NEURODEVELOPMENTAL FUNCTION OF HCFC1

项目摘要 HCFC 1突变导致以钴胺素先天性缺陷为特征的多发性先天性异常综合征 代谢、顽固性癫痫、智力残疾和运动功能障碍。尽管对氟氯烃1 在这些神经损伤中，描述HCFC 1在大脑发育过程中的功能的机制 尚未完全阐明。HCFC 1编码一种转录辅因子蛋白，已知其调节 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 在早期脑发育过程中扩增并分化成脑中所有主要的细胞类型（即神经元， 神经胶质）。为了开始阐明HCFC 1在NPC扩增中的假定机制，我们创建了Co 60等位基因 其在斑马鱼HCFC 1a直系同源物中引入功能缺失突变。通过免疫组化 标记和细胞计数，我们证明了Co 60等位基因（Co 60/+）的杂合子携带者增加， NPC的扩散。接下来，我们使用Co 60/+全脑匀浆的转录组学来揭示14倍的 asxl 1的表达增加，asxl 1是一种对细胞增殖和AKT活化至关重要的转录因子 信号我们发现，在Co 60/+突变体中，抑制AKT的上游激活剂PI 3 K， 依赖性NPC过度增殖。此外，初步的蛋白质印迹和密度测定分析，我们的 突变体证实了AKT（Thr 308）的过度磷酸化。我们接下来使用染色质免疫沉淀来证实 人HCFC 1与斑马鱼asxl 1启动子的直接结合。总之，这些发现间接地联系到 首次发现hcfc 1a功能和asxl 1表达与AKT激活和NPC增殖有关。还剩哪些尚待 理解的是来自hcfc 1a突变体的分离的NPC中AKT信号传导的水平， 下游信号分子，如mTOR，调节增殖。完成这项研究将有助于确定 新的分子途径，调节HCFC 1下游的大脑发育， 它的失调是如何导致神经发育障碍的在此奖项的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、高级成像和行为分析。 神经科学此外，我的目标是提高我的技能，并获得专业发展， 手稿审查和准备，建立和维护我的科学网络，并完成我的论文 research.

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.