MicroRNA101 and the Termination of Early Phase Neural Development

MicroRNA101 与早期神经发育的终止

• 批准号: 8823230
• 负责人: Darwin K BERG
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8823230
• 关键词: AMPA Receptors; Accounting; Acute; Address; Adult; Amyloid; Automobile Driving; Biological Neural Networks; Brain; Calcium; Chloride Ion; Chlorides; Complex; Development; Epilepsy; Event; Failure; Frequencies; Future; Goals; Image; Individual; Intervention; Investigation; Life; Mediating; Messenger RNA; MicroRNAs; N-Methyl-D-Aspartate Receptors; Nervous system structure; Neuronal Differentiation; Neurons; Outcome; Pathway interactions; Phase; Play; Population; Porifera; Process; Proteins; Regulation; Regulatory Pathway; Reporting; Role; Schedule; Shapes; Signal Transduction; Site; Slice; Synapses; Synaptic plasticity; System; Testing; Therapeutic; Time; Work; cohort; gamma-Aminobutyric Acid; in vivo; inhibitor/antagonist; insight; interest; nervous system development; nervous system disorder; neurodevelopment; neuronal growth; patch clamp; postnatal; prevent; public health relevance; regenerative; synaptogenesis; transmission process

DESCRIPTION (provided by applicant): The developing nervous system undergoes a major transition in early postnatal life when it shifts from a period of exuberant synapse formation to a subsequent period of synaptic pruning and network consolidation. Synaptic numbers approach a maximum during this time, the rules governing synaptic plasticity change, AMPA and NMDA receptor compositions are altered, and GABAergic signaling shifts from being depolarizing/excitatory to hyperpolarizing/inhibitory, in addition to other changes. Mechanisms driving this profound transition are poorly understood at best. Interesting candidates for doing so are microRNAs (miRs) because they can target many different mRNAs at the same time for blockade or destruction, thereby producing a "regulatory hub" to coordinate complex changes across large systems. Our preliminary evidence indicates that miR-101 is an attractive candidate for executing this transition. It is abundant, increases substantially at the relevant time, and remains high in the adult. Using antagonists to block miR-101 function in vivo reveals significant aberrations: substantial increases are seen in spontaneous synchronized activity across neuronal populations and is accompanied by increases in synaptic number and excitatory synaptic input to neurons. Blocking miR-101 also appears to delay maturation of GABAergic signaling, enabling it to be depolarizing at later times. Importantly, our preliminary results using target site blockers to protect specific mRNAs suggest that miR-101 achieves its effects by acting on multiple targets. One is likely to be NKCC1, the chloride transporter responsible for GABA being depolarizing. But other miR-101 targets appear to be important as well, suggesting that terminating the depolarizing phase of GABAergic signaling is not by itself sufficient to account for all the major changes comprising the developmental transition. We will test the role of miR-101 in mediating the transition in nervous system development by using antagonists, sponges, and target site blockers to prevent its action in vivo while assessing the consequences for synaptic activity and network function. Calcium fluors in acute slices will report the frequency and extent of spontaneous coordinated activity across neuronal populations, as well as total activity and numbers of participating neurons. Patch-clamp recording will reveal type and amount of synaptic activity while immunostaining will quantify synapses. By selecting individual miR-101 targets for protection, it will be possible to dissect th contributions of different pathways to the transition and to evaluate the consequences of defective regulation. This will provide new insight into mechanisms driving the transition, reveal the role of miR-101 in particular, and likely have biomedical relevance by revealing the vulnerability of the system to individual regulatory pathways being compromised, producing, for example, greater propensity for epileptic seizure-like events.

描述（由申请人提供）：发育中的神经系统在产后早期经历了重大转变，从突触形成旺盛时期转变为突触形成期。 随后的突触修剪和网络巩固时期。在此期间，突触数量接近最大值，控制突触可塑性变化的规则、AMPA 和 NMDA 受体组成发生改变，GABA 信号从去极化/兴奋性转变为超极化/抑制性，此外还有其他变化。人们对推动这一深刻转变的机制知之甚少。这样做的有趣候选人 是 microRNA (miR)，因为它们可以同时针对许多不同的 mRNA 进行封锁或破坏，从而产生一个“调节中心”来协调大型系统中的复杂变化。我们的初步证据表明 miR-101 是执行这一转变的一个有吸引力的候选者。它很丰富，在相关时间大幅增加，并且在成人中保持较高水平。在体内使用拮抗剂阻断 miR-101 功能揭示了显着的异常：神经元群体的自发同步活动显着增加，并伴随着突触数量和神经元兴奋性突触输入的增加。阻断 miR-101 似乎还会延迟 GABA 信号的成熟，使其能够在以后去极化。重要的是，我们使用靶点阻断剂保护特定 mRNA 的初步结果表明 miR-101 通过作用于多个靶点来实现其效果。其中一个可能是 NKCC1，它是负责 GABA 去极化的氯离子转运蛋白。但其他 miR-101 靶点似乎也很重要，这表明终止 GABA 信号转导的去极化阶段本身不足以解释发育转变中的所有主要变化。我们将通过使用拮抗剂、海绵和靶位点阻断剂来测试 miR-101 在介导神经系统发育转变中的作用，以防止其在体内发挥作用，同时评估对突触活动和网络功能的影响。急性切片中的钙荧光将报告神经元群体自发协调活动的频率和程度，以及参与神经元的总活动和数量。膜片钳记录将揭示突触活动的类型和数量，而免疫染色将量化突触。通过选择单个 miR-101 目标进行保护，将有可能剖析不同途径对转变的贡献并评估有缺陷的调节的后果。这将为推动这一转变的机制提供新的见解，特别是揭示 miR-101 的作用，并且可能具有生物医学相关性，因为揭示了系统对个体调节途径受到损害的脆弱性，从而产生例如癫痫发作样事件的更大倾向。