Development of SK channel modulators as therapeutic agents for ataxia

该项目的长期目标是开发脊髓小脑共济失调（SCA）的新型治疗药物。对于SCA没有有效的治疗或治愈，这在很大程度上是由于对疾病发病机制的认识不足。在这个项目中，我们将重点关注脊髓小脑共济失调2型（SCA 2）。SCA 2是一种常染色体显性遗传性神经退行性疾病，由ataxin-2（Atxn 2）蛋白中的polyQ扩增引起。小脑浦肯野细胞（PC）是SCA 2患者的主要病理部位。在此之前，我们的实验室发现PC中异常的神经元钙信号在SCA的发病机制中起重要作用。先前的研究也表明，在共济失调小鼠模型中，PC的神经元活性是异常的。我们证明了小电导钙激活钾（SK）通道的正变构调节剂（PAM）可以使SCA 2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SCA 2的治疗剂，并可能用于其他共济失调。该项目汇集了两个实验室的互补技能和专门知识，以发现此类制剂。为了实现这一目标，我们将重点关注以下具体目标：R21阶段目标1。鉴定新的亚型选择性SK PAM候选物。我们将确定SK2通道的晶体结构与原型亚型选择性PAM的复杂性。我们将获得的结构信息，将用于执行虚拟高通量筛选亚型选择性SK PAM。第二阶段目标。验证新型SK PAM的功能活性。我们将使用基于荧光的高通量测定和电生理学方法来测试SK PAM候选物。第三章.研究新型SK PAM对SK通道的特异性。我们将使用电生理记录评估SK PAM候选物对SK通道相对于其他K+、Na+和Ca 2+通道的特异性。第四章.研究新型SK PAM对SCA 2小鼠小脑PC活性的影响。在这些研究中，我们将进行离体小脑切片电生理记录PC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满足的需求。这些分子还将为了解SK通道在健康和疾病中的生物学功能提供新颖独特的研究工具。因此，该项目将对基础和转化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.