GABA activation of the M-current

这项阿尔茨海默病(AD)补充建议是基于雅培实验室最近的两项发现。 形成亲本R01基础的第一个发现是KCNQ2/3电压门控钾(Kv)。 通道由γ-氨基丁酸(GABA)直接激活，GABA是脑内主要的抑制性神经递质 脊椎动物中枢神经系统，其敏感性与最敏感的典型GABAA受体相当。这 新描述的监管形式潜在地具有很大的影响力，因为KCNQ2/3通道是主要的 哺乳动物大脑中一种强大的抑制力量-神经元M-电流的分子相关性。第二 我们的最新发现是阿尔茨海默氏症前体蛋白(APP)切割产物C99，两者 与KCNE家族的Kv通道调节亚基相似，本身调节多个Kv 渠道，包括KCNQ家族的渠道。具体来说，我们发现C99与KCNQ2/3在 在体外与KCNQ2/3形成物理复合物，在体外抑制KCNQ2/3的功能。我们现在打算 研究C99在体内对KCNQ2/3通道的调节与AD的相关性，作为亲本的逻辑延伸 R01，给它带来了一个广告的视角。我们提出了两个具体目标，旨在更全面地理解 C99、APP和其他APP裂解产物抑制在AD病理中的新的潜在作用 神经元KCNQ通道。我们计划利用Kcnq3和Kcnq5 GABA结合位点突变小鼠系WE 为父母R01开发的，与特征良好的3xTg-AD小鼠模型相结合，模拟 人类阿尔茨海默病的许多特征。在目标1中，我们将检验神经元M-电流被抑制的假设。 3xTg-AD小鼠脑。这一假设是基于我们的体外数据，这些数据表明神经元数量增加 浓度的C99，以及可能的其他APP裂解产物，将抑制神经元M电流并改变其 体内的门控动力学。我们还将确定3xTg-AD小鼠的大脑是否改变了GABA对 M-Current，将此目标与父级R01紧密联系在一起，但父级R01缺少AD重点 R01。在目标2中，我们将测试破坏M电流的GABA调节会加剧行为的假设 AD中的变化。我们将量化Kcnq3-W265L和Kcnq5-W270L GABA结合位点的行为变化 在野生型和3xTg-AD背景下为亲本R01培育的突变小鼠，并将它们与 纯合子3xTg-AD小鼠，以确定KCNQ通道GABA调节的中断是否会加剧AD 发病机制。本AD补充资料的总体目标是：(I)确定C99在AD早期是否积累 与我们的体外研究所建议的M-电流下调有关，以及(Ii)确定这一点如何 影响神经元放电；(Iii)确定阻止GABA对KCNQ通道的调制是否会加剧 阿尔茨海默病患者的神经元和M电流放电缺陷，以及行为异常。本副刊中的研究 有可能确定AD认知功能障碍的新治疗方向。

This Alzheimer’s disease (AD) supplement proposal is based on two recent discoveries from the Abbott lab. The first discovery, which forms the basis of the parent R01, is that KCNQ2/3 voltage-gated potassium (Kv) channels are directly activated by γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in vertebrate CNS, with a sensitivity comparable to that of the most sensitive canonical GABAA receptors. This newly described form of regulation is potentially highly influential because KCNQ2/3 channels are the primary molecular correlate of neuronal M-current, a powerful inhibitory force in mammalian brain. The second discovery is our more recent finding that the Alzheimer’s precursor protein (APP) cleavage product, C99, both bears similarities to the KCNE family of Kv channel regulatory subunits, and itself regulates multiple Kv channels, including those of the KCNQ family. Specifically, we found that C99 co-localizes with KCNQ2/3 in vivo, forms physical complexes with KCNQ2/3 in vitro, and inhibits KCNQ2/3 function in vitro. We now intend to study the relevance to AD of C99 regulation of KCNQ2/3 channels in vivo, as a logical extension of the parent R01, bringing an AD perspective to it. We propose two Specific Aims directed towards a fuller understanding of the novel potential role in AD pathology of C99, APP and potentially other APP cleavage product inhibition of neuronal KCNQ channels. We plan to utilize the Kcnq3 and Kcnq5 GABA binding-site mutant mouse lines we developed for the parent R01, in conjunction with the well-characterized 3xTg-AD mouse model that mimics many features of human AD. In Aim 1, we will test the hypothesis that neuronal M-current is inhibited in the 3xTg-AD mouse brain. This hypothesis is based on our in vitro data, that suggest increased neuronal concentrations of C99, and potentially other APP cleavage products, will inhibit neuronal M-current and alter its gating kinetics in vivo. We will also determine whether 3xTg-AD mouse brains have altered GABA regulation of M-current, linking this aim tightly with the parent R01 but with an AD focus that was absent from the parent R01. In Aim 2, we will test the hypothesis that disrupted GABA regulation of M-current exacerbates behavioral changes in AD. We will quantify behavioral changes in Kcnq3-W265L and Kcnq5-W270L GABA binding-site mutant mice bred for the parent R01, in both a wild-type and 3xTg-AD background, and compare them to homozygous 3xTg-AD mice, to determine if disruption of GABA modulation of KCNQ channels exacerbates AD pathogenesis. The overall goals of this AD supplement are to (i) determine if C99 accumulation in early AD correlates with downregulation of M-current, as suggested by our in vitro studies and (ii) determine how this affects neuronal firing; (iii) identify whether preventing GABA modulation of KCNQ channels exacerbates neuronal and M-current firing defects, and behavioral abnormalities, in AD. The studies in this supplement have the potential to identify novel therapeutic directions for cognitive dysfunction in AD.