A screening assay for chemicals that affect the differentiation of human neural cells.

影响人类神经细胞分化的化学物质的筛选试验。

• 批准号: 9016112
• 负责人: Michael Rountree
• 金额: $ 22.5万
• 依托单位: NZUMBE EPIGENETICS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9016112
• 关键词: Acute; Adult; Affect; Antibodies; Astrocytes; Autistic Disorder; Biological Assay; Brain; Cell Differentiation Induction; Cell Differentiation process; Cell Line; Cells; Chemical Exposure; Chemicals; Complex; DNA Methylation; Development; Disease; Environment; Environmental Exposure; Epigenetic Process; Exposure to; Fluorescence; Fluorescent Antibody Technique; Funding; Gene Expression; Glial Fibrillary Acidic Protein; Growth Factor; Health; Histone Deacetylation; Human; In Vitro; Institutes; Label; Lead; Life; Measures; Mission; Mus; Neonatal; Neuroglia; Neuronal Differentiation; Neurons; Oligodendroglia; Oregon; Outcome; Phase; Ploidies; Population; Positioning Attribute; Process; Reader; Relative (related person); Risk; Schizophrenia; Staging; Staining method; Stains; Stem cells; Testing; Time; TimeLine; Trichostatin A; Valproic Acid; Work; base; bisphenol A; cell type; commercialization; disorder risk; early life exposure; epigenome; epigenomics; high throughput screening; in vivo; induced pluripotent stem cell; inhibitor/antagonist; interest; methylazoxymethanol; nerve stem cell; nervous system disorder; nestin protein; neural circuit; neurodevelopment; neuron development; neurotoxicology; novel; programs; public health relevance; relating to nervous system; response; screening; toxicant

 DESCRIPTION (provided by applicant): Human brain development represents a highly vulnerable time to chemical exposures because immature cells are actively undergoing differentiation into more complex cell types (e.g., neurons, astrocytes, oligodendroglia) to form neural circuits. A major reason for this vulnerability is that neuronal differentiation requires critical time-dependent changes to the epigenome (i.e., neuroepigenetics), and environmental exposures pose acute risks to the epigenomes of actively differentiating cells. Significantly, abnormal brain develop from epigenomic perturbation is believed to be an underlying cause of several neurological disorders including autism and schizophrenia. Despite this risk, however, the identity of chemicals present in our environment that affect human neuronal differentiation and do so by perturbing developing epigenomes is lagging. Nzumbe Inc. is responding to RFA-ES-15-005 entitled "Novel Assays for Screening the Effects of Chemical Toxicants on Cell Differentiation" with an application to develop a screening assay to identify chemicals that interfere with the process of neural differentiation. Our focus will be on chemicals that perturb epigenetic mechanisms because of their potential to stably and aberrantly affect brain development. We will use markers of neuronal and glial cell differentiation to identify effects from chemical exposures. Two specific aims are proposed. The first aim will use human neuroprogenitor/neural stem cells (hNPCs) derived from human iPS cells to establish time points of interest as the hNPCs differentiate into cell subtypes recapitulating normal human brain development. This work will be performed with hNPCs undergoing proliferation and differentiation in 96-well plates to facilitate automated screening. Fluorescent-labeled antibodies will allow for automated screening to distinguish the different cell subtypes that arise at each time point tested and their relative proportions in the total cell population. The work proposed in aim 2 will test the effects of five chemicals with known and suspected epigenetic activities for their ability to perturb normal differentiation, as described in aim 1. These chemicals include two well- characterized epigenetic inhibitors, 5-deoxyazacytdine and trichostatin A, which inhibit DNA methylation and histone deacetylation, respectively, as initial controls. Next we will test two chemicals on the tox-21 list, valproic acid and Bisphenol A, which can perturb epigenomes and alter brain development, and a third chemical, methylazoxymethanol, which has clear effects on neuronal development and is both a metabolite and precursor of several chemicals on the tox-21 list. The proposed work will identify discrete time points and cell-specific markers that can be used to screen for aberrant changes in neuronal differentiation following exposures to chemicals with epigenetic activities. This work will lead to a well-defined assay that can be commercialized in a phase II funded program.

 描述（由申请人提供）：人类大脑发育是一个非常容易受到化学暴露影响的时期，因为未成熟的细胞正在积极分化成更复杂的细胞类型（例如神经元、星形胶质细胞、少突胶质细胞）以形成神经回路。这种脆弱性的主要原因是神经元分化需要表观基因组（即神经表观遗传学）发生关键的时间依赖性变化，而环境暴露对活跃分化细胞的表观基因组构成严重风险。值得注意的是，表观基因组扰动导致的大脑发育异常被认为是包括自闭症和精神分裂症在内的多种神经系统疾病的根本原因。然而，尽管存在这种风险，但对我们环境中存在的影响人类神经元分化并通过扰乱发育中的表观基因组来实现这一作用的化学物质的识别仍然滞后。 Nzumbe Inc. 正在响应题为“筛选化学毒物对细胞分化影响的新颖测定法”的 RFA-ES-15-005，并申请开发筛选测定法来识别干扰神经分化过程的化学物质。我们的重点将是扰乱表观遗传机制的化学物质，因为它们有可能稳定和异常地影响大脑发育。我们将使用神经元和神经胶质细胞分化的标记来识别化学暴露的影响。提出了两个具体目标。第一个目标是使用源自人类 iPS 细胞的人类神经祖细胞/神经干细胞 (hNPC) 来建立感兴趣的时间点，因为 hNPC 会分化成重现正常人类大脑发育的细胞亚型。这项工作将利用在 96 孔板中进行增殖和分化的 hNPC 进行，以促进自动筛选。荧光标记抗体 将允许自动筛选以区分在每个测试时间点出现的不同细胞亚型及其在总细胞群中的相对比例。中提出的工作 目标 2 将测试五种具有已知和可疑表观遗传活性的化学物质干扰正常分化的能力，如目标 1 中所述。这些化学物质包括两种 充分表征的表观遗传抑制剂 5-脱氧氮杂胞苷和曲古抑菌素 A 作为初始对照，分别抑制 DNA 甲基化和组蛋白脱乙酰化。接下来我们将测试两个 tox-21 清单上的化学物质丙戊酸和双酚 A 可以扰乱表观基因组并改变大脑发育，而第三种化学物质甲基偶氮甲醇对神经元发育有明显影响，并且是 tox-21 清单上几种化学物质的代谢物和前体。拟议的工作将确定离散时间点和细胞特异性标记物，这些标记物可以 用于筛选暴露于具有表观遗传活性的化学物质后神经元分化的异常变化。这项工作将产生一种明确的检测方法，可以在第二阶段资助的项目中商业化。