Scalable Assays for Morphological Analysis of Mammalian Neurons

哺乳动物神经元形态学分析的可扩展测定

• 批准号: 8192511
• 负责人: Bernardo L Sabatini
• 金额: $ 42.25万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8192511
• 关键词: Algorithms; Alzheimer' s Disease; Antibodies; Autistic Disorder; Axon; Benchmarking; Biochemical Pathway; Biological Assay; Cell Line; Cell physiology; Cells; Classification; Collaborations; Collection; Communities; Complex; Computer software; Data; Data Analyses; Data Set; Dendrites; Development; Disease; Ensure; Excitatory Synapse; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Image; Image Analysis; In Vitro; Inhibitory Synapse; Institutes; Label; Lead; Machine Learning; Measurement; Mediating; Mental Retardation; Methods; Mining; Mitochondria; Monitor; Morphology; Mus; Neurobiology; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuroglia; Neurons; Organelles; Parkinson Disease; Pathway Analysis; Pathway interactions; Phenotype; Predisposition; Procedures; Process; Protocols documentation; RNA Interference; Relative (related person); Screening procedure; Structure; Subfamily lentivirinae; Synapses; System; Techniques; Time; Transfection; Validation; Viral; Work; base; bioimaging; brain cell; density; design; fluorescence imaging; follow-up; genome-wide; high throughput screening; medical schools; neuropsychiatry; open source; prototype; scale up; small hairpin RNA; synaptogenesis; tissue culture; tool

DESCRIPTION (provided by applicant): Medium and high-throughput assays (i.e., screens) have generally not been applied to mammalian neurons because of the difficulties in culturing them in large numbers and because of the low efficiency with which the genetic makeup of neurons can be altered. Furthermore, because many aspects of neuronal function can only be assayed with electrophysiological assays, follow-up analysis and validation of screening hits is difficult. We propose to use automated imaging approaches to analyze synapse number and neuronal structure in vitro in a scalable format. We have implemented tissue culture and immunostaining approaches to monitor the number and types of synapses formed onto neurons in multi-well plates. We will couple this analysis with lentivirus mediated introduction of short-hairpin RNAs to induce RNA interference against genes expressed in neurons. This will be performed in concert with transcriptional analysis of neurons to determine the key changes in gene expression that correlate with structural and synaptic changes. The proposal represents a significant collaboration between several groups with expertise in functional analysis of neurons, automated analysis of images, viral mediated manipulation of gene expression, and whole-genome transcriptional analysis. We hope that our work will lead, for the first time, to a turn-key and robust method of analysis of neuron and synapse structure suitable for scalable, whole-genome analysis. Such a system will permit the unbiased and systematic analysis of pathways involved in neuropsychiatric diseases including neurodegenerative diseases such as Alzheimer's and Parkinson's as well as neurodevelopmental disorders such as mental retardation and autism. PUBLIC HEALTH RELEVANCE: Massively parallel analysis of cells in many conditions has allowed the discovery of key pathways that control cell function. Unfortunately, these techniques have not been applied to neurons due to difficulties in handling, manipulating, and analyzing large numbers of brain cells. We propose to develop imaging-based techniques to analyze neurons in dishes at a high throughput in order to find pathways that control their development and susceptibility to disease.

描述（由申请人提供）：中和高通量测定（即筛选）通常未应用于哺乳动物神经元，因为大量培养哺乳动物神经元存在困难，而且改变神经元基因组成的效率较低。此外，由于神经元功能的许多方面只能通过电生理学测定来测定，因此筛选命中的后续分析和验证很困难。我们建议使用自动成像方法以可扩展的格式在体外分析突触数量和神经元结构。我们已经实施了组织培养和免疫染色方法来监测多孔板中神经元上形成的突触的数量和类型。我们将将此分析与慢病毒介导的短发夹 RNA 引入相结合，以诱导 RNA 干扰神经元中表达的基因。这将与神经元转录分析一起进行，以确定与结构和突触变化相关的基因表达的关键变化。该提案代表了几个在神经元功能分析、图像自动分析、病毒介导的基因表达操纵和全基因组转录分析方面具有专业知识的小组之间的重要合作。我们希望我们的工作将首次带来一种适用于可扩展的全基因组分析的神经元和突触结构分析的交钥匙且稳健的方法。这样的系统将允许对涉及神经精神疾病的途径进行公正和系统的分析，包括阿尔茨海默氏症和帕金森氏症等神经退行性疾病以及智力低下和自闭症等神经发育障碍。 公共健康相关性：在许多条件下对细胞进行大规模并行分析可以发现控制细胞功能的关键途径。不幸的是，由于处理、操纵和分析大量脑细胞存在困难，这些技术尚未应用于神经元。我们建议开发基于成像的技术以高通量分析培养皿中的神经元，以找到控制其发育和疾病易感性的途径。