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了自适应的细胞反应，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）转录引发。 miRNA包括源自DNA的大型调节分子，但不转化为蛋白质。它们通过抑制其翻译或增强其降解来调节蛋白质编码的mRNA的表达。由于单个miRNA调节了5-30个靶mRNA的表达，因此miRNA转录可能会影响各种细胞反应。 microRNA调节发育可塑性和凋亡的各个方面。它们也与微生物防御和肿瘤发生有关。在这里，我们将鉴定小鼠絮凝物中的miRNA，其转录受到持续的光动物诱发的攀爬纤维活性调节。我们将确定其mRNA表达被鉴定的miRNA抑制的基因。我们将检验以下假设：小脑絮凝中miRNA子集的转录是通过光动力诱发的攀岩纤维活性调节的。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。拟议的研究有三个目标：首先，我们将使小鼠长期（24h）单向HOKS暴露。 24小时后，我们将去除刺激的小鼠的絮凝物，将RNA提取并从两个絮凝物并排在miRNA微阵列上并排运行样品。我们将确定通过杂交组织化学诱导miRNA变化的细胞类型。我们将使用“实时” RT-PCR用于整个絮状RNA样品，分析MIRNA诱导变化的时间过程。一旦确定了诱导的miRNA变化的细胞来源，我们将使用激光捕获显微镜获得RNA的细胞特异性样品。 其次，我们将通过将特定的miRNA抑制剂微注射到小脑中，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，这是设计抑制剂的特定miRNA的靶标。 MRNA增强将与基于微注射抑制剂的互补miRNA核苷酸基序进行比较。 第三，我们将以14-3-3-8的蛋白质来表征其他蛋白质的磷酸化，这些蛋白质在响应于HOK的情况下在絮凝物中受到差异调节。我们已经表明，在HOK期间，在同侧的絮状肌中，14-3-3-8 mRNA的转录降低到PXA方向刺激的眼睛。我们的初步数据表明，14-3-3-8受差异表达的miRNA，miR335负调节。 MiR335的转录在同侧吞噬的HOK中增加到PXA方向刺激的眼睛。因此，MiR335可以解释观察到的HOKS诱导的14-3-3-8表达抑制作用。我们将通过鉴定其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。 拟议研究的最终目标是识别由单个miRNA调节的蛋白质家族。它还将确定有希望的特定分子靶标，用于患有各种脊椎动物共济失调患者的诊断和治疗干预。 公共卫生相关性：拟议的研究将阐明RNA的一部分如何促进小脑运动的运动。它将显示如何根据经验更改细胞功能所需的蛋白质。它可以确定特定的分子靶标，用于患有各种脊椎动物共济失调患者的诊断和治疗干预。