Vestibulo-cerebellar contribution to spatial adaptation

描述(由申请人提供)：持续的传入信号引起适应性细胞反应，最终可能是突触可塑性的基础。一种这样的适应性反应可以通过微RNA(MiRNA)转录来启动。MiRNAs由一大类调控分子组成，这些分子来自DNA，但不能翻译成蛋白质。它们通过抑制蛋白质编码的mRNAs的翻译或促进其降解来调节mRNAs的表达。由于单个miRNA调控5-30个靶mRNA的表达，因此miRNA转录可以影响广泛的细胞反应。MicroRNAs调控发育可塑性和细胞凋亡的各个方面。它们还与微生物防御和肿瘤发生有关。在这里，我们将识别小鼠小叶中的miRNAs，其转录受持续的光动力诱发的攀升纤维活动的调节。我们将确定其mRNA表达被已识别的miRNAs抑制的基因。我们将检验这一假设，即小脑小叶中miRNAs的子集的转录受光动力诱发的攀升纤维活动的调节。这些miRNAs通过抑制mRNAs的翻译或促进其降解来调节小脑中几个蛋白质编码mRNAs的表达。这项拟议的研究有三个目标：第一，我们将使小鼠暴露于长期(24小时)单向HOKS。HOKS后24小时，去除刺激小鼠的小叶，提取RNA，并在miRNA微阵列上并排运行。我们将通过杂交组织化学鉴定引起miRNA变化的细胞类型。我们将使用实时RT-PCR对整个絮状RNA样本分析miRNAs诱导变化的时间过程。一旦确定了诱导的miRNA变化的细胞来源，我们将使用激光捕获显微镜来获得细胞特定的RNA样本。其次，我们将通过将特定的miRNA抑制剂注射到小脑中，将miRNA与细胞功能联系起来。显微注射的miRNA抑制剂应该增加作为特定miRNAs靶标的mRNAs的表达，而这些miRNAs是为特定miRNAs设计的。MRNA增强将与基于显微注射抑制剂的互补miRNA核苷酸基序的预测进行比较。第三，我们将表征一种蛋白质14-3-3-8，它依赖于其他蛋白质的磷酸化，这些蛋白质在小叶中对HOKS有不同的调节。我们已经证明，在HOKS期间，在PXA方向刺激的眼睛同侧的小叶中，14-3-3-8mRNA的转录减少。我们的初步数据表明，14-3-3-8受到差异表达的miRNA miR335的负调控。在HOKS期间，在PXA方向刺激的眼同侧小叶中，miR335的转录增加。因此，miR335可以解释HOKS诱导的14-3-3-8表达的抑制。我们将通过鉴定小脑中与14-3-3-8相互作用的其他蛋白质来阐明14-3-3-8的功能。我们将使用这些技术来测试其他miRNAs对小脑细胞功能的抑制作用。这项拟议研究的最终目标是识别受单个miRNAs调控的蛋白质家族。它还将确定有希望的特定分子靶点，用于诊断和治疗各种类型的脊髓小脑性共济失调患者。 与公共健康相关：这项拟议的研究将阐明RNA的一个子集如何有助于小脑的运动学习。它将展示细胞功能所需的蛋白质是如何根据经验变化的。它可以为不同类型的脊髓小脑性共济失调患者的诊断和治疗干预确定特定的分子靶点。

DESCRIPTION (provided by applicant): Sustained afferent signals evoke adaptive cellular responses that may ultimately underlie synaptic plasticity. One such adaptive response could be initiated by micro-RNA (miRNA) transcription. miRNAs comprise a large family of regulatory molecules that are derived from DNA, but not translated into proteins. They modulate the expression of protein-coding mRNAs by repressing their translation or enhancing their degradation. Since a single miRNA modulates expression of 5-30 target mRNA's, miRNA transcription could influence a wide array of cellular responses. MicroRNAs regulate aspects of developmental plasticity and apoptosis. They are also implicated in microbial defense and oncogenesis. Here we will identify miRNAs in the mouse flocculus whose transcription is modulated by sustained optokinetically-evoked climbing fiber activity. We will identify the genes whose mRNA expression is repressed by the identified miRNAs. We will test the hypothesis that transcription of a subset of miRNAs in the cerebellar flocculus is modulated by optokinetically-evoked climbing fiber activity. These miRNAs regulate the expression of several protein-coding mRNAs in the cerebellum by repressing their translation or enhancing their degradation. The proposed research has three objectives: First, we will expose mice to long-term (24h) unidirectional HOKS. After 24h of HOKS we will remove the flocculi of stimulated mice, extract the RNA and run samples from the two flocculi side by side on a miRNA microarray. We will identify cell types in which changes of miRNA are induced with hybridization histochemistry. We will analyze the time course of induced changes in miRNAs using 'real time' RT-PCR for whole floccular RNA samples. Once the cellular origin of the induced miRNA change is determined, we will use laser-capture microscopy to obtain cell specific samples of RNA. Second, we will link miRNAs to cellular function by microinjecting specific miRNA inhibitors into the cerebellum. The microinjected miRNA inhibitors should increase the expression of the mRNAs that are targets of the specific miRNAs for which the inhibitors are designed. mRNA enhancement will be compared with predictions based on complementary miRNA nucleotide motifs of the microinjected inhibitor. Third, we will characterize a protein, 14-3-3-8, on the phosphorylation of other proteins that are differentially regulated in the flocculus in response to HOKS. We have already shown that during HOKS the transcription of 14-3-3-8 mRNA decreases in the flocculus ipsilateral to the eye stimulated in the PxA direction. Our preliminary data show that 14-3-3-8 is negatively regulated by a differentially expressed miRNA, miR335. The transcription of miR335 increases during HOKS in the flocculus ipsilateral to the eye stimulated in the PxA direction. Consequently, miR335 could account for the observed HOKS-induced repression of 14-3-3-8 expression. We will clarify the functions of 14-3-3-8 by identifying other proteins with which it interacts in the cerebellum. We will use these techniques to test the repressive effects of other miRNAs on cellular function in the cerebellum. The ultimate goal of the proposed research is tol identify families of proteins regulated by single miRNAs. It will also identify promising specific molecular targets for diagnostic and therapeutic intervention in patients with various categories of spinocerebellar ataxias. PUBLIC HEALTH RELEVANCE: The proposed research will clarify how a subset of RNA contributes to motor learning by the cerebellum. It will show how proteins necessary for cellular function are changed in accordance with experience. It may identify specific molecular targets for diagnostic and therapeutic intervention in patients with various categories of spinocerebellar ataxias.