Characterization of a novel CUL9 signaling axis involved in the early phases of corticogenesis
参与皮质生成早期阶段的新型 CUL9 信号轴的表征
基本信息
- 批准号:9760539
- 负责人:
- 金额:$ 2.97万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2019
- 资助国家:美国
- 起止时间:2019-04-01 至 2020-05-31
- 项目状态:已结题
- 来源:
- 关键词:3-DimensionalAdaptor Signaling ProteinAgingBehaviorBindingBiological AssayBrainCUL9 geneCell Cycle ProgressionCell Cycle RegulationCell Differentiation processCellsCerebral cortexComplexCoupledCullin ProteinsDataDevelopmentDifferentiation AntigensDiseaseES01FamilyGenetic TranscriptionGlutamatesGoalsHomologous GeneHumanImmunoblottingIn VitroKnockout MiceLeadLigaseLightLiteratureMaintenanceMediatingMegalencephalyModelingMolecularMutateMutationNeurodevelopmental DisorderNeuroectodermNeuronal DifferentiationNeuronsNuclear Pore ComplexPhasePhenotypePlayProcessPropertyProsencephalonProteinsRecombinant ProteinsRegulationResearchRoleSignal TransductionStem cellsTestingUbiquitinUbiquitinationanaphase-promoting complexautism spectrum disorderdevianthippocampal pyramidal neuronhuman pluripotent stem cellinsightknock-downmembermutantnerve stem cellneurogenesisnovelpluripotencyrelating to nervous systemself-renewalstem cell differentiationstem cell fatetranscription factorubiquitin ligaseubiquitin-protein ligase
项目摘要
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develop enlarged brains (megalencephaly) and display autism-like behavior (unpublished observations).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.
项目摘要
E3泛素连接酶的cullin环连接酶(CRL)家族在控制基本发育中起关键作用,
并且一些CRL涉及早期神经发育过程的调节。然而,Cullin-9
(CUL9),已被证明是具有难以捉摸的功能的独特成员。CRL通常形成大的复合体,
泛素化一组特定的底物。CUL9尚未显示形成大的复合物,并且仅具有两个
识别的底物。我们的数据提供了强有力的证据,CUL9可能调节转录因子的关键,
神经干细胞在皮层分化的早期神经诱导阶段的命运。我发现CUL9蛋白
在皮层神经元能分化的神经诱导阶段,其水平急剧增加。我怀疑
高水平的CUL9表达可能是增殖的、自我更新的人类肿瘤细胞分化所必需的。
具有多向分化潜能的神经前体细胞(hNPC)。CUL9基因敲除小鼠偶尔
发展出增大的大脑(巨脑畸形)并表现出类似自闭症的行为(未发表的观察结果)。
巨脑畸形通常是发育中的大脑皮层中NPC池维护不当的结果
导致皮质生成异常。NPC转录因子如SOX 2和PAX6水平的调节是
在NPC决定自我更新或差异化时至关重要。我的初步数据和现有文献提供了
这是我假设的一个强有力的前提,即CUL9及其底物的适当调节是必需的,
从人多能干细胞(hPSC)分化自我更新和多能hNPC。在目标1中,
将定义CUL9在维持hNPC的自我更新和多能性中的作用。我的初步研究
表明CUL9缺失的hPSC形成扩大的胚状体和较少的神经突起,
管腔扩张。将评估CUL9耗尽的细胞形成自我更新的多能hNPC的能力。
此外,由CUL9耗尽的细胞形成的神经突起的数量、管腔大小和组织将与CUL9耗尽的细胞形成的神经突起的数量、管腔大小和组织结构有关。
要量化。在目标2中,我将阐明CUL9控制hNPC分化的分子机制。
我的初步数据表明,具有耗尽的CUL9水平的hNPC表达升高的SOX 2水平,
PAX6蛋白。我将通过体外泛素化试验验证SOX 2和/或PAX6是否为CUL9底物。然后
将鉴定CUL9靶向的残基。我还证明了CUL9和后期促进因子
复合物/环体(APC/C)相互作用,以及APC/C底物衔接蛋白CDC 20耗竭
同源物1(CDH1)导致增加的CUL9蛋白水平。因此,我将描述CUL9-APC/C
相互作用并确定CUL9是否是APC/C-CDH1底物。一种不能与CUL9相互作用的突变体构建体
将鉴定APC/C的表达及其对神经玫瑰花结形成的影响,以及SOX 2和
将确定PAX 6蛋白水平和转录活性。这些研究的结果可能揭示了一个
新的CUL9-APC/C信号轴需要适当分化的hNPC;我的研究也可以揭示
光进入潜在的机制,当中断可能导致巨脑畸形和自闭症谱系障碍。
项目成果
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Natalya Anne Ortolano其他文献
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