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: 10478894
• 负责人: Edgar Orlando Correa-Colón
• 金额: $ 4.68万
• 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/10478894
• 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.

项目总结 这项提议的总体目标是确定发生在神经元和 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 合成、包装和释放神经递质必须与突触后能力相协调 神经元呈现正确的神经递质受体。揭示了分子机制 协调这些过程可能具有生物医学意义，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。致信地址 这种知识差距，线虫代表了一个理想的模型来研究这些机制，因为它已知 连接体、强大的遗传学和单细胞分辨率分析。利用这些工具，在进化上 保守的转录因子UNC-30/PITX1-3已被证明控制神经元通讯 在神经索GABA能运动神经元(MNS)和体壁肌肉之间通过直接激活 GABA生物合成基因(如UNC-47/VGAT、UNC-25/GAD)的表达。我们得到的数据表明 由GABA MNS产生的一种分泌型突触组织者UNC-30或MADD-4S的遗传缺失导致 突触后区域(肌肉)上聚集的GABA受体(GABAR)显著减少。而且，我们的 初步数据显示，缺乏UNC-30基因活性的动物表现出MADD-4S表达减少 GABA MNS。因此，我们假设UNC-30控制着建立和维持 通过直接激活MADD-4S和GABA生物合成基因来实现功能性突触。要评估这一点 假设，在目标1中，我将使用MADD-4S特异性报告程序来确定UNC-30是否直接调节 MADD-4S的表达。我还将进行跨物种救援实验，以确定北卡罗来纳大学的S 在调节MADD-4S表达中的作用在系统发育中是保守的。在《目标2》中，我将利用 生长素诱导的降解系统在整个发育和发育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。而且，我们初步的 数据表明，UNC-30也是防止GABA MN采用替代神经元身份所必需的， 提示双重作用：MADD-4S和GABA生物合成基因的激活者，以及替代基因的抑制者 身份基因。在目标3中，我将检验这样一个假设，即不同的监管因素决定了北卡罗来纳大学30岁以下S的双重身份 角色。这一建议意义重大，因为人类PITX和GABA生物合成基因的突变与 神经退行性疾病。此外，它还解决了神经元发育领域中的一个长期存在的问题 和疾病：神经元如何建立和维持功能性突触。这项工作在技术上是创新的 它使用强大的遗传方法和实施新的方法来研究体内的转铁蛋白功能。这 该项目将提供一个极好的培训机会，以支持我成为一名多产、 独立研究人员。