NEURODEVELOPMENTAL FUNCTION OF HCFC1

HCFC1 的神经发育功能

• 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
• 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

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.

项目摘要 HCFC1的突变引起多个先天性异常综合征，其特征是先天性钴胺的误差 代谢，顽固性癫痫，智力障碍和运动功能障碍。尽管对HCFC1有含义 在这些神经系统障碍中，描述了大脑发育过程中HCFC1功能的机制 尚未完全阐明。 HCFC1编码已知调节的转录co因子蛋白 包括神经前体细胞（NPC）的各种祖细胞的细胞增殖。 NPC经历了快速 早期大脑发育过程中的扩展并分化为大脑中所有主要细胞类型（即神经元， 神经胶质）。为了开始在NPC扩展中阐明HCFC1的假定机制，我们创建了CO60等位基因 这引入了斑马鱼HCFC1A直系同源物中功能突变的丧失。通过免疫组织化学 标记和细胞计数，我们证明了CO60等位基因的杂合载体（CO60/+）增加了 NPC的扩散。接下来，我们使用CO60/+全脑匀浆的转录组学揭示了14倍 ASXL1表达的增加，一种转录因子对于细胞增殖和激活至关重要 信号。我们发现抑制Akt的上游激活剂PI3K恢复了ASXL1的co60/+突变体 依赖的NPC过度增殖。此外，我们的初步蛋白质印迹和光密度分析我们 突变体证实AKT的高磷酸化（THR308）。接下来，我们使用染色质免疫沉淀来确认 人类HCFC1与斑马鱼ASXL1启动子的直接结合。这些发现一起间接地链接了 首次具有Akt激活和NPC增殖的HCFC1A函数和ASXL1表达。还有什么 理解是源自HCFC1A突变体的孤立的NPC中的Akt信号传导水平，哪个AKT 下游信号分子（如MTOR）调节增殖。这项研究的完成将有助于确定 新的分子途径，将HCFC1下游调节脑发育和指定机制定为 它的失调如何导致神经发育障碍。在该奖项的F99阶段，我寻求 为了获得分子生物学技术的工作知识，包括蛋白质隔离，西方 免疫印迹，荧光激活细胞分选（FACS），流式细胞仪，高级成像和行为 神经科学。此外，我旨在完善自己的技能并获得授予技巧的专业发展， 手稿审查和准备，建立和维护我的科学网络，并确定论文 研究。