Identifying targets that lower APP as a therapeutic strategy for Alzheimer's disease

项目摘要 AD是最常见的神经退行性疾病，其病理特征在于细胞外聚集的 Aβ [淀粉样前体蛋白（APP）的裂解产物]和神经元内神经元缠结， 过度磷酸化的tau蛋白因此，AD属于神经退行性蛋白病的较大家族，其 包括疾病如帕金森病（PD）和多聚谷氨酰胺疾病如亨廷顿病（HD）， 脊髓小脑性共济失调1型（SCA 1）。AD药物开发面临的众多挑战之一是缺乏 能够改变疾病进程的新靶点。抗体是目前正在开发的药物的主要目标， 在临床试验中并没有产生任何实际效益。这项工作的目标是阐明可能的 调节APP水平的脂质途径的致病作用，可能有助于 AD.大脑对APP的稳态水平敏感的想法基于两条主要证据。 首先，对其他神经退行性蛋白病如PD和SCA1的研究已经确定， 是致病的。其次，人类遗传学研究表明， APP的额外副本（例如，21三体或APP基因座重复的人）发展为早发性AD，但罕见病例 部分三体21（PT 21），其中APP不包含在三体片段中，未显示AD神经病理学。 因为降低疾病驱动蛋白的水平在SCA1小鼠模型中显示出益处， 测试降低异常升高的APP水平是否可以预防Aβ生成并减轻疾病。到 Zoghbi实验室在人类中进行了平行的高通量shRNA筛选， 细胞和苍蝇。两种筛选的综合数据确定了一些基因，当它们被抑制时， 表达荧光标记APP的转基因细胞系中的APP长度水平和减轻的果蝇毒性 然后进一步测试这些候选物调节内源性APP的能力， 令人惊讶的是，我发现了两个基因，ACSL3和SLC27A1，它们在人类细胞中起作用。 同样的途径来调节脂肪酸代谢。为了确定是否有其他的基因落入这个 为了研究这条通路，我回到了最初的基于细胞的筛选数据，发现了十几个额外的基因， 参与脂质代谢，这在果蝇基因组中是不存在的，因此以前没有得到验证。 其中三个，ACOT8，ACADL和ACAD10已经在人类细胞中成功验证。整体 本提案的目的是在AD的人类细胞模型（Aim 1）和小鼠中测试这五种候选物 AD模型（目的2），以确定全长APP水平是否可以降低，APP加工是否改变，淀粉样蛋白是否可以改变， 病理学减轻，脂质代谢和APP之间的潜在机制联系被发现。的 APP调节因子的发现将为了解APP生物学开辟新的领域，将为治疗 这将为APP增加的AD病例提供一个靶点，并将为可能增加APP的基因提供丰富的来源。 对AD的脆弱性。

Project Summary AD, the most common neurodegenerative disease, is characterized pathologically by extracellular aggregation of Aβ [a cleavage product of amyloid precursor protein (APP)] and intraneuronal neurofibrillary tangles consisting of hyperphosphorylated tau. As such, AD belongs to the larger family of neurodegenerative proteopathies, which includes diseases such as Parkinson's (PD) and polyglutamine diseases such as Huntington's (HD) and spinocerebellar ataxia type 1 (SCA1). One of the many challenges facing drug development for AD is the lack of new targets capable of modifying the disease course. Ab is the main target for drugs currently in the pipeline, but it has not yielded any tangible benefits in clinical trials. The goal of this work is to elucidate the possible pathogenic role of a lipid pathway which regulates the levels of APP and may contribute to the development of AD. The idea that the brain is sensitive to the steady-state levels of APP is based on two major lines of evidence. First, studies of other neurodegenerative proteopathies such as PD and SCA1 have established that elevated levels of the disease-driving protein are pathogenic. Second, human genetic studies show that individuals carrying an extra copy of APP (e.g., those with Trisomy 21 or APP locus duplication) develop early-onset AD, but rare cases of partial trisomy 21(PT21), in which APP is not included in the trisomic segment, show no AD neuropathology. Because lowering the levels of a disease-driving protein have shown benefits in mouse models of SCA1, it is worth testing whether lowering abnormally elevated APP levels could prevent Aβ generation and mitigate disease. To identify novel regulators of APP, the Zoghbi lab performed parallel high-throughput shRNA screens in human cells and flies. Combined data from both screens identified a number of genes that, when inhibited, lowered full- length APP levels in a transgenic cell line expressing a fluorescently-labeled APP and mitigated toxicity in flies overexpressing human APP. These candidates were then further tested for their ability to regulate endogenous APP levels in human cells and surprisingly, I identified two genes, ACSL3 and SLC27A1, which function in the same pathway to regulate fatty acid metabolism. To determine if there were additional genes that fell into this pathway, I went back to the original primary cell-based screen data and uncovered a dozen additional genes involved in lipid metabolism which are absent in the fly genome and were therefore not previously validated. Three of these, ACOT8, ACADL, and ACAD10 have now been successfully validated in human cells. The overall objective of this proposal is to test these five candidates in a human cellular model of AD (Aim 1) and in mouse models of AD (Aim 2) to determine if full-length APP levels can be lowered, APP processing altered, amyloid pathology mitigated, and a potential mechanistic link between lipid metabolism and APP uncovered. The discovery of APP regulators will open new areas to understand APP biology, will provide potential therapeutic targets for cases of AD in which APP is increased, and will afford a rich source for genes that might increase vulnerability to AD.