High-Throughput Phenotypic Assay for Compounds That Influence Mitochondrial Health Using iPSC-Derived Human Neurons.
High-Throughput Phenotypic Assay for Compounds That Influence Mitochondrial Health Using iPSC-Derived Human Neurons.
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DOI：
10.1177/24725552211000671
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发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Davis RL
中科院分区：
文献类型：
--
作者：
MacMullen C;Davis RL
迫切需要开发高通量测定以鉴定为患有神经退行性疾病的个体提供治疗的化合物。大多数脑疾病，包括神经退行性疾病，共有线粒体功能障碍的共同神经病理学，其可导致神经元凋亡、活性氧（ROS）的过度产生和这些疾病的其他细胞神经病理学特征。具有在TetOn启动子控制下的神经生成素-2转录因子的稳定基因组插入的人iPSC可以在暴露于多西环素的3天内分化成兴奋性人神经元（i3神经元）。这些神经元已被用于开发和验证的线粒体动力学和功能的参数，使用两种化合物，已知促进小鼠神经元，4-羟基查耳酮和2，4-二羟基查耳酮的线粒体伸长的活细胞测定。该试验涉及将神经元接种在384孔微量滴定板中，用已知或未知物质处理它们，然后使用慢病毒载体捕获神经元线粒体的形态信息以表达神经元靶向荧光报告基因。与对照相比，暴露于这两种化合物24小时的i3神经元培养物表现出显著降低的圆形度和显著增加的长度，这两个形态学参数与线粒体健康增加相关。该测定是快速的，在一周的i3神经元培养后或一个月后（如果神经元与星形胶质细胞共培养）获得结果。该活细胞线粒体表型测定可用于高通量筛选或作为通过其他高通量筛选活动获得的化合物的正交测定。
There is a critical need to develop high throughput assays to identify compounds that offer therapy for individuals suffering from neurodegenerative diseases. Most brain disorders, including neurodegenerative diseases, share the common neuropathology of mitochondria dysfunction, which can lead to apoptosis of neurons, over-production of reactive oxygen species (ROS), and other cellular neuropathologies characteristic of these diseases. Human iPSCs with a stable genomic insertion of the neurogenin-2 transcription factor under the control of the TetOn promoter can be differentiated into excitatory human neurons (i3Neurons) within 3 days of exposure to doxycycline. These neurons have been used to develop and validate a live cell assay for parameters of mitochondrial dynamics and function using two compounds known to promote mitochondrial elongation in mouse neurons, 4-hydroxychalcone and 2,4-dihyrdroxychalcone. The assay involves plating the neurons in 384 well microtiter plates, treating them with known or unknown substances, and then capturing morphological information for the neuronal mitochondria using a lentivirus vector to express a mitochondrial-targeted fluorescence reporter. The i3Neuron cultures exposed to these two compounds for 24hr exhibit significantly decreased circularity and significantly increased length compared to controls, two morphological parameters correlated with increased mitochondrial health. The assay is rapid, with results obtained after a one week-long i3Neuron culture or one month if neurons are co-cultured with astrocytes. This live cell, mitochondrial phenotypic assay can be used for high-throughput screening or as an orthogonal assay for compounds obtained via other high throughput screening campaigns.
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DOI：
10.1042/bj20081386
发表时间：
2009-01-01
期刊：
The Biochemical journal
影响因子：
--
作者：
Murphy MP
通讯作者：
Murphy MP
影响因子：
13.6
作者：
Varkuti, Boglarka H.;Kepiro, Miklos;Davis, Ronald L.
通讯作者：
Davis, Ronald L.
DOI：
10.1186/1747-5341-4-2
发表时间：
2009-01-15
期刊：
Philosophy, ethics, and humanities in medicine : PEHM
影响因子：
--
作者：
Shanks N;Greek R;Greek J
通讯作者：
Greek J
影响因子：
5.1
作者：
Hoffmann, D.;Schott, J. W.;Schambach, A.
通讯作者：
Schambach, A.
DOI：
10.1124/jpet.112.192138
发表时间：
2012-09-01
影响因子：
3.5
作者：
Johri, Ashu;Beal, M. Flint
通讯作者：
Beal, M. Flint