Drug Discovery for Spinocerebellar Ataxia Type 2 (SCA2)

描述（由申请人提供）：神经退行性疾病代表着不断增加的社会和经济负担，世卫组织估计表明，到2040年，它们将取代癌症成为第二大死亡原因。在神经退行性疾病的研究中，已经发现了大量的通路，但它们与各自的人类疾病的直接和主要相关性一直难以证明，通路仍然难以定位。对于多聚谷氨酰胺疾病，有明确的证据表明，各自基因的表达水平影响表型，小鼠模型中的关闭表达逆转了已经建立的运动表型。我们选择了常染色体显性聚谷氨酰胺（polyQ）疾病来证明小分子可以在转录或mRNA稳定水平上下调疾病基因的表达。脊髓小脑性共济失调2型（SCA2）是一种多系统神经退行性疾病，由ataxin-2蛋白中多q结构域的显性突变引起。SCA2患者发展为进行性共济失调，随后在其他神经系统中丧失功能。部分患者出现明显的帕金森症状。与许多神经退行性疾病类似，没有已知的症状或疾病改善治疗方法。该研究的主要目的是鉴定抑制ATXN2表达的化合物，并在SCA2小鼠模型中测试它们的功效。为此，我们开发了一种基于细胞的实验，并使用相同的荧光素酶表达结构与体内小鼠模型配对。这将使我们能够将化合物从NIH化学基因组学中心（NCGC）进行的高通量筛选快速推进到小鼠的功效和毒性测试。提出了四个具体目标：1)在NCGC中使用30万种化合物进行HTS， 2)在SCA2患者淋巴细胞中进行化合物的体外测试，3)在ATXN2-荧光素酶转基因小鼠中进行化合物的体内测试，以及4)在人源化SCA2小鼠BAC模型中测试化合物改善ATXN2表型的能力。我们的方法利用了ATXN2-荧光素酶转基因，其荧光素酶基因两侧是ATXN2基因的上游和下游部分。小鼠模型可以快速测试化合物在体内的功效，包括通过血脑屏障，以及化合物的进一步发展，以在具有复制人类SCA2的形态学，生化和运动表型的小鼠模型中进行测试。这项工作将为神经退行性疾病的治疗开辟新天地，证明了用化合物靶向主导作用突变基因的可行性。我们的目标是鉴定小分子，不仅针对基因表达水平，而且通过3'-UTR确定mRNA的稳定性。我们的重点是小分子和广泛和快速的hts后测试，在许多啮齿动物模型和人类细胞系。

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. In neurodegenerative disease research, a wealth of pathways has been uncovered, but their direct and primary relevance to the respective human disease have been difficult to prove and pathways have remained difficult to target. For polyglutamine diseases, there is clear evidence that expression levels of the respective genes affect the phenotype and shutting-off expression in mouse models reverses already established motor phenotypes. We have chosen an autosomal dominant polyglutamine (polyQ) disease to demonstrate the proof-of-principle that small molecules can be used to downregulate expression of a disease gene at the transcriptional or mRNA stability level. Spinocerebellar ataxia type 2 (SCA2) is a multisystem neurodegenerative disease caused by a dominantly-acting mutation leading to expansion of a polyQ domain in the ataxin-2 protein. SCA2 patients develop progressive ataxia and later lose function in other neuronal systems. Prominent parkinsonian signs develop in some. Similar to many neurodegenerative disorders, no symptomatic or disease-modifying treatments are known. The primary objective of the proposed research is to identify compounds inhibiting ATXN2 expression and to test them for efficacy in SCA2 mouse models. To this end, we have developed a cell-based assay paired with an in vivo mouse model using identical luciferase expression constructs. This will allow us to progress compounds rapidly from a high-throughput screen conducted at NIH Chemical Genomics Center (NCGC) to efficacy and toxicity testing in mice. Four specific aims are proposed: 1) HTS with 300,000 compounds at the NCGC, 2) In vitro testing of compounds in SCA2 patient lymphoblasts, 3) In vivo testing of compounds in ATXN2-luciferase transgenic mice, and 4) testing the ability for compounds to ameliorate an ATXN2 phenotype in a humanized SCA2 mouse BAC model. Our approach takes advantage of an ATXN2-luciferase transgene with luciferase gene flanked by the upstream and downstream portions of the ATXN2 gene. Mouse models are in place to test rapidly the efficacy of compounds in vivo including passage of the blood brain barrier, and further progression of compounds to test in mouse models with morphological, biochemical, and motor phenotypes replicating human SCA2. The proposed work will break new ground for treatment of neurodegenerative diseases by demonstrating feasibility of targeting dominant-acting mutated genes with compounds. We aim to identify small molecules not only targeting gene expression levels, but also mRNA stability via the 3'-UTR. Our focus is on small molecules and extensive and rapid post HTS-testing in a number of rodent models as well as human cell lines. PUBLIC HEALTH RELEVANCE: Neurodegenerative diseases are not only a major worldwide health problem, but owing to the large number affected and long course of illness, a significant economic threat. We are using SCA2, a debilitating and terminal disorder, as a model to examine novel approaches to identify disease-modifying compounds. Our approach of tightly coupling in vitro and in vivo screens can used to target other neurodegenerative diseases caused by dominant-acting gene mutations.