Generation of Neurons by Force-Mediated Epigenetic Mechanisms through Manipulation of Intrinsic Mechanoregulators

通过操纵内在机械调节器通过力介导的表观遗传机制产生神经元

• 批准号: 10664507
• 负责人: Chrystian Junqueira Alves
• 金额: $ 24.04万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664507
• 关键词: 3-Dimensional; AMOT gene; Acceleration; Actomyosin; Adhesions; Affect; Architecture; Cell Line; Cell Nucleus; Cells; Cellular Structures; Central Nervous System Diseases; Chromatin; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Consumption; Cytoskeleton; Data; Disease model; Doxycycline; Drug Screening; Electrophysiology (science); Elements; Epigenetic Process; Gene Expression; Generations; Genes; Genetic Epistasis; Genetic Transcription; Genome; Goals; Histones; Human; Investigation; Knock-out; Lamins; Link; Maps; Mechanics; Mediating; Mediator; Mentored Research Scientist Development Award; Mentors; Methods; Modeling; Morphology; Neuronal Differentiation; Neurons; Nuclear; Nuclear Envelope; Nuclear Structure; Pathway interactions; Phosphorylation; Proliferating; Property; Proteins; Publications; Regulation; Role; Signal Transduction; Specific qualifier value; Testing; Time; Training; United States National Institutes of Health; Writing; axon guidance; cell replacement therapy; drug discovery; epigenomics; gain of function; genomic locus; human embryonic stem cell; human pluripotent stem cell; improved; interest; knock-down; loss of function; mechanical force; mechanotransduction; nerve stem cell; nervous system disorder; neural; neurotransmitter release; novel; physical property; plexin; receptor; response; skills; small hairpin RNA; stem cells; transcriptomics; transmission process

PROJECT SUMMARY There is a great interest in deriving induced neuronal (iN) cells from human pluripotent stem cells (hPSCs) for central nervous system disease modeling and drug discovery. However, current differentiation methods are laborious and inefficient, thus, identifying new ways to improve neuronal differentiation is desirable. Strikingly, in my most recent publication I discovered that knockout of the axon guidance receptor Plexin-B2 in hNPCs reduced cell stiffness, increased Yes-Associated Protein 1 (YAP) phosphorylation, and accelerated neuronal differentiation. As a critical downstream mechanism, my preliminary data points to a pathway of Plexin-B2 altered mechanoregulation affecting lamin-mediated nuclear envelope architecture and localization of repressive histone marks at genomic loci containing neuron-specific genes. Based on these data, I will investigate an intrinsic ‘force-based’ method to generate iNs by manipulating Plexin-B2, Angiomotin (AMOT; a mechanosensitive component of Hippo pathway), and YAP in both 2D and 3D neuronal cultures and examine the link between Plexin-B2 and Hippo/YAP mechano-signaling in regulating lamin-mediated nuclear physical properties, force transmission to the nuclear envelope, and changes in chromatin accessibility during neural induction. To carry out this investigation, I propose the following aims: #1. Investigate the impact of Plexin-B2-altered cell mechanics on neuronal differentiation, by optimizing the differentiation method to generate iNs by both Plexin-B2 knockdown and knockout in multiple hPSC lines. I will test the robustness, stability, and functionality of iNs by evaluating transcriptional identity, neuronal lineage, maturation, and survival markers, electrophysiological properties, and neurotransmitter release. #2. Investigate a mechanochemical loop between Plexin-B2 and YAP during neuronal differentiation, by performing a comprehensive gain- and loss-of-function epistasis studies to map out the relationships between Plexin-B2, YAP, and AMOT during neural induction and evaluate their impact on enhancing neuronal differentiation, maturation, survival, electrophysiological functionality, and expression of major neuronal lineage markers. #3. Investigate lamin-mediated nuclear architecture and chromatin interactions in Plexin-YAP mechano-regulation of neuronal differentiation, by performing a comprehensive gain- and loss-of-function epistasis studies to validate lamin as the mediator of a Plexin-B2 and Hippo/YAP intrinsically-regulated mechano-axis to promote neuronal differentiation. I will then evaluate the role of Plexin-B2 / Hippo/YAP / lamins in releasing genomic loci harboring neuron-specific genes from repressive chromatin at nuclear periphery to interior regions with open chromatin. This K01 Award will provide me training in neuronal electrophysiology, transcriptomics and epigenomics, lab management and mentoring skills, as well as time and mentoring to develop and write my first NIH-R01. My long-term goal is to investigate new mechano-responsive elements (MREs) underlying cell fate choices to generate specific iNs for disease modelling and potential cell replacement therapy for neurological diseases.

项目摘要 对于从人多能干细胞（hPSC）衍生诱导的神经元（iN）细胞以用于治疗癌症存在极大的兴趣。 中枢神经系统疾病建模和药物发现。然而，当前的区分方法是 因此，需要鉴定改善神经元分化的新方法，这是费力且低效的。引人注目的是，在 我最近发表的文章发现，hNPC中轴突导向受体丛蛋白B2的敲除 降低细胞硬度、增加Yes相关蛋白1（雅普）磷酸化并加速神经元 分化作为一个关键的下游机制，我的初步数据指出，丛蛋白-B2的途径改变， 机械调节影响核纤层蛋白介导的核膜结构和抑制性组蛋白的定位 在含有神经元特异性基因的基因组位点标记。 基于这些数据，我将研究一种内在的“基于力”的方法，通过操纵 2D和3D中的丛蛋白-B2、血管动蛋白（AMOT; Hippo通路的机械敏感组分）和雅普 神经元培养，并检查丛蛋白-B2和Hippo/雅普机械信号传导在调节神经元的细胞凋亡中的联系。 核纤层蛋白介导的核物理性质，向核膜的力传递， 神经诱导过程中的染色质可及性。为了进行这项研究，我提出以下目标：#1。 研究丛蛋白-B2改变的细胞力学对神经元分化的影响，通过优化 在另一个实施方案中，使用分化方法在多个hPSC系中通过丛蛋白-B2敲低和敲除两者来产生iN。我会 通过评估转录身份，神经元谱系， 成熟和存活标志物、电生理学特性和神经递质释放。#2.探讨 在神经元分化过程中丛蛋白-B2和雅普之间的机械化学环， 全面的功能获得和丧失上位性研究，以绘制丛蛋白-B2，雅普， 和AMOT，并评估它们对增强神经元分化，成熟， 存活、电生理功能和主要神经元谱系标志物的表达。#3.探讨 核纤层蛋白介导的核结构和染色质相互作用在丛蛋白-YAP机械调控中的作用 神经元分化，通过进行全面的获得和丧失功能上位性研究，以验证 核纤层蛋白作为丛蛋白-B2和Hippo/雅普内在调节的机械轴的介导物，促进神经元 分化然后，我将评估丛状蛋白-B2/Hippo/雅普/核纤层蛋白在释放携带Hippo基因的基因组位点中的作用。 神经元特异性基因从核周边的抑制性染色质到具有开放染色质的内部区域。 这个K01奖将为我提供神经元电生理学，转录组学和表观基因组学方面的培训， 实验室管理和指导技能，以及时间和指导，以开发和编写我的第一个NIH-R01。 我的长期目标是研究细胞命运选择的新的机械反应元件（MRE）， 生成用于疾病建模和神经系统疾病的潜在细胞替代疗法的特定iN。