Regulation of Cortical Progenitor Mitosis

本研究旨在阐明顶端极性复合体蛋白在皮质祖细胞有丝分裂中的新功能。 揭示了Pals1(蛋白)基因突变引起小头畸形的一种新的发病机制 与LIN7 1相关，也称为MPP5)。PALS1，最近才被认为是一个基因 负责人类的小头畸形，编码进化上保守的顶极的一个分量 很复杂。我们使用不同CRE驱动程序的多个Pals1遗传模型始终显示出压倒性优势 由于细胞活力受损而造成的皮质细胞损失。然而，细胞和分子缺陷背后的 在Pals1突变体中发现的大量细胞死亡仍不清楚。通过大量的时间推移成像，我们发现 Pals1的缺失导致有丝分裂进程异常延长，这与越来越多的证据一致 有丝分裂异常是小头畸形的重要原因。值得注意的是，有丝分裂细胞的静态分析 Pals1缺陷祖细胞的图像和时间推移成像显示内化细胞的出现 有丝分裂细胞内的核。这种不同寻常的细胞行为模仿了牙槽突起，这是一种细胞自相残杀，被 为了促进或抗肿瘤的目的，肿瘤细胞吞噬活的邻近细胞。目前还不清楚这是不是 异常细胞事件可能是其他疾病的致病因素，如小头畸形症。因此，鼠标 其皮质前体细胞具有齿突样突起的模型将为研究 小头畸形的发病机制。我们的初步研究表明，细胞内(CIC)结构 代表一个动态和移动的细胞实体，与延长的有丝分裂和异常高度相关 在细胞质分裂中。与肿瘤细胞一样，ROCK抑制完全消除CIC结构并恢复正常 有丝分裂的长度。此外，我们检测到P53靶标P21在Pals1突变体和 研究发现，p53的基因消除产生了显著的皮质大小挽救，以及大量的 减少CIC结构和细胞死亡。这些观察结果使我们假设Pals1的损失 诱导对核分裂缺陷负责的CIC病理，损害基因组内容物和皮质 通过Rho-ROCK和P53的异常激活使细胞存活。为了测试这一点，我们将确定 CIC结构的生物发生、维持和消除及其对有丝分裂和随后的影响 皮质细胞的基因组完整性和命运(Aim1)。接下来，我们将研究Pals1的删除/减少是如何引起的 Rho-Rock异常活化所致牙周病(AIM2)。最后，我们将描述P53激活对 Pals1突变祖细胞的CIC形成和Rho-Rock调节(Aim3)。目前的研究提供了一种 关于Pals1突变如何导致如此戏剧性的皮质表型的重要分子和细胞线索 完全没有大脑皮层和海马体。此外，我们的研究将首次 证实皮质祖细胞可以发生齿突样突起，这提供了一种新的致病机制 内生细胞自相残杀导致小头畸形。

This proposal is to delineate the novel function of apical polarity complex proteins in cortical progenitor mitosis and uncover a new pathogenic mechanism of microcephaly caused by genetic mutation of Pals1 (protein associated with Lin7 1, also known as MPP5). PALS1, which has been incriminated only recently as a gene responsible for microcephaly in humans, encodes a component of the evolutionarily conserved apical polarity complex. Our multiple Pals1 genetic models using different Cre drivers consistently demonstrate overwhelming cortical cell loss because of compromised cell viability. However, the cellular and molecular defects behind the massive cell death found in Pals1 mutants remain unknown. Through extensive time-lapse imaging, we found that Pals1 loss causes abnormally lengthened mitotic progression, consistent with accumulating evidence that anomalies of mitosis are a significant cause of microcephaly. Remarkably, analyses of mitotic cells in static images and time-lapse imaging of Pals1-deficient progenitors revealed the emergence of internalized cells with nuclei inside of mitotic cells. This unusual cellular behavior mimics entosis, which is cell cannibalism utilized by tumor cells to engulf live neighboring cells for pro- or anti-tumorigenic purposes. It is unknown whether this extraordinary cellular event can be pathogenic in other diseases such as microcephaly. Therefore, the mouse model with an entosis-like process in its cortical progenitors will provide important new insights into the pathogenic mechanisms of microcephaly. Our preliminary study demonstrated that cell-in-cell (CIC) structures represent a dynamic and mobile cellular entity that is highly associated with lengthened mitosis and abnormalities in cytokinesis. As in tumor cells, ROCK inhibition completely abrogates CIC structures and restores the normal length of mitosis. Furthermore, we detected a striking increase of the P53 target, P21, in the Pals1 mutants and found that genetic elimination of P53 produces a remarkable rescue of cortical size along with substantial reductions of CIC structures and cell death. These observations lead us to hypothesize that Pals1 loss induces CIC pathology responsible for mitotic defects that compromise genomic content and cortical cell viability through the abnormal activation of Rho-ROCK and p53. To test this, we will determine the biogenesis, maintenance, and elimination of CIC structures and how they impact mitosis and subsequent genomic integrity and fate of cortical cells (Aim1). Next, we will study how Pals1 deletion/reduction causes entosis through abnormal Rho-ROCK activation (Aim2). Finally, we will delineate the effect of P53 activation on CIC formation and Rho-ROCK regulation in Pals1 mutant progenitors (Aim3). The current study provides an important molecular and cellular clue as to how Pals1 mutation causes such a dramatic cortical phenotype as the complete absence of the cerebral cortex and hippocampus. Furthermore, for the first time, our study will establish that an entosis like process can occur in cortical progenitors, providing a novel pathogenic mechanism by which entotic cell cannibalism produces microcephaly.