Deciphering the role of microRNAs in neuronal maturation using live imaging

除其他因素外，大脑的最佳功能取决于神经元的兴奋性和抑制性活动之间的微妙平衡。GABA是一种参与细胞交流的神经递质或化学信使，是大脑中抑制活动的主要参与者。事实上，正常情况下，当GABA与神经元结合时，该神经元的活动受到抑制。然而，有趣的是，对于未成熟的神经元，这种神经递质发挥相反的作用，具有兴奋功能。在神经元成熟过程中，GABA的作用逐渐由兴奋性转变为抑制性。这个关键时期被认为在发育中起着重要的作用，它的时间可以改变一个人的神经发育轨迹，确实对大脑功能有持久的影响。虽然导致这种变化的蛋白质是众所周知的，但调控它们的机制却鲜为人知。众所周知，rna是必不可少的细胞构建模块，携带包括蛋白质表达在内的所有功能的关键信息。在20世纪90年代初，研究人员发现了一类新的小RNA分子，称为microRNA。从那以后，对这一课题的研究表明，这些分子对神经元的存活和功能至关重要。microrna通过精细调节细胞中的蛋白质表达而起作用。在人类中，已经鉴定出超过2000种不同的microrna，每种microrna控制着几十种具有不同功能的蛋白质。我们的假设是，其中一些microrna在神经元中GABA作用的兴奋性到抑制性转换的微调中是重要的。为了研究microrna在这一过程中的作用，我们需要能够在改变细胞中单个microrna的数量的同时，监测GABA对活神经元活性的影响。在这个研究项目中，我们将使用小鼠神经元原代培养来测试单个细胞的发育成熟作为单个microrna数量的函数。未成熟神经元通常会被GABA激活，而成熟神经元则不会。我们把这个项目分成三个不同的目标。首先，我们将确定哪些microrna在神经元成熟中发挥重要作用。其次，我们将评估调节这些microrna的数量对神经元活动的影响。最后，我们将开发一种新的方法，结合最先进的RNA测序方法和多种光学显微镜技术，将单个神经元的分子含量与其活动联系起来。这个程序将允许更好地理解microrna在神经元成熟微调中的作用。这种多学科的努力将需要在单细胞分辨率上研究microrna作用的创新策略的发展，并将最终阐明多种情况下的神经发育障碍。

The optimal functioning of the brain depends, among other things, on a delicate balance between the excitatory and inhibitory activity of neurons. GABA, a neurotransmitter or chemical messenger involved in cellular communication, is the major player of inhibitory activity in the brain. Indeed, normally, when GABA binds to a neuron, the activity of this neuron is inhibited. However, it is interesting to note that for immature neurons, this neurotransmitter rather plays an opposite role and has an excitatory function. During the neuronal maturation, the action of GABA will gradually change from excitatory to inhibitory. This critical period is believed to play an important role in development and its timing can modify the neurodevelopmental trajectory of an individual and indeed have lasting effects on the brain functions. Whereas the proteins responsible for this change are well known, the mechanisms that regulate them are much less understood. RNAs are known to be essential cellular building blocks that carry crucial information for all functions, including protein expression. In the early 1990's, researchers discovered a new class of small RNA molecules, called microRNA. Research on this subject has since shown that these molecules are essential for neuron survival and function. MicroRNAs operate by finely regulating the protein expression in cells. In humans, over two thousand different microRNAs have been identified, each controlling a few dozen proteins with diverse functions. Our hypothesis is that some of these microRNAs are important in the fine-tuning of the excitatory to inhibitory switch of GABA action in neurons. In order to study the role of microRNAs in this process, we need to be able to monitor the effects of GABA on the activity of living neurons while varying the amount of individual microRNAs in the cells. For this research program, we will use mouse neuronal primary culture to test the developmental maturation of single cells as a function of the number of individual microRNAs. Immature neurons are usually excited by the action of GABA while mature neurons are not. We have separated this project into three distinct objectives. Firstly, we will identify which microRNAs play a significant role in neuronal maturation. Secondly, we will evaluate the effect of modulating the number of these microRNAs on neuronal activity. Finally, we will develop a new methodology, combining state-of-the art RNA sequencing approach and multiple optical microscopy techniques, to relate the molecular content of single neurons to their activity. This program will allow a better understanding of the role of microRNAs in the fine-tuning of neuronal maturation. This multidisciplinary effort will require the development of innovative strategies to study the action of microRNAs at the single cell resolution and will ultimately shed light on neurodevelopmental disorders in a multitude of contexts.