MicroRNA101 and the Termination of Early Phase Neural Development

描述(申请人提供)：发育中的神经系统在出生后早期经历了一个重要的转变，当它从活跃的突触形成时期转变为 随后的突触修剪和网络巩固阶段。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发生改变，AMPA和NMDA受体组成发生变化，GABA能信号从去极化/兴奋性转变为超极化/抑制性，此外还有其他变化。推动这一深刻转变的机制充其量也是知之甚少。这样做的有趣候选者 是microRNAs(MiRs)，因为它们可以同时针对许多不同的mRNAs进行封锁或破坏，从而产生一个“监管中心”，以协调跨大系统的复杂变化。我们的初步证据表明，miR-101是执行这一过渡的有吸引力的候选者。它是丰富的，在相关的时间大幅增加，并保持在成年人的高水平。在体内使用拮抗剂阻断miR-101功能显示出显著的异常：神经元群体的自发同步活动大幅增加，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似乎也延迟了GABA能信号的成熟，使其能够在以后的时间去极化。重要的是，我们使用靶点阻滞剂保护特定mRNAs的初步结果表明，miR-101是通过作用于多个靶点而达到其效果的。一个很可能是NKCC1，负责GABA去极化的氯离子转运体。但其他miR-101靶点似乎也很重要，这表明终止GABA能信号的去极化阶段本身并不足以解释组成发育转变的所有主要变化。我们将通过使用拮抗剂、海绵和靶点阻滞剂来阻止miR-101在体内的作用，同时评估其对突触活动和网络功能的影响，以测试miR-101在神经系统发育过程中的中介作用。急性切片中的钙荧光将报告神经元群体之间自发协调活动的频率和程度，以及参与的神经元的总活动和数量。膜片钳记录将显示突触活动的类型和数量，而免疫染色将对突触进行量化。通过选择单独的MIR-101目标进行保护，将有可能剖析不同途径对过渡的贡献，并评估监管缺陷的后果。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，并可能通过揭示系统对个别调节途径被破坏的脆弱性而具有生物医学相关性，例如，产生更大的癫痫样事件的倾向。

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.