Defective abscission and apoptosis of neural progenitor cells in a novel model of microcephaly

新型小头畸形模型中神经祖细胞的缺陷性脱落和凋亡

• 批准号: 9977259
• 负责人: Jessica Neville Little
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977259
• 关键词: Affect; Age; Apoptosis; Birth; Brain; Cell Line; Cell division; Cells; Cellular Morphology; Cerebral cortex; Cultured Cells; Cytokinesis; DNA Damage; Data; Defect; Development; Embryo; Epithelial; Epithelium; Event; Excision; Family member; Genes; Genetic; Germ-Line Mutation; Human; Image; Impairment; In Vitro; Individual; Intellectual functioning disability; Kinesin; Knowledge; Lead; Location; Mediating; Microcephaly; Mitosis; Modeling; Mus; Mutant Strains Mice; Mutation; Neurodevelopmental Disorder; Neurons; Organ; Organism; Pathogenesis; Phase; Phenotype; Prevention; Preventive measure; Preventive treatment; Process; Protein p53; Proteins; Public Health; Regulation; Research; Role; Shapes; Structure; TP53 gene; Techniques; Testing; Thick; Time; Tissues; Viral; apical membrane; body system; brain malformation; cell type; daughter cell; experimental study; in vivo; insight; mouse model; mutant; nerve stem cell; neuroepithelium; novel; postnatal; prevent; response; stem cell proliferation

When neural progenitor cells (NPCs) fail to divide correctly at the right time and location, neurodevelopmental disorders can occur, including gross abnormalities in the size and structure of the brain and more subtle defects in neuronal layering and connectivity. One result of impaired NPC division in the cerebral cortex is microcephaly. The majority of the genetic causes of microcephaly in humans are due to mutations that affect parameters of mitosis in NPCs, but recently defects in cytokinesis have been implicated as well. A novel model of microcephaly studied here, resulting from the loss of the kinesin-6 family member Kif20b, has defects specifically in abscission, the last step in the cytokinesis phase of NPC division. Kif20b protein is detected in the midbodies of NPCs, a structure that connects the two daughter cells' apical membranes until mediating abscission. In Kif02b-/- cortex as well as in Kif20b-/- dissociated NPCs, midbody abnormalities, signifying an abnormal abscission process, and apoptosis have been observed. A common mechanism in the pathogenesis of many genetic and viral causes of microcephaly is p53-dependent apoptosis. However, it is not known whether p53 is activated to cause apoptosis in response to defective abscission in any cell type. To test whether apoptosis in Kif20b-/- mice is p53 dependent, we created mice double mutant for Kif20b and p53. New preliminary data shows that the survival, apoptosis and decreased cortical thickness, at least at early ages, in Kif20b-/- mice are rescued by p53 co-deletion. However, the relationship between midbody defects and p53 activation in Kif20b-/- NPCs is unknown. Additionally, the importance of abscission regulation to produce a cortex of normal size and structure, apart from apoptosis prevention, is unclear. Correct midbody alignment is important for epithelial structure in other organisms, and midbody inheritance has been shown to influence cell fate, but the functional relevance of these observations for cortical development have not been demonstrated. The survival of double mutant mice for many months past birth provides an opportunity to examine the consequences of abnormal abscission for postnatal cortical development. My specific hypothesis is that p53 is activated in response to impaired abscission in Kif20b-/- NPCs to cause apoptosis, and that this response is important to prevent abnormalities in cortical size and structure. To test this hypothesis, I propose to study Kif20b-/- p53-/- mice in vivo and Kif20b-/- NPCs cultured in vitro with and without inhibition of p53. With these techniques and using fixed and live imaging experiments, I will complete the following aims: Aim 1) Determine the relationship between abscission defects and p53-dependent apoptosis in Kif20b-/- mice. Aim 2) Investigate the consequence of Kif20b loss for NPCs when p53 is co-deleted. The proposed research is significant in that it will bridge the gap in our knowledge of how apoptosis is regulated in response to abscission defects and whether this is mediated by p53. Additionally, the research will provide insight on normal abscission in NPCs and the consequences for its perturbation in vivo and in vitro.

当神经前体细胞（NPC）不能在正确的时间和位置正确分裂时，神经发育 可能会发生紊乱，包括大脑大小和结构的严重异常，以及更微妙的异常。 神经元分层和连接的缺陷。NPC在大脑皮层分裂受损的一个结果是， 小头畸形人类小头畸形的大多数遗传原因是由于基因突变， 参数的有丝分裂的NPC，但最近在胞质分裂的缺陷也有牵连。 这里研究的一种新的小头畸形模型，是由于驱动蛋白-6家族成员Kif 20 b的丢失造成的， 特别是在鼻咽癌分裂的胞质分裂阶段的最后一步，即分裂中的缺陷。Kif 20 b蛋白是 在NPC的中间体中检测到，这是一种连接两个子细胞顶膜的结构， 调解和解在Kif 02 b-/-皮质以及Kif 20 b-/-解离的NPC中， 表明异常的凋亡过程，并且已经观察到凋亡。中的一种常见机制， 小头畸形的许多遗传和病毒原因的发病机制是p53依赖性细胞凋亡。但不 已知p53是否被激活以响应任何细胞类型中的缺陷性凋亡而引起凋亡。测试 无论Kif 20 b-/-小鼠中的细胞凋亡是否是p53依赖性的，我们创建了Kif 20 b和p53的小鼠双突变体。新 初步数据显示，至少在早期年龄， 通过p53共缺失拯救Kif 20 b-/-小鼠。然而，中间体缺陷和p53之间的关系 Kif 20 b-/-NPC中的激活是未知的。此外，生产的重要性， 正常大小和结构的皮质，除了细胞凋亡的预防，是不清楚的。正确的身体中部对齐是 对其他生物体上皮结构也很重要，而且已显示中间体遗传影响细胞 命运，但这些观察结果对皮质发育的功能相关性尚未得到证实。 双突变小鼠在出生后存活数月提供了一个机会， 出生后皮质发育异常的后果。我的假设是p53 在Kif 20 b-/-NPC中，Kif 20 b-/- NPCs被激活以响应受损的凋亡而引起凋亡，并且这 反应对于防止皮质大小和结构的异常是重要的。为了验证这个假设，我 建议在体内研究Kif 20 b-/- p53-/-小鼠和在体外培养的Kif 20 b-/-NPC， 第53页。通过这些技术，并使用固定和实时成像实验，我将完成以下目标： 目的1）探讨Kif 20 b-/-小鼠凋亡缺陷与p53依赖性细胞凋亡的关系。 目的2）研究p53基因共缺失对NPC Kif 20 b基因缺失的影响。 这项研究的意义在于它将填补我们对细胞凋亡的认识上的差距。 调节响应于解离缺陷，以及这是否由p53介导。此外，研究将 提供了对NPC中的正常分解及其在体内和体外扰动的后果的见解。