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

描述(申请人提供)：我们已经证明，过表达野生型人类转甲状腺素(TTR)基因可以抑制经过充分研究验证的人类阿尔茨海默病(AD)转基因模型的神经病理和行为表型。我们还发现，TTR在体外与A2相互作用，抑制寡聚体和纤维的形成，并抑制组织培养的细胞毒性；在体内，TTR-A2复合体与APP23和一些人AD脑的抗血清共同免疫共沉淀。我们还注意到，在APP23小鼠模型中，70%的AD大脑中的人类皮质神经元用抗TTR抗体染色(相比之下，年龄匹配的对照组大脑中只有10%)，几乎所有的海马神经元和皮质神经元都是如此。APP23小鼠原代海马神经元的TTRmRNA(通过qtPCR检测)比对照组高10倍，在海马区裂解产物中的比例甚至更高。这些发现，再加上观察到，当APP23毒株与小鼠TTR基因敲除背景杂交时，病理变化似乎加速，表明TTR可能是AD类型神经退行性疾病的一种正常生理性神经元防御机制。相反，TTR过度表达对AD特征的抑制可能被视为药理作用。我们的建议旨在寻找小分子，以促进神经元起源的细胞中TTRs的转录和蛋白质生产。为此，我们开发了报告构建体，其中人类TTR启动子驱动高斯荧光素酶报告基因，该报告基因在我们机构可用的高通量筛选模式下很容易检测到。初步研究表明，该检测方法在各种条件下都是可靠的、可重复性的和稳定的。我们建议使用该方法来连续筛选一组可用的分子文库(Maybridge、Lopac、MLPCN、Scripps佛罗里达)，以确定在神经元定义的细胞中最有效、特异性最低的TTR转录增强子。我们将利用我们实验室建立的细胞生物学、蛋白质组和微阵列技术来表征它们的作用机制，并最终测试它们抑制APP23转基因阿尔茨海默病小鼠模型所显示的神经病理和行为表现的能力。 与公共卫生相关：我们已经能够通过将一种明显有益的基因的许多副本插入阿尔茨海默病小鼠中，来抑制在经过良好验证的阿尔茨海默病小鼠模型中出现的特征斑块的出现和相关的学习困难。我们现在希望找到一种化合物，可以用在小鼠(最终是人类)身上，增加神经元中有益蛋白的产生，从而使它们对AD蛋白的毒性作用具有抵抗力。

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.