxolone selectively activate delta GABAA receptors rather than gamma GABAA receptors. In addition, ethanol in the range of 1-2 glasses of wine has been found to activate those delta GABAA receptors that use beta3 subunits. We hypothesize that activation of delta GABAA receptors by THIP, muscimol, ganaxolone, and low-dose alcohol will suppress tremor in the harmaline model of essential tremor in wild-type but not littermate delta knockout mice. In addition, the tremor-suppressing effect of alcohol in wild-type mice should be reversed by the alcohol antagonist Ro15-4513, providing further evidence that alcohol is suppressing tremor via a GABAA receptor. We use the harmaline tremor model, which has many similarities to tremor of essential tremor. Motion power is digitally analyzed to provide measures of motion in the tremor bandwidth as a ratio of overall motion power. Our pilot data indicate that THIP, muscimol, ganaxolone, and low-dose alcohol all suppress harmaline tremor in wild-type but not in delta knockout mice. The effect of low-dose alcohol was blocked by Ro15-4513. We propose to replicate these findings (Aim 1). The delta subunit is associated with either alpha 4 or with alpha 6 in GABAA receptors. The alpha4- delta receptors mediate sedation and motor impairment when activated, whereas no behavior has previously been found associated with alpha6-delta receptor activation. Our pilot experiments showed that THIP and low-dose alcohol suppress harmaline tremor in wild-type but not in alpha6 knockout mice, indicating the alpha6-delta receptor mediates tremor suppression. We propose to replicate this finding and test ganaxolone as well (Aim 2). GABAA receptors containing alpha6-delta are found only in the cerebellar cortex, where they associate with beta3, beta2 or beta1. The next step is to investigate whether cerebellar beta3 is important for tremor suppression by these delta-receptor acting drugs by breeding mice with beta3 missing in the part of the cerebellum where alpha6 is expressed (Aim 3). It is anticipated that beta3 will be critical for low-dose alcohol's anti-tremor action, but itmay not be critical for THIP or ganaxolone. Should appropriate strains of knockout mice become available, we will test whether beta2 or beta1 are important for mediating the anti-tremor action of THIP and ganaxolone. These experiments are expected to lead to the identification of a GABAA receptor subtype, alpha6-beta2/3- delta, found only in the cerebellar cortex that will constitute a molecular target to which anti-tremor therapies can be designed to activate. Such a therapy, through its high selectivity, is anticipated to be potent and well tolerated. As the first drug to be developed specifically for essential tremor from laboratory research, it would be expected to confer greater efficacy than existing drugs, preventing disability and loss of occupation in veterans with essential tremor.