Vestibulo-cerebellar contribution to spatial adaptation

前庭小脑对空间适应的贡献

• 批准号: 7934470
• 负责人: NEAL H BARMACK
• 金额: $ 32.4万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-26 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934470
• 关键词: Accounting; Apoptosis; Categories; Cell physiology; Cells; Cerebellum; Code; Complex; DNA; Data; Diagnostic; Eye; Family; Fiber; Genes; Genetic Transcription; Goals; Histocytochemistry; Ipsilateral; Lasers; Learning; Link; Messenger RNA; MicroRNAs; Microinjections; Microscopy; Molecular Target; Mus; Neurons; Nucleotides; Patients; Phosphorylation; Protein Family; Protein Kinase C; Proteins; Purkinje Cells; RNA; Repression; Research; Reverse Transcriptase Polymerase Chain Reaction; Running; Sampling; Serine; Side; Signal Transduction; Spinocerebellar Ataxias; Synaptic plasticity; System; Techniques; Testing; Therapeutic Intervention; Time; Translating; Translations; base; cell type; design; developmental plasticity; experience; inhibitor/antagonist; mRNA Expression; microbial; motor learning; protein expression; public health relevance; response; tumorigenesis

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.

描述（由申请人提供）：持续的传入信号引起适应性细胞反应，这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小 RNA (miRNA) 转录启动。 miRNA 包含一大类源自 DNA 的调节分子，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蛋白质编码 mRNA 的表达。由于单个 miRNA 调节 5-30 个目标 mRNA 的表达，因此 miRNA 转录可以影响多种细胞反应。 MicroRNA 调节发育可塑性和细胞凋亡的各个方面。它们还涉及微生物防御和肿瘤发生。在这里，我们将鉴定小鼠绒球中的 miRNA，其转录受到持续光动诱发的攀爬纤维活性的调节。我们将鉴定其 mRNA 表达被已鉴定的 miRNA 抑制的基因。我们将检验这样的假设：小脑小叶中 miRNA 子集的转录受到光动诱发的攀爬纤维活动的调节。这些 miRNA 通过抑制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。拟议的研究有三个目标：首先，我们将小鼠长期（24 小时）单向 HOKS 暴露。 HOKS 24 小时后，我们将去除受刺激小鼠的絮状物，提取 RNA 并在 miRNA 微阵列上并排运行两个絮状物的样本。我们将通过杂交组织化学鉴定诱导 miRNA 变化的细胞类型。我们将使用“实时”RT-PCR 对整个絮状 RNA 样品分析 miRNA 诱导变化的时间过程。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来获取细胞特异性的 RNA 样本。 其次，我们将通过将特定的 miRNA 抑制剂显微注射到小脑中，将 miRNA 与细胞功能联系起来。显微注射的 miRNA 抑制剂应增加 mRNA 的表达，这些 mRNA 是抑制剂设计的特定 miRNA 的靶标。 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，它对其他蛋白质的磷酸化进行表征，这些蛋白质在絮球中响应 HOKS 受到差异调节。我们已经表明，在 HOKS 期间，在 PxA 方向刺激的眼睛同侧的小球中 14-3-3-8 mRNA 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 方向刺激的眼睛同侧小球中，miR335 的转录增加。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 miRNA 对小脑细胞功能的抑制作用。 本研究的最终目标是鉴定受单个 miRNA 调控的蛋白质家族。它还将确定有希望的特定分子靶标，用于对各类脊髓小脑共济失调患者进行诊断和治疗干预。 公共健康相关性：拟议的研究将阐明 RNA 的一个子集如何促进小脑的运动学习。它将展示细胞功能所需的蛋白质如何根据经验发生变化。它可以确定对各类脊髓小脑共济失调患者进行诊断和治疗干预的特定分子靶点。