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

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 可能会发生疾病，包括大脑大小和结构的严重异常，以及更细微的 神经元分层和连通性缺陷。大脑皮层鼻咽癌分裂受损的一个结果是 小头畸形。人类小头畸形症的大多数遗传原因是由于影响 NPC的有丝分裂参数，但最近也涉及到细胞质分裂的缺陷。 在这里研究的一种新的小头畸形模型，是由于Kinesin-6家族成员Kif20b的丢失导致的，已经 特别是在鼻咽癌分裂的胞质分裂阶段的最后一步--脱落中存在缺陷。Kif20b蛋白是 在神经母细胞的中体中检测到，这种结构连接两个子细胞的顶膜，直到 调停离任。在Kif02b-/-皮质以及Kif20b-/-分离的NPC中，中体异常， 表明脱落过程异常，并观察到细胞凋亡。一种常见的机制在 小头畸形症的许多遗传和病毒原因的发病机制是P53依赖的细胞凋亡。然而，它并不是 已知P53是否被激活以引起任何细胞类型中有缺陷的脱落而导致的细胞凋亡。为了测试 Kif20b-/-小鼠的细胞凋亡是否依赖于P53，我们创造了Kif20b和P53的双突变小鼠。新的 初步数据显示，至少在早期，小鼠的存活、细胞凋亡和皮质厚度减少 Kif20b-/-小鼠通过p53共缺失而获救。然而，中体缺陷与P53的关系 Kif20b-/-NPC中的激活尚不清楚。此外，离职规则对于产生一个 正常大小和结构的皮质，除了防止细胞凋亡外，尚不清楚。正确的上半身对齐方式是 对其他生物的上皮结构很重要，中体遗传已被证明影响细胞 命运，但这些观察结果与大脑皮层发育的功能相关性尚未得到证实。 双突变小鼠在出生后几个月的存活提供了一个检查 异常脱落对出生后皮质发育的影响。我的具体假设是P53 Kif20b-/-NPC中的脱落受损而被激活，从而导致细胞凋亡，而这 反应对于防止皮质大小和结构的异常很重要。为了检验这一假设，我 建议研究Kif20b-/-P53-/-小鼠体内和体外培养的Kif20b-/-NPC在有和没有抑制的情况下 P53。使用这些技术，使用固定和现场成像实验，我将完成以下目标： 目的1)探讨Kif20b-/-小鼠脱落缺陷与P53依赖性细胞凋亡的关系。 目的2)研究P53基因缺失对鼻咽癌Kif20b基因缺失的影响。 这项拟议的研究意义重大，因为它将弥合我们对细胞凋亡如何理解的鸿沟。 对脱落缺陷的反应以及这是否由P53介导。此外，这项研究将 洞察神经母细胞的正常脱落及其在体内和体外的扰动后果。

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.