Microcephaly in an RNAi mouse with reduced sulfation

硫酸化减少的 RNAi 小鼠中的小头畸形

• 批准号: 7976774
• 负责人: NANCY B SCHWARTZ
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-13 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976774
• 关键词: Address; Adult; Affect; Apoptosis; Biological Process; Brain; Cell Culture Techniques; Cell Death; Cell Proliferation; Cell Survival; Cells; DNA Damage; Development; Developmental Delay Disorders; Embryo; Enzymes; Epilepsy; Event; Foundations; Generations; Genetic; Genetic Recombination; Goals; Human; Inorganic Sulfates; Investigation; Lead; Measures; Mediating; Metabolism; Microcephaly; Modification; Mus; Neocortex; Neuronal Differentiation; Neurotransmitters; Oxidative Stress; PAPS synthetase; Pattern; Phenotype; Physiology; Production; Protein Isoforms; Proteoglycan; RNA Interference; Reactive Oxygen Species; Research; Research Personnel; Role; Signal Pathway; Solid; Steroids; Structure; System; Tetracyclines; Tissues; Transgenic Animals; Transgenic Mice; Unspecified or Sulfate Ion Sulfates; Xenobiotics; brain size; critical period; extracellular; in vivo; insight; knock-down; macromolecule; malformation; mouse model; nerve stem cell; nestin protein; neurodevelopment; neurogenesis; novel; overexpression; research study; sulfation

DESCRIPTION (Provided by Applicant): To study the role of cellular sulfation in embryonic brain development, the investigators have developed a novel inducible RNAi system to reduce the cellular levels of PAPS, the universal sulfate donor in the cell. They have generated transgenic mice which can be induced through Cre-mediated recombination to express an RNAi hairpin targeting one of two PAPS synthesizing enzymes, PAPSS1, which is abundantly expressed in brain during neurogenesis. Crossing the inducible PAPSS1 RNAi mice to Nestin-Cre mice result in expression of the RNAi hairpin in neural progenitor cells beginning at approximately E9.5. Mice from these matings survive to adulthood but have brains that are markedly smaller (microcephaly) than control littermates. Preliminary characterization of the PAPSS1 RNAi brain reveals reduced PAPS synthetase activity and a narrow window of apoptosis early in neurogenesis. The cause of the apoptosis event is unknown, though the investigators have evidence that it may be due to oxidative stress. The objective of this application is to begin to elucidate the mechanism by which cellular sulfation influences cell survival in early brain development. Towards this goal, the investigators will first characterize the PAPSS1 brain specific knock-down phenotype by examining total PAPSS activity, cell proliferation, cell death, and laminar patterning. Second, to explore the cause of cortical cell death in the PAPSS1 RNAi mouse and how it might lead to the observed reduction in brain size, they will analyze indicators of oxidative stress and formation of reactive oxygen species (ROS), as well as determine whether oxidative stress may be causing DNA damage in the PAPSS1 knock-down. Furthermore, changes in extracellular proteoglycan sulfation (a major use of the PAPS generated by PAPS synthetase) will be assessed to determine whether proteoglycan sulfation can affect ROS levels during neuronal differentiation. The proposed studies will provide a comprehensive understanding of the phenotypic consequences of reduced PAPSS1 expression, and a substantial foundation for subsequent investigation into the roles of sulfation in the developing brain. PROJECT NARRATIVE: Malformations arising during cortical development are increasingly recognized as important causes of epilepsy and developmental delay. This application makes use of mouse models to elucidate the function of sulfation during brain cortical development. Since smaller brain size is the main phenotype in the proposed transgenic animals, the investigators anticipate that these studies will lead to a better understanding of microcephaly in humans.

描述（由申请人提供）：为了研究细胞硫酸化在胚胎脑发育中的作用，研究者开发了一种新的诱导型RNAi系统，以降低细胞中通用硫酸盐供体PAPS的细胞水平。 他们已经产生了转基因小鼠，这些小鼠可以通过Cre介导的重组来诱导表达针对两种PAPS合成酶之一的RNAi发夹，PAPSS 1，其在神经发生期间在大脑中大量表达。 将诱导型PAPSS 1 RNAi小鼠与Nestin-Cre小鼠杂交导致RNAi发夹在神经祖细胞中的表达在大约E9.5开始。 这些交配的小鼠存活至成年，但其大脑明显小于对照组小鼠（小头畸形）。 PAPSS 1 RNAi脑的初步表征揭示了PAPS合成酶活性降低和神经发生早期细胞凋亡的狭窄窗口。 细胞凋亡事件的原因尚不清楚，尽管研究人员有证据表明这可能是由于氧化应激。 本申请的目的是开始阐明细胞硫酸化影响早期脑发育中细胞存活的机制。 为了实现这一目标，研究人员将首先通过检查总PAPSS活性、细胞增殖、细胞死亡和层状模式来表征PAPSS 1脑特异性敲低表型。 其次，为了探索PAPSS 1 RNAi小鼠皮质细胞死亡的原因以及它如何导致观察到的脑体积减小，他们将分析氧化应激和活性氧（ROS）形成的指标，并确定氧化应激是否可能导致PAPSS 1敲低中的DNA损伤。 此外，将评估细胞外蛋白聚糖硫酸化（PAPS合成酶产生的PAPS的主要用途）的变化，以确定蛋白聚糖硫酸化是否会影响神经元分化期间的ROS水平。 拟议的研究将提供PAPSS 1表达减少的表型后果的全面理解，并为后续研究硫酸化在发育中的大脑中的作用奠定了坚实的基础。 项目叙述：皮质发育过程中出现的畸形越来越被认为是癫痫和发育迟缓的重要原因。 本申请利用小鼠模型来阐明脑皮质发育过程中硫酸化的功能。 由于较小的大脑尺寸是拟议的转基因动物的主要表型，研究人员预计，这些研究将导致更好地了解人类的小头畸形。