Development of SK channel modulators as therapeutic agents for ataxia

该项目的广泛和长期目标是开发治疗脊髓小脑性共济失调(SCA)的新型治疗剂。目前尚无有效的治疗或治愈SCA的方法，这在很大程度上是由于对该病的发病机制认识不足。在这个项目中，我们将重点研究脊髓小脑型共济失调2(SCA2)。SCA2是一种常染色体显性遗传性神经退行性疾病，由ataxin-2(Atxn2)蛋白的多聚Q扩展引起。小脑浦肯野细胞(PC)是SCA2患者的主要病理部位。在此之前，我们的实验室发现了PC中神经元钙信号的异常，这些信号在SCAS的发病机制中起着重要作用。以前的研究还表明，在共济失调小鼠模型中，PC的神经元活动异常。我们证明小电导钙激活钾(SK)通道的阳性变构调节剂(PAM)可以使SCA2小鼠PC的异常放电模式正常化，并减轻行为和神经病理表型。这些结果表明，SK PAM可以作为治疗SCA2和其他共济失调的潜在有用的药物。该项目汇集了两个实验室发现此类病原体的互补技能和专门知识。为了实现这一目标，我们将专注于以下具体目标：R21阶段目标1.寻找新的亚型选择性SK PAM候选者。我们将确定SK2通道与原型亚型选择性PAM的复合体的晶体结构。我们将获得用于对亚型选择性SK PAM进行虚拟高通量筛选的结构信息。R21阶段目标2.验证新型SK PAM的功能活性。我们将使用基于荧光的高通量检测和电生理方法来检测SK PAM候选者。R33期目标3.研究新型SK PAM对SK通道的特异性。我们将使用电生理记录来评估SK PAM候选SK通道相对于其他K+、Na+和Ca~(2+)通道的特异性。R33期目标4.研究新型SK PAM对SCA2小鼠小脑PC活性的影响。在这些研究中，我们将进行体外小脑切片电生理记录PC的活动。由此产生的亚型选择性SK PAM将为未来的临床前和临床开发提供关键线索，满足共济失调患者的关键和未得到满足的需求。这些分子还将为理解SK通道在健康和疾病中的生物学功能提供新颖和独特的研究工具。因此，拟议的项目将对基础神经科学和翻译神经科学产生重大影响。

The broad, long-term objective of the project is to develop novel therapeutic agents for spinocerebellar ataxias (SCAs). There is no effective treatment or cure for SCAs, in large part due to insufficient understanding of the disease pathogenesis. In this project, we will focus on spinocerebellar ataxia type 2 (SCA2). SCA2 is an autosomal dominant genetic neurodegenerative disorder caused by polyQ expansion in ataxin-2 (Atxn2) protein. Cerebellar Purkinje cells (PCs) are the main locus of pathology in SCA2 patients. Previously, our laboratory discovered abnormal neuronal calcium signaling in PCs, which play an important role in the pathogenesis of SCAs. Previous studies also suggest that neuronal activity of PCs is abnormal in ataxic mouse models. We demonstrated that positive allosteric modulators (PAMs) of small conductance calcium-activated potassium (SK) channels can normalize the abnormal firing pattern of PCs in SCA2 mice and alleviate behavioral and neuropathological phenotypes. These results suggest that SK PAMs can be potentially useful as therapeutic agents for the treatment of SCA2 and possibly for other ataxias. This project brings together the complementary skills and expertise of two laboratories for the discovery of such agents. To achieve this goal, we will focus on the following specific aims: R21 Phase Aim 1. To identify novel subtype-selective SK PAM candidates. We will determine the crystal structure of SK2 channels in complex with the prototype subtype-selective PAMs. We will obtain structural information that will be used to perform virtual high throughput screening for subtype-selective SK PAMs. R21 Phase Aim 2. To validate functional activity of novel SK PAMs. We will use fluorescence-based high throughput assay and electrophysiological methods to test SK PAM candidates. R33 Phase Aim 3. To investigate the specificity of novel SK PAMs for SK channels. We will evaluate the specificity of the SK PAM candidates for SK channels over other K+, Na+ and Ca2+ channels using electrophysiological recordings. R33 Phase Aim 4. To investigate the effects of novel SK PAMs on the activity of cerebellar PCs in SCA2 mice. We will perform ex vivo cerebellar slice electrophysiological recordings of PC activity in these studies. The resulting subtype-selective SK PAMs will provide critical leads for future preclinical and clinical development, fulfilling the critical and unmet needs in ataxia patients. These molecules will also offer novel and unique research tools for understanding the biological function of SK channels in health and disease. Thus, the proposed project will have significant impact in basic and translational neuroscience.