Stimulators of Neuronal Transthyretin (TTR) Transcription as Alzheimer's Therapy

描述（由申请人提供）：我们已经证明，野生型人转甲状腺素（TTR）基因的过表达可以抑制经过充分研究验证的人阿尔茨海默病（AD）转基因模型的神经病理和行为表型。我们还发现，在体外，TTR与A2相互作用可抑制低聚物和纤维的形成以及组织培养细胞毒性，在体内，TTR-A2复合物可从APP23和一些人AD大脑中与抗血清共同免疫沉淀到这两种蛋白。我们还注意到，来自AD大脑的70%的人类皮质神经元被抗ttr抗体染色（相比之下，10%的年龄匹配的对照大脑），APP23小鼠模型中几乎所有的海马和皮质神经元都被抗ttr抗体染色。APP23小鼠海马原代神经元中TTR mRNA（通过qtPCR）比对照高10倍，海马裂解物中的比例甚至更高。这些发现，再加上观察到当APP23菌株交叉到小鼠TTR敲除背景时，病理变化似乎会加速，这表明TTR可能是AD型神经退行性疾病的正常生理神经元防御机制。相反，TTR过表达对AD特征的抑制可能是药理学上的。我们的建议是针对寻找小分子，将加强TTR转录和蛋白质生产的神经元来源的细胞。为此，我们开发了报告基因构建，其中人类TTR启动子驱动高斯荧光素酶报告基因，该基因很容易在我们机构可用的高通量筛选模式中检测到。初步研究表明，该方法在各种条件下都是可靠的、可重复的和稳定的。我们建议使用该试验连续筛选一组可用的分子文库（Maybridge， LOPAC， MLPCN, Scripps Florida），以确定25个最有效，特异性最小毒性的TTR转录增强子。我们将利用我们实验室建立的细胞生物学、蛋白质组学和微阵列技术来表征它们的作用机制，并最终测定它们抑制APP23转基因AD小鼠模型所显示的神经病理和行为表现的能力。

DESCRIPTION (provided by applicant): We have shown that over-expression of a wild type human transthyretin (TTR) gene can suppress the neuropathologic and behavioral phenotypes of a well-studied validated transgenic model of human Alzheimer's disease (AD). We have also shown that TTR interacts with A2 in vitro to inhibit oligomer and fibril formation and tissue culture cytotoxicity and in vivo with TTR-A2 complexes being co-immunoprecipitable from APP23 and some human AD brains with anti-sera to either protein. We have also noted that 70% of human cortical neurons from AD brains stain with an anti-TTR antibody (compared with 10% of age matched control brains) as do almost all the hippocampal and cortical neurons in the APP23 mouse model. TTR mRNA (by qtPCR) is 10 times higher in primary hippocampal neurons from APP23 mice than from controls and the ratio is even greater in hippocampal lysates. These findings coupled with the observation that the pathologic changes appear to be accelerated when the APP23 strain is crossed onto a mouse TTR knockout background suggest that TTR may be a normal physiologic neuronal defense mechanism in neurodegenerative disease of the AD type. In contrast the suppression of the features of AD by TTR over-expression may be viewed as pharmacologic. Our proposal is directed at finding small molecules that will enhance TTR transcription and protein production in cells of neuronal origin. To that end we have developed reporter constructs in which the human TTR promoter drives a Gaussia luciferase reporter gene which is readily detectable in a high throughput screening mode available at our institution. Pilot studies have shown that the assay is robust, reproducible and stable under a variety of conditions. We propose to use the assay to serially screen a group of available molecular libraries (Maybridge, LOPAC, MLPCN, Scripps Florida) to identify the 25 most potent, specific least toxic enhancers of TTR transcription in neuronally defined cells. We will characterize their mechanism of action using cell biologic, proteomic and microarray techniques established in our laboratory and ultimately assay their capacity to suppress the neuropathologic and behavioral manifestations displayed by the APP23 transgenic mouse model of AD. PUBLIC HEALTH RELEVANCE: We have been able to suppress the appearance of the characteristic plaques and the associated learning difficulties seen in a well-validated mouse model of Alzheimer's disease by genetically inserting many copies of an apparently beneficial gene into the AD mice. We now wish to find compounds that can be administered to the mice (and ultimately humans) that will increase the production of the beneficial protein in neurons thus rendering them resistant to the toxic effects of the AD protein.