NEURODEVELOPMENTAL FUNCTION OF HCFC1

HCFC1 的神经发育功能

基本信息

批准号：
10541351
负责人：
Victoria L Castro
金额：
$ 4.14万
依托单位：
UNIVERSITY OF TEXAS EL PASO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10541351
关键词：
AKT inhibition Achievement Adult Alleles Animals Award Behavior Behavioral Binding Brain Brain Diseases Cell Count Cell Differentiation process Cell Proliferation Cell Separation Cobalamin Data Densitometry Development Disease Epilepsy FRAP1 gene Fellowship Fishes Flow Cytometry Fluorescence-Activated Cell Sorting Future Gene Expression Genes Genetic Transcription Goals Hereditary Disease Human Image Impairment In Vitro Intellectual functioning disability Intractable Epilepsy Knowledge Label Larva Link Manuscripts Metabolism Molecular Molecular Biology Mus Mutation Neurodevelopmental Disorder Neuroglia Neurologic Neurons Neurosciences Neurosphere Nonsense Mutation Orthologous Gene Pathway interactions Pentylenetetrazole Phase Phenotype Phosphorylation Postdoctoral Fellow Predisposition Preparation Proliferating Proteins Proteomics Proto-Oncogene Proteins c-akt Reporter Research Role S phase Science Seizures Signal Transduction Signaling Molecule Sirolimus Syndrome Techniques Testing Time Training Training Programs Transgenic Organisms Western Blotting Zebrafish brain abnormalities cell type chromatin immunoprecipitation congenital anomaly critical period host cell factor C1 in vivo inhibitor knock-down loss of function mutation mTOR Inhibitor mTOR inhibition motor disorder mouse model mutant nerve stem cell neuron development novel prevent professor promoter single-cell RNA sequencing skills stem cells therapeutic target transcription factor transcriptomics upstream kinase

项目摘要

Project Summary Mutation of HCFC1 causes a multiple congenital anomaly syndrome characterized by inborn errors of cobalamin metabolism, intractable epilepsy, intellectual disability, and motor dysfunction. Despite an implication for HCFC1 in these neurological impairments, a mechanism describing the function of HCFC1 during brain development has not been completely elucidated. HCFC1 encodes for a transcriptional co-factor protein known to regulate cellular proliferation of various progenitor cells including neural precursor cells (NPC). NPCs undergo rapid expansion during early brain development and differentiate into all the major cell types in the brain (i.e. neurons, glia). To begin to elucidate a putative mechanism for HCFC1 in NPC expansion, we created the Co60 allele which introduces a loss of function mutation in the zebrafish hcfc1a ortholog. Through immunohistochemical labeling and cell counts, we demonstrated that heterozygous carriers of the Co60 allele (Co60/+) had increased proliferation of NPCs. We next used transcriptomics of Co60/+ whole brain homogenates to reveal a 14-fold increase in the expression of asxl1, a transcription factor critical for cell proliferation and activation of AKT signaling. We found that inhibition of PI3K, an upstream activator of AKT, in Co60/+ mutants restored asxl1 dependent NPC over proliferation. Moreover, preliminary western blotting and densitometry analysis of our mutants confirm hyperphosphorylation of AKT (Thr308). We next used chromatin immunoprecipitation to confirm a direct binding of human HCFC1 to the zebrafish asxl1 promoter. Together, these findings indirectly link for the first time hcfc1a function and asxl1 expression with AKT activation and NPC proliferation. What remains to be understood is the level of AKT signaling in isolated NPCs derived from hcfc1a mutants and which AKT downstream signaling molecules, like mTOR, regulate proliferation. Completion of this study will help to identify novel molecular pathways that regulate brain development downstream of HCFC1 and pinpoint mechanisms as to how its dysregulation contributes to neurodevelopmental disorders. During the F99 phase of this award, I seek to gain working knowledge of techniques in molecular biology that include protein isolation, western immunoblotting, fluorescence activated cell sorting (FACS), flow cytometry, advanced imaging, and behavioral neuroscience. Additionally, I aim to refine my skills and obtain professional development in grantsmanship, manuscript review and preparation, build and maintain my science network, and finalize my dissertation research.

项目概要 HCFC1突变导致多种先天性异常综合征，其特征是钴胺素先天性缺陷新陈代谢、顽固性癫痫、智力障碍和运动功能障碍。尽管对 HCFC1 有影响在这些神经损伤中，描述了 HCFC1 在大脑发育过程中的功能的机制尚未完全阐明。 HCFC1 编码一种已知可调节的转录辅因子蛋白包括神经前体细胞（NPC）在内的各种祖细胞的细胞增殖。 NPC 经历快速在早期大脑发育过程中扩张并分化为大脑中的所有主要细胞类型（即神经元、胶质细胞）。为了开始阐明 HCFC1 在 NPC 扩增中的假定机制，我们创建了 Co60 等位基因这在斑马鱼 hcfc1a 直向同源物中引入了功能丧失突变。通过免疫组化标记和细胞计数，我们证明 Co60 等位基因 (Co60/+) 的杂合携带者有所增加 NPC 的增殖。接下来，我们使用 Co60/+ 全脑匀浆的转录组学揭示了 14 倍 asxl1 的表达增加，asxl1 是一种对细胞增殖和 AKT 激活至关重要的转录因子发信号。我们发现，在 Co60/+ 突变体中抑制 PI3K（AKT 的上游激活剂）可以恢复 asxl1 依赖NPC过度增殖。此外，我们的初步蛋白质印迹和光密度分析突变体证实了 AKT (Thr308) 的过度磷酸化。接下来我们使用染色质免疫沉淀来确认人 HCFC1 与斑马鱼 asxl1 启动子的直接结合。总之，这些发现间接链接到首次 hcfc1a 功能和 asxl1 表达与 AKT 激活和 NPC 增殖有关。还剩下什么了解了源自 hcfc1a 突变体的分离 NPC 中 AKT 信号传导的水平，以及哪个 AKT 下游信号分子，如 mTOR，调节增殖。完成本研究将有助于确定调节 HCFC1 下游大脑发育的新分子途径，并查明机制其失调如何导致神经发育障碍。在这个奖项的 F99 阶段，我寻求获得分子生物学技术的工作知识，包括蛋白质分离、西方免疫印迹、荧光激活细胞分选 (FACS)、流式细胞术、先进成像和行为学神经科学。此外，我的目标是提高我的技能并获得资助方面的专业发展，稿件审查和准备，建立和维护我的科学网络，并完成我的论文研究。