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，最终可能成为突触可塑性的基础。一种这样的适应性反应可以由微rna （miRNA）转录启动。mirna包含一个来自DNA的调控分子家族，但不翻译成蛋白质。它们通过抑制蛋白质编码mrna的翻译或增强其降解来调节其表达。由于单个miRNA可调节5-30个靶mRNA的表达，因此miRNA转录可影响广泛的细胞反应。microrna调节发育可塑性和细胞凋亡。它们还与微生物防御和肿瘤发生有关。在这里，我们将在小鼠小叶中鉴定mirna，其转录受持续光动力诱发的攀援纤维活性的调节。我们将鉴定其mRNA表达被鉴定的mirna抑制的基因。我们将验证小脑小叶中mirna子集的转录是由光动力学诱发的攀爬纤维活性调节的假设。这些mirna通过抑制其翻译或增强其降解来调节小脑中几种蛋白质编码mrna的表达。该研究有三个目标：首先，我们将小鼠暴露于长期（24小时）单向HOKS中。HOKS 24h后，我们将去除受刺激小鼠的絮凝体，提取RNA，并将两个絮凝体的样品并排放在miRNA微阵列上。我们将用杂交组织化学鉴定miRNA变化诱导的细胞类型。我们将使用“实时”RT-PCR对整个絮状RNA样本分析诱导mirna变化的时间过程。一旦确定了诱导miRNA变化的细胞起源，我们将使用激光捕获显微镜获得细胞特异性RNA样本。其次，我们将通过向小脑微注射特异性miRNA抑制剂将miRNA与细胞功能联系起来。微注射的miRNA抑制剂应该增加作为特定miRNA靶点的mrna的表达，而这些抑制剂是为这些特定miRNA设计的。mRNA增强将与基于微注射抑制剂的互补miRNA核苷酸基序的预测进行比较。第三，我们将描述一种蛋白14-3-3-8，它与其他蛋白的磷酸化有关，这些蛋白在对HOKS的反应中在小叶中受到差异调节。我们已经证明，在HOKS过程中，在PxA方向刺激的眼睛同侧小叶中14-3-3-8 mRNA的转录减少。我们的初步数据显示14-3-3-8受到差异表达的miRNA miR335的负调控。miR335的转录在HOKS期间在PxA方向刺激的眼睛同侧小叶中增加。因此，miR335可以解释观察到的hoks诱导的14-3-3-8表达抑制。我们将通过识别与它在小脑中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mirna对小脑细胞功能的抑制作用。提出的研究的最终目标是确定由单个mirna调节的蛋白质家族。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共济失调患者。

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.