The Alzheimer's Therapeutics Screening Assay: a high-throughput drug-discovery platform utilizing neurons and microglia derived from human induced pluripotent stem cells and Kinetic Image Cytometry

阿尔茨海默病(AD)是导致痴呆症的最常见原因，随着年龄的增长，阿尔茨海默氏症(AD)的发病率不断增加，目前有550万人 在美国，没有治愈的方法。这个第一阶段的项目将启动阿尔茨海默氏症治疗的开发 筛选试验(ATSA)，它将利用来自人类诱导的神经元、小胶质细胞和星形胶质细胞 多能干细胞。迫切需要一种新的测试系统，因为以前FDA批准的治疗方法 治疗阿尔茨海默病的效果有限和暂时，而且自2003年以来没有批准过治疗阿尔茨海默病的新药；许多 从那时起失败的临床试验表明，目前的临床前模型不能充分预测AD。AS AD患者大脑中积累的淀粉样β蛋白(A)斑块，制药公司已经 穷尽地瞄准β-淀粉样蛋白级联通路进行药物发现。然而，虽然其中许多 药物减少了斑块，在临床试验中没有一种药物有效地减缓或防止记忆丧失。功能性 磁共振(FMRI)研究表明，遗忘性轻度认知障碍(AMCI)的受试者 AD的前驱症状，在斑块形成之前显示海马神经元过度活跃，提示 神经元钙调节失调可能是阿尔茨海默病的基础。与此相关的是，小胶质细胞，大脑的常驻免疫细胞， 在AD中被激活，并分泌谷氨酸和炎性细胞因子，可能改变神经元钙离子。 此外，阿尔茨海默病的最大遗传风险是载脂蛋白E(APOE4)的亚型4和蛋白水解物 APOE4片段导致细胞内钙调节失调，并对神经元和小胶质细胞产生毒性。为 在ATSA中，将发展HiPSC-神经元、-MG和-星形胶质细胞共培养和AD- 添加相关的应力(例如，APOE4片段或A寡聚体)，并监测细胞在 使用动态图像细胞术的钙(或电压)，这是一种量化钙的数字显微镜方法 逐个细胞的瞬时活动。细胞形态(突起、突触点状)及相关生物标志物 也将被量化。ATSA将是多功能的，第二阶段的目标将包括利用患者的IPSCs 代表两性的遗传性或散发性AD患者将被纳入，以进一步询问与AD相关的 小路。一旦开发完成，ATSA平台将能够每天测试数百种化合物 找出那些对AD相关压力有好处的人。ATSA将由Vala Sciences销售 Inc.与制药公司签约研究，开发治疗AD的新疗法，并用于 Vala研究人员的内部药物发现。

Alzheimer’s Disease (AD), the most common cause of dementia, increases with age, afflicts 5.5 million people in the US, and there is no cure. This phase I project will initiate development of the Alzheimer’s Therapeutics Screening Assay (ATSA), which will utilize neurons, microglia, and astrocytes, derived from human induced pluripotent stem cells. A new testing system is desperately needed as previously FDA-approved therapeutics for AD have limited and temporary effects, and no new drug has been approved for AD since 2003; the many failed clinical trials since then illustrate that current preclinical models are not sufficiently predictive for AD. As amyloid beta peptide (A) plaques accumulate in the brains of AD patients, pharmaceutical companies have exhaustively targeted the beta-amyloid cascade pathway for drug-discovery. However, while many of these agents reduced plaques, none effectively slowed or prevented memory loss in clinical trials. Functional magnetic resonance (fMRI) studies show that subjects with amnestic mild cognitive impairment (aMCI), a prodromal symptom of AD, display hyperactive hippocampal neurons prior to plaque formation, suggesting that dysregulation of neuronal calcium may underlie AD. Relatedly, microglia, the resident immune cells of the brain, are activated in AD and secrete glutamate and inflammatory cytokines that may alter neuronal calcium. Furthermore, the greatest genetic risk for AD is isoform 4 of apolipoprotein E (APOE4), and proteolytic fragments of APOE4 cause dysregulation of intracellular calcium and are toxic to neurons and microglia. For the ATSA, methods will be developed in which hiPSC-neurons, -MG, and -astrocytes are cocultured and AD- relevant stresses (e.g., APOE4 fragments or A oligomers) added, and the cells monitored for alterations in calcium (or voltage) using Kinetic Image Cytometry, a digital microscopy method that quantifies calcium transient activity on a cell-by-cell basis. Cell morphology (neurites, synaptic puncta) and relevant biomarkers will also be quantified. The ATSA will be versatile, and phase II goals will include utilizing iPSCs from patients with inherited or sporadic AD, representing both genders, will be incorporated to further interrogate AD-relevant pathways. Once developed, the ATSA platform will enable testing of hundreds of compounds per day to identify those with beneficial effects against AD related stresses. The ATSA will be marketed by Vala Sciences Inc for contract research to pharmaceutical companies developing novel therapeutics for AD, and used for internal drug-discovery by Vala researchers.