Characterization of miRNAs on neural development and plasticity

NMDAR-LTD通常与脊柱收缩和丢失有关。LTD期间突触强度和树突的变化可以维持较长时间。突触的长期结构和功能修饰是大脑中信息存储的基本细胞底物(Hofer，Mr ic-Flogel等人)。2009年，Fu，Yu等人。2012年)。它不仅是正常大脑发育和功能的中心，而且已经被证明在包括精神障碍在内的大脑疾病的病理生理学中发挥着重要作用。LTD的结构和功能可塑性的机制部分重叠。例如，它们都需要NMDA受体、钙调蛋白和肌动蛋白解聚，但AMPA受体内吞和蛋白磷酸酶1只参与突触抑制。然而，尽管对突触可塑性进行了深入的研究，但与LTD相关的脊柱重塑的分子机制，特别是脊髓变化的长期维持，仍然很大程度上是不清楚的。 在本综述期间，我们系统地研究了miRNAs在LTD中的作用以及与之相关的脊柱重塑。我们使用下一代深度测序来鉴定在LTD中差异表达的miRNAs，以及一个生物信息学工具(我们内部开发的)来揭示由它们丰富的基因和细胞过程。值得注意的是，我们发现LTD诱导导致了miRNA转录本的全球变化，对细胞过程具有潜在的多效性影响，其中许多过程调节突触的属性和功能。 为了评估LTD中miRNA表达变化的生理后果，我们利用海马片的电生理学和原代培养的活神经元的时移成像，测试了两个差异表达的miRNAs miR-191和miR-135对突触和脊髓可塑性的影响。我们发现，这两个miRNAs都是诱导LTD、持续脊柱收缩和延迟消除脊柱所必需的，但都不是诱导脊柱收缩所必需的。然而，miR-191和miR-135在LTD维持中的作用尚不清楚，因为当它们的表达被干扰时，LTD在细胞中不被诱导。未来需要对其他假定的LTD miRNAs进行研究，以确定miRNAs在LTD维持中的作用。 在研究miR-191和miR-135调节脊柱重塑的机制时，我们发现它们的靶基因原调蛋白2(miR-191)和复合蛋白-1和-2(miR-135)在脊柱可塑性中起中介作用。有趣的是，我们发现：1)脊椎长期收缩需要平移。考虑到较小的脊椎含有相对较少的AMPA受体和突触后密度蛋白，这是一个令人惊讶的发现；2)肌动蛋白解聚是以持续的、依赖蛋白质合成的方式调节的，以支持长期的脊柱可塑性。肌动蛋白解聚是一种众所周知的触发细胞重构的机制。然而，我们的研究表明，在脊柱重建的初始阶段，肌动蛋白细胞骨架需要不断地修饰，以维持细胞的形态变化；3)LTD诱导减少AMPA受体的胞吐。AMPA受体胞吐是LTP的主要机制，在LTP中不被认为是改变的。然而，我们的数据显示，AMPA受体胞吐功能被LTD诱导抑制，以支持持久的脊柱重塑。

NMDAR-LTD is usually associated with spine shrinkage and loss. The changes in synaptic strength and dendritic spines during LTD can be maintained for prolonged periods of time. Long-lasting structural and functional modification of synapses is an essential cellular substrate for information storage in the brain (Hofer, Mrsic-Flogel et al. 2009, Fu, Yu et al. 2012). Not only is it central to normal brain development and function, it has also been shown to play an important role in the pathophysiology of brain disorders, including psychiatric disorders. The mechanisms underlying the structural and functional plasticity in LTD overlap partially. For instance, they both require NMDA receptors, calcineurine and actin depolymerization, but AMPA receptor endocytosis and protein phosphatase 1 are only involved in synaptic depression. Despite intensive study of synaptic plasticity, however, the molecular mechanisms underlying spine remodeling associated with LTD, especially long-term maintenance of changes in spines, are still largely unclear. During the current review period, we have systematically investigated the role of miRNAs in LTD and spine remodeling associated with it. We used next-generation deep sequencing to identify miRNAs differentially expressed in LTD, along with a bioinformatics tool (which we developed in-house) to reveal genes and cellular processes enriched by them. Remarkably, we found that LTD induction leads to a global change in miRNA transcriptomes with potentially pleiotropic effects on cellular processes, many of which modulate the properties and functions of synapses. To evaluate the physiological consequence of miRNA expression changes in LTD, we tested the effects of two differentially expressed miRNAs, miR-191 and miR-135, on synaptic and spine plasticity using electrophysiology in hippocampal slices and time-lapse imaging of live neurons in primary cultures. We found that both miRNAs are necessary for LTD induction, persistence of spine shrinkage and delayed spine elimination, but neither is required for induction of spine shrinkage. The roles of miR-191 and miR-135 in LTD maintenance, however, are unclear as LTD is not induced in cells when their expression is perturbed. Future studies characterizing other putative LTD miRNAs are needed to determine the role of miRNAs in LTD maintenance. While investigating the mechanisms by which miR-191 and miR-135 regulate spine remodeling, we found that their target genes tropomodulin 2 (for miR-191) and complexin-1 and -2 (for miR-135) mediate their functions in spine plasticity. Intriguingly, we found that: 1) translation is required for long-lasting spine shrinkage. This is a surprising finding given that smaller spines contain relatively fewer AMPA receptors and postsynaptic density proteins; 2) actin depolymerization is regulated in a sustained, protein synthesis-dependent manner to support prolonged spine plasticity. Actin depolymerization is a well-known mechanism to trigger cell remodeling. Our study, however, shows that actin cytoskeleton needs to be continuously modified beyond the initial phase of spine restructuring to maintain the morphological change of cells; 3) AMPA receptor exocytosis is reduced by LTD induction. AMPA receptor exocytosis is a predominant mechanism for LTP, and is not thought to be altered in LTD. However, our data show that AMPA receptor exocytosis is suppressed by LTD induction to support long-lasting spine remodeling.