The conserved transcription factor UNC-30/PITX1-3 coordinates synaptogenesis and cell identity in C. elegans motor neurons

保守转录因子 UNC-30/PITX1-3 协调线虫运动神经元的突触发生和细胞身份

• 批准号: 10314649
• 负责人: Edgar Orlando Correa-Colón
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314649
• 关键词: Address; Adoption; Adult; Alleles; Alzheimer' s Disease; Anabolism; Animals; Auxins; Binding Sites; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Consensus; Data; Defect; Development; Disease; Event; GABA Receptor; Gene Activation; Gene Expression; Genes; Genetic; Genetic Screening; Goals; Health; Human; Knowledge; Life; Link; Maintenance; Methods; Modeling; Molecular; Motor Neurons; Muscle; Mutate; Mutation; Nematoda; Nerve; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotransmitter Receptor; Neurotransmitters; Parkinson Disease; Phylogeny; Process; Protein Isoforms; Proteins; Regulation; Reporter; Research Personnel; Resolution; Role; Side; Synapses; System; Technical Expertise; Testing; Transgenic Organisms; Work; base; connectome; experimental study; gene repression; genetic approach; genome editing; in vivo; innovation; loss of function; molecular marker; mutant; neuron development; novel; postsynaptic; postsynaptic neurons; presynaptic neurons; prevent; receptor; synaptic function; synaptogenesis; tool; training opportunity; transcription factor

PROJECT SUMMARY The overall goal of this proposal is to determine the molecular mechanisms that take place in neurons and enable them to establish and maintain functional synapses. For this, the ability of presynaptic neurons to synthesize, package, and release a neurotransmitter must be coordinated with the ability of postsynaptic neurons to present the correct neurotransmitter receptors. Revealing the molecular mechanism that coordinates these processes could have biomedical implications since defects in the establishment and maintenance of functional synapses are linked to severe neurodegenerative disorders in humans. To address this knowledge gap, C. elegans represents an ideal model to study those mechanisms due to its known connectome, powerful genetics, and single-cell resolution analysis. Leveraging these tools, the evolutionarily conserved transcription factor (TF) UNC-30/PITX1-3 has been shown to control neuronal communication between nerve cord GABAergic (GABA) motor neurons (MNs) and body-wall muscle by directly activating the expression of GABA biosynthesis genes (e.g., unc-47/VGAT, unc-25/GAD). We obtained data that shows genetic loss of either unc-30 or madd-4S, a secreted synaptic organizer produced by GABA MNs, results in a dramatic reduction of GABA-Receptor (GABAR) clustering on the postsynaptic domain (muscle). Moreover, our preliminary data shows that animals lacking unc-30 gene activity show reduction of madd-4S expression in GABA MNs. Therefore, we hypothesize that UNC-30 controls both the establishment and maintenance of functional synapses by directly activating madd-4S and GABA biosynthesis genes. To evaluate this hypothesis, in Aim 1 I will use madd-4S specific reporters to determine whether UNC-30 directly regulates madd-4S expression. I will also conduct cross-species rescue experiments to determine whether UNC-30’s role in regulating madd-4S expression is conserved across phylogeny. In Aim 2 I will take advantage of the auxin-inducible degron system to selectively deplete UNC-30 at a particular stage throughout development and determine whether UNC-30 is required to maintain the functionality of synapses. Moreover, our preliminary data suggest UNC-30 is also required to prevent the adoption of alternative neuronal identities in GABA MNs, suggesting a dual role: activator of madd-4S and GABA biosynthesis genes, and repressor of alternative identity genes. In Aim 3, I will examine the hypothesis that distinct regulatory factors dictate UNC-30’s dual role. This proposal is significant because human mutations in PITX and GABA biosynthesis genes are linked to neurodegenerative disorders. Also, it addresses a long-standing question in the fields of neuron development and disease: how do neurons establish and maintain functional synapses. This work is technically innovative in its use of powerful genetic approaches and implementing novel methods to study TF function in vivo. This project will provide an excellent training opportunity to support my long-term goal of becoming a productive, independent researcher.

项目概要 该提案的总体目标是确定神经元和神经元中发生的分子机制 使它们能够建立和维持功能性突触。为此，突触前神经元的能力 神经递质的合成、包装和释放必须与突触后的能力相协调 神经元呈现正确的神经递质受体。揭示分子机制 协调这些过程可能会产生生物医学影响，因为建立和 功能性突触的维持与人类严重的神经退行性疾病有关。致地址 由于这一知识差距，秀丽隐杆线虫代表了研究这些机制的理想模型，因为它已知 连接组、强大的遗传学和单细胞分辨率分析。利用这些工具，进化 保守转录因子 (TF) UNC-30/PITX1-3 已被证明可以控制神经元通讯 通过直接激活神经索 GABA 能 (GABA) 运动神经元 (MN) 和体壁肌肉之间的 GABA 生物合成基因（例如 unc-47/VGAT、unc-25/GAD）的表达。我们获得的数据显示 unc-30 或 madd-4S（一种由 GABA MN 产生的分泌性突触组织者）的遗传缺失会导致 突触后域（肌肉）上的 GABA 受体（GABAR）聚集显着减少。此外，我们的 初步数据表明，缺乏 unc-30 基因活性的动物在体内表现出 madd-4S 表达的减少 GABA MN。因此，我们假设 UNC-30 控制着 通过直接激活 mad​​d-4S 和 GABA 生物合成基因来实现功能性突触。为了评价这一点 假设，在目标 1 中，我将使用 madd-4S 特定记者来确定 UNC-30 是否直接调节 madd-4S 表达。我还将进行跨物种救援实验，以确定 UNC-30 是否 调节 madd-4S 表达的作用在整个系统发育过程中都是保守的。在目标 2 中，我将利用 生长素诱导的降解决定子系统，可在整个开发和生产的特定阶段选择性地消耗 UNC-30 确定是否需要 UNC-30 来维持突触的功能。此外，我们初步 数据表明 UNC-30 还需要防止 GABA MN 中采用替代神经元身份， 表明具有双重作用：madd-4S 和 GABA 生物合成基因的激活剂，以及替代基因的阻抑剂 身份基因。在目标 3 中，我将检验以下假设：不同的监管因素决定了 UNC-30 的双重性 角色。这项提议意义重大，因为人类 PITX 和 GABA 生物合成基因的突变与 neurodegenerative disorders.此外，它还解决了神经元发育领域长期存在的问题 和疾病：神经元如何建立和维持功能性突触。这项工作在技术上具有创新性 它利用强大的遗传方法和实施新方法来研究体内 TF 功能。这 项目将提供一个极好的培训机会，以支持我成为一名富有成效、 独立研究员。