Microcephaly in an RNAi mouse with reduced sulfation

硫酸化减少的 RNAi 小鼠中的小头畸形

基本信息

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
项目类别：
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

项目摘要

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，研究人员开发了一种新型的诱导型RNAi系统，以降低PAP的细胞水平，即细胞中的通用硫酸盐供体。它们已经产生了转基因小鼠，该小鼠可以通过CRE介导的重组诱导，以表达针对两种PAPS合成酶之一的RNAi发夹，PAPSS1，该酶在神经发生过程中在大脑中大量表达。将诱导型PAPSS1 RNAi小鼠跨越到嵌套-CRE小鼠中导致RNAi发夹在神经祖细胞中的表达，始于大约E9.5。这些胶水的小鼠生存到成年，但大脑明显小（小头畸形），而不是对照同窝仔。 PAPSS1 RNAi脑的初步表征揭示了PAPS合成酶活性的降低和神经发生早期凋亡的狭窄窗口。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所致。该应用的目的是开始阐明细胞硫酸会影响早期大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PAPS活动，细胞增殖，细胞死亡和层状图案来表征PAPSS1脑特异性敲低表型。其次，为了探索PAPSS1 RNAi小鼠中皮质细胞死亡的原因，以及它如何导致观察到的脑大小的减小，他们将分析氧化应激和形成活性氧（ROS）（ROS）的指标，并确定氧化应激是否可能导致PAPSS 1敲击中的DNA损伤。此外，将评估细胞外蛋白聚糖硫化（PAPS合成酶产生的PAP的主要用途）的变化，以确定蛋白聚糖硫酸化在神经元分化过程中是否会影响ROS水平。拟议的研究将对PAPSS1表达降低的表型后果提供全面的理解，并为随后研究硫酸化在发育中的大脑中的作用提供了实质性的基础。项目叙述：皮质发育期间引起的畸形越来越被认为是癫痫和发育延迟的重要原因。该应用利用小鼠模型来阐明脑皮质发育过程中硫酸化的功能。由于较小的大脑大小是提出的转基因动物的主要表型，因此研究人员预计这些研究将使人们对人类的小头畸形有更好的了解。