RNA Editing-Mediated Regulation of Serotonin 2C Receptor Density

RNA 编辑介导的血清素 2C 受体密度调节

基本信息

批准号：
9105183
负责人：
Hussain Jinnah
金额：
$ 2.82万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9105183
关键词：
Adenosine Affect Affinity Affinity Chromatography Amino Acids Animals Antidepressive Agents Antipsychotic Agents Anxiety Autopsy Behavioral Biological Models Biology Bipolar Disorder Brain Brain region Cell physiology Cells Clustered Regularly Interspaced Short Palindromic Repeats Coupling Diagnosis Disease Drug Targeting Dysthymic Disorder Epitopes Event Exhibits Failure to Thrive Functional disorder GTP-Binding Proteins Genes Growth Human Hyperphagia In Vitro Inosine Investigation Ligands Major Depressive Disorder Mass Spectrum Analysis Mediating Mental Depression Messenger RNA Metals Methods Molecular Profiling Monitor Moods Movement Mus Muscle hypotonia Mutant Strains Mice Neonatal Nervous System Physiology Neurons Obesity Obsessive compulsive behavior Obsessive-Compulsive Disorder Patients Pattern Phenotype Physiological Positioning Attribute Process Property Protein Isoforms Proteins Proteomics RNA RNA Editing RNA Processing Reaction Recording of previous events Regulation Research Research Personnel Resolution Role Sampling Schizophrenia Serotonin Receptor 5-HT2C Shipping Ships Signal Transduction Sleep Specificity Structure System Techniques Transcript Variant Weaning Wild Type Mouse base brain tissue extracellular feeding food consumption genome editing insight interest mouse model mutant neuropathology neuropsychiatric disorder neuropsychiatry novel protein expression public health relevance receptor receptor density receptor expression receptor function response selected ion monitoring serotonin receptor suicide victim therapeutic development

项目摘要

DESCRIPTION (provided by applicant): The 2C subtype of serotonin receptor (5HT2C) is widely expressed in the brain and has been implicated in several neuropsychiatric disorders, including major depressive disorder, dysthymia, obsessive-compulsive disorder, anxiety, bipolar disorder, and schizophrenia. Transcripts encoding the 5HT2C receptor can be differentially modified by up to five adenosine-to-inosine editing events, a process responsible for the cell-specific expression of as many as 24 protein isoforms that differ by up to three amino acids within the second intracellular loop of the receptor, a region essential for G protein coupling efficacy. In vitro, the fully edited 5HT2C isoform (5HT2C-VGV) exhibits reduced constitutive activity and altered subcellular localization in comparison to the genomically-encoded isoform (5HT2C-INI). Thus, 5HT2C editing may represent a critical regulatory mechanism by which neurons modulate their response to changing extracellular signals by altering the efficacy and specificity of receptor:G protein interactions. Moreover, alterations in 5HT2C editing have been observed in patients diagnosed with schizophrenia and bipolar disorder, in suicide victims with a history of major depression, and in response to antidepressant and antipsychotic treatments, suggesting that improper editing of 5HT2C transcripts may be a contributing factor in neuropsychiatric illness. The long term objectives of the proposed research are to define the cellular mechanisms that regulate 5HT2C expression and signaling, as well as possible relation- ships between 5HT2C editing and neuropathologies. Recent studies have revealed that genetically modified mice solely expressing the fully edited 5HT2C receptor isoform (5HT2C-VGV) produce an unprecedented 40- to 70-fold increase in receptor density without a concurrent change in steady-state 5HT2C mRNA. Thus, RNA editing has dramatic consequences on the expression of 5HT2C protein through uncharacterized post- transcriptional mechanism(s). To evaluate whether a disparity exists between edited 5HT2C transcripts and their encoded protein products in wild-type mice, affinity purification methods wil be used to isolate 5HT2C receptors, followed by a mass spectrometric proteomic analysis to quantify the relative expression levels of 5HT2C protein isoforms. A recently developed CRISPR/Cas-based approach will be used to generate mutant mice in which epitope tag(s) have been introduced into to the endogenous 5HT2C locus, thus enabling a more effective purification of 5HT2C protein for subsequent comparisons of mRNA and protein isoform distribution in discrete brain regions. Identification of disparities between 5HT2C RNA and protein isoform expression will have important implications for human studies of disease-related alterations in 5HT2C RNA editing, in which inferences about receptor isoform expression and function have been based solely upon edited mRNA distribution profiles. Furthermore, these findings will raise the possibility that editing of other mRNA transcripts may similarly affect subsequent protein expression levels, thus expanding the role for this RNA processing event in mammalian biology.

描述（由适用提供）：5-羟色胺受体（5HT2C）的2C亚型在大脑中广泛表达，并且已在几种神经精神疾病中隐含，包括重度抑郁症，心理症，强迫症，强迫症，强迫症，焦虑症，焦虑症，双相情感障碍和精神病。编码5HT2C受体的转录本可以通过多达五个腺苷对肌苷编辑事件进行不同的修饰，这是一个导致多达24种蛋白质同工型细胞特异性表达的过程，该过程在受体的第二个细胞内环内最多三个氨基酸不同，这是G蛋白质的必不可少的区域，是G蛋白的必不可少的区域。在体外，与基因组编码的同工型（5HT2C-INI）相比，完全编辑的5HT2C同工型（5HT2C-VGV）表现出降低的组成性活性并改变了亚细胞定位。这是5HT2C编辑可能代表了一种关键的调节机制，通过改变受体的效率和特异性：G蛋白相互作用，神经元通过该机制调节其对改变细胞外信号的反应。此外，在被诊断出患有精神分裂症和躁郁症的患者中观察到了5HT2C编辑的改变，在具有严重抑郁症病史的自杀性违规行为中，以及响应抗抑郁药和抗精神病药物的响应，这表明5HT2C转录物的编辑不当可能是神经精神疾病的5HT2C转录。拟议研究的长期目标是定义调节5HT2C表达和信号传导的细胞机制，以及可能的关系 - 5HT2C编辑与神经病理学之间的关系。最近的研究表明，仅表达完全编辑的5HT2C受体同工型（5HT2C-VGG）的一般修饰的小鼠产生了前所未有的40至70倍的受体密度增加而没有稳态5HT2C mRNA的同时变化。这就是RNA编辑通过未表征的转录后机制对5HT2C蛋白的表达产生巨大后果。为了评估在野生型小鼠中编辑的5HT2C转录物与其编码蛋白质产物之间是否存在差异，将使用亲和力纯化方法分离5HT2C受体，然后进行质谱蛋白质组学分析以量化5HT2C蛋白质相对的相对表达水平。最近开发的基于CRISPR/CAS的方法将用于生成突变小鼠，其中表位标签已引入内源性5HT2C基因座中，从而使5HT2C蛋白更有效地纯化5HT2C蛋白，以便随后比较离散大脑区域中mRNA和蛋白质同工分布。 5HT2C RNA和蛋白质同工型表达之间差异的鉴定将对5HT2C RNA编辑中与疾病相关的变化的研究具有重要意义，在5HT2C RNA编辑中，有关受体同工型表达和功能的信息仅基于编辑的mRNA分布分布。此外，这些发现将提高其他mRNA转录物的编辑可能会类似地影响随后的蛋白质表达水平，从而扩大哺乳动物生物学中这种RNA加工事件的作用。