Microcephaly in an RNAi mouse with reduced sulfation

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的诱导RNAi系统，以降低细胞中普遍的硫酸盐供体PAPS的细胞水平。他们已经培育出转基因小鼠，通过cre介导重组诱导其表达一种RNAi发夹，该发夹靶向两种PAPS合成酶之一的PAPSS1，该酶在神经发生时在大脑中大量表达。将可诱导的PAPSS1 RNAi小鼠与nesting - cre小鼠杂交，在大约E9.5时开始在神经祖细胞中表达RNAi发夹。这些交配的老鼠存活到成年，但它们的大脑明显比对照组小（小头畸形）。PAPSS1 RNAi脑的初步表征显示，PAPS合成酶活性降低，神经发生早期凋亡窗口窄。细胞凋亡事件的原因尚不清楚，尽管研究人员有证据表明它可能是由于氧化应激。本应用的目的是开始阐明细胞硫酸化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测总PAPSS活性、细胞增殖、细胞死亡和层流模式来表征PAPSS1脑特异性敲除表型。其次，为了探索PAPSS1 RNAi小鼠皮质细胞死亡的原因及其如何导致观察到的脑体积缩小，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在PAPSS1敲除中引起DNA损伤。此外，将评估细胞外蛋白多糖硫酸化（PAPS合成酶产生的PAPS的主要用途）的变化，以确定蛋白多糖硫酸化是否会影响神经元分化过程中的ROS水平。拟议的研究将提供对PAPSS1表达减少的表型后果的全面理解，并为后续研究硫酸盐在发育中的大脑中的作用奠定坚实的基础。

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.