Characterization of a novel CUL9 signaling axis involved in the early phases of corticogenesis

项目总结 E3泛素连接酶家族中的剔除环连合酶(CRL)在控制基本发育过程中起着关键作用。 一些CRL被认为与早期神经发育过程的调节有关。然而，库林-9 (CUL9)，已被证明是一个独特的成员，具有难以捉摸的功能。CRL通常形成大型复合体， 泛化一组特定的底物。CUL9还没有被证明形成大的复合体，只有两个 已确定的底物。我们的数据提供了强有力的证据，证明CUL9可能调节关键的转录因子 神经干细胞在皮质分化的早期神经诱导阶段的命运。我已经证明了CUL9蛋白 在皮质谷氨酸能分化的神经诱导阶段，水平急剧上升。我怀疑 CUL9的高水平表达可能是增殖、自我更新的人类分化所必需的 具有多向分化潜能的神经前体细胞。零星CUL9基因敲除小鼠 出现大脑增大(巨脑)和表现出自闭症样行为(未发表的观察结果)。 巨脑通常是由于发育中的大脑皮层中的NPC池维护不当所致。 导致皮质醇生成异常。鼻咽癌转录因子水平的调节，如SOX2和PAX6，是 对全国人大决定自我更新或差异化至关重要。我的初步数据和目前的文献提供了 我的假设有一个强有力的前提，即CUL9及其底物需要适当的调节 从人多能干细胞(HPSCs)分化出自我更新和多能的hNPC。在目标1中，我 将确定CUL9在维持hNPC自我更新和多能性方面的作用。我的初步研究 证明CUL9缺失的hPSCs形成扩大的类胚体和较少的神经花环 扩张的管腔。将评估CUL9耗竭细胞形成自我更新的多潜能hNPC的能力。 此外，CUL9耗尽细胞形成的神经花环的数量、管腔大小和组织将 被量化。在目标2中，我将阐明CUL9调控hNPC分化的分子机制。 我的初步数据显示，CUL9水平耗尽的hNPC表现出SOX2和SOX2水平的升高 Pax6蛋白。我将通过体外泛素化试验来验证SOX2和/或PAX6是否为CUL9底物。那么， CUL9的目标残留物将被识别出来。我还演示了CUL9和后期促进 复合体/环体(APC/C)相互作用，以及APC/C底物接头蛋白CDC20耗尽 同源基因1(CDH1)导致CUL9蛋白水平增加。因此，我将描述CUL9-APC/C 并确定CUL9是否是APC/C-CDH1的底物。一种不能相互作用的CUL9突变结构 随着APC/C的鉴定及其表达对神经花环形成的影响，SOX2和SOX2 将测定Pax6蛋白水平和转录活性。这些研究的结果可能揭示出 正确分化hNPC所需的新的CUL9-APC/C信号轴；我的研究也可能 光转化为潜在的机制，当被破坏时，可能会导致巨脑症和自闭症谱系障碍。

PROJECT SUMMARY The cullin-ring ligase (CRL) family of E3 ubiquitin ligases play a critical role in the control of basic development, and some CRLs have been implicated in regulation of early neurodevelopmental processes. However, Cullin-9 (CUL9), has proven to be a unique member with an elusive function. CRLs generally form large complexes that ubiquitinate a set of specific substrates. CUL9 has not been shown to form large complexes and has only two identified substrates. Our data provide strong evidence that CUL9 may regulate transcription factors key for neural stem cell fate in the early neural induction phase of cortical differentiation. I have shown that CUL9 protein levels drastically increase during the neural induction phase of cortical glutamatergic differentiation. I suspect that high levels of CUL9 expression may be required for the differentiation of proliferative, self-renewing human neural precursor cells (hNPCs) with multipotent differentiation potential. CUL9 knockout mice sporadically develop enlarged brains (megalencephaly) and display autism-like behavior (unpublished observations). Megalencephaly is often the result of improper maintenance of the NPC pool in the developing cerebral cortex leading to abnormal corticogenesis. Regulation of NPC transcription factor levels, such as SOX2 and PAX6, is critical in an NPC’s decision to self-renew or differentiate. My preliminary data and the current literature provide a strong premise for my hypothesis that proper regulation of CUL9 and its substrates is required for the differentiation of self-renewing and multipotent hNPCs from human pluripotent stem cells (hPSCs). In Aim 1, I will define the role of CUL9 in maintaining self-renewal and multipotency of hNPCs. My preliminary studies demonstrate that CUL9 depleted hPSCs form enlarged embryoid bodies and fewer neural rosettes with an expanded lumen. The ability of CUL9 depleted cells to form self-renewing, multipotent hNPCs will be assessed. Additionally, the number, lumen size, and organization of neural rosettes formed from CUL9 depleted cells will be quantified. In Aim 2, I will clarify the molecular mechanism underlying CUL9 control of hNPC differentiation. My preliminary data demonstrate that hNPCs with depleted CUL9 levels express elevated levels of SOX2 and PAX6 protein. I will verify if SOX2 and/or PAX6 are CUL9 substrates by in vitro ubiquitination assays. Then, the residues targeted by CUL9 will be identified. I have also demonstrated that CUL9 and the Anaphase Promoting Complex/Cyclosome (APC/C) interact, and that depletion of the APC/C substrate adaptor protein CDC20 Homolog 1 (CDH1) results in increased CUL9 protein levels. Thus, I will characterize the CUL9-APC/C interaction and determine if CUL9 is a substrate of APC/C-CDH1. A CUL9 mutant construct unable to interact with the APC/C will be identified and the effect of its expression on neural rosette formation, and SOX2 and PAX6 protein levels and transcriptional activity will be determined. The results of these studies may reveal a novel CUL9-APC/C signaling axis required for the proper differentiation of hNPCs; my studies could also shed light into potential mechanisms that when disrupted can lead to megalencephaly and Autism Spectrum Disorder.