LOCATION-DEPENDENT SIGNALING OF MGLU5 IN MODELS OF SYNAPTIC PLASTICITY USING CRISPR-TARGETED MICE

使用 CRISPR 靶向小鼠的突触可塑性模型中 MGLU5 的位置依赖性信号传导

• 批准号: 9375216
• 负责人: KAREN L O'MALLEY
• 金额: $ 19.06万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375216
• 关键词: Address; Affect; Agonist; Alleles; Animal Model; Animals; Autistic Disorder; Behavior; Behavioral Paradigm; Biological Assay; Biological Models; C-terminal; CRISPR/Cas technology; Cancer Therapy Evaluation Program; Cell surface; Cells; Clinical Research; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Corpus striatum structure; Couples; DNA; Data; Development; Disease; Disease model; Drug Targeting; Epitopes; Exhibits; Exploratory/Developmental Grant; Foundations; Fragile X Syndrome; Future; G-Protein-Coupled Receptors; GRM1 gene; GRM5 gene; Gene Targeting; Generations; Genetic; Genetic Models; Glutamate Transporter; Glutamates; Goals; Hippocampus (Brain); Human; Immediate-Early Genes; Immunohistochemistry; Impairment; In Vitro; Injection of therapeutic agent; Intellectual functioning disability; Intracellular Membranes; Knock-in; Knockout Mice; Knowledge; Lead; Learning; Link; Location; Long-Term Depression; MAPK3 gene; Mediating; Memory; Messenger RNA; Metabotropic Glutamate Receptors; Modeling; Motor; Mouse Strains; Mus; N-terminal; Nature; Neurons; Neurotransmitters; Nuclear Envelope; Obsessive-Compulsive Disorder; Oligonucleotides; Outcome; Pain; Pathway interactions; Permeability; Pharmacology; Phosphorylation; Play; Process; Protein Biosynthesis; Receptor Signaling; Regulation; Reporting; Research; Research Personnel; Rewards; Risk; Role; Signal Pathway; Signal Transduction; Slice; Specificity; Synapses; Synaptic plasticity; System; Testing; Therapeutic; Transgenic Organisms; Translations; Treatment Efficacy; Universities; Validation; Variant; Washington; Work; anxiety-like behavior; autism spectrum disorder; chronic pain; developmental disease; egg; expectation; experimental study; genetic approach; improved; improved outcome; in vivo; insight; memory process; metabotropic glutamate receptor 5; mouse model; new therapeutic target; novel therapeutics; offspring; pain behavior; pre-clinical; quisqualate; receptor; receptor function; response; social anxiety; success; synaptogenesis; therapeutic candidate; therapeutic target; tool; vector

The metabotropic glutamate receptor, mGlu5, plays a fundamental role in many neuronal processes including synapse formation, synaptic plasticity, and changes in synaptic efficacy. Not surprisingly, impaired mGlu5 signaling is implicated in disorders of synaptogenesis such as Fragile X Syndrome (FXS), autism, and obsessive compulsive disorder (OCD). Indeed, genetically or pharmacologically blocking mGlu5 function robustly improves animal models of these disorders; however exploratory clinical trials have exhibited varying degrees of success. Rather than invalidating mGlu5 as a therapeutic target, such results highlight the need for a better understanding of receptor function including its cell and location specificity. For example, we have shown that 60-90% of mGlu5 is located on intracellular membranes where it couples to distinct signaling systems versus its cell surface counterpart. Importantly, intracellular mGu5 is sufficient for establishing hippocampal and striatal long term depression, a form of synaptic learning and memory that is dysfunctional in FXS, autism and OCD. The objective of the proposed research is to develop animal models that will enable testing of both location-specific mGlu5 signaling and the ability of candidate therapeutics to affect receptors on the intracellular membranes versus the cell surface. Our central hypothesis is that the effects of signaling by mGlu5 in vivo are “location dependent”. In Aim 1, we will take advantage of CRISPR technology to generate two mouse strains: one will incorporate a short C-terminal tag on mGlu5 that will target the receptor solely to the ER and nuclear membranes; and the other will incorporate a short N-terminal epitope on mGlu5 that will target the receptor solely to the cell surface. In Aim 2, we will use these unique animals to capitalize on our recent observation suggesting that intracellular mGlu5, but not cell-surface-localized mGlu5, is critical in synaptic models of learning and memory. We hypothesize that blocking intracellular-restricted mGlu5 will inhibit changes in synaptic plasticity underlying learning and memory as well as motor, social and anxiety-like behaviors whereas blocking cell-surface-restricted mGlu5 will not do so. This would be the first report of location-specific functions of intracellular versus cell surface-localized mGlu5 in vivo. Because mGlu5 is one of a growing number of receptors that signal from inside the cell, the proposed experiments will enhance knowledge of other intracellular receptors as well. Future in vivo studies targeting drugs to intracellular versus cell-surface-localized receptors are expected to lead to the development of new and effective therapeutic tools for FXS, autism, OCD, and other mGlu5-modulated disorders.

代谢型谷氨酸受体mGlu 5在许多神经元中起着重要作用。 包括突触形成、突触可塑性和突触功效变化的过程。 毫不奇怪，受损的mGlu 5信号转导与突触发生障碍有关， 脆性X综合征（FXS），自闭症和强迫症（OCD）。的确， 基因或基因阻断mGlu 5功能， 然而，探索性临床试验已经显示出不同程度的成功。 这样的结果不是使mGlu 5作为治疗靶点无效，而是强调了对治疗靶点的需要。 更好地了解受体功能，包括其细胞和位置特异性。比如说， 我们已经表明，60-90%的mGlu 5位于细胞内膜上，在那里它与 不同的信号系统与其细胞表面对应物。重要的是，细胞内mGu 5是 足以建立海马和纹状体的长期抑制，一种突触形式， 在FXS自闭症和强迫症中有功能障碍的学习和记忆。的目的 拟议的研究是开发动物模型，这将使测试这两个特定位置 mGlu 5信号传导和候选治疗剂影响细胞内受体的能力 膜与细胞表面。我们的中心假设是， mGlu 5在体内是“位置依赖性的”。在目标1中，我们将利用CRISPR技术 以产生两种小鼠品系：一种将在mGlu 5上掺入短的C-末端标签， 靶向受体只对ER和核膜;和其他将纳入一个 mGlu 5上的短N-末端表位，其将受体仅靶向细胞表面。在Aim中 2，我们将利用这些独特的动物来利用我们最近的观察表明， 细胞内mGlu 5，而不是细胞表面定位的mGlu 5，是关键的突触模型， 学习和记忆。我们假设阻断细胞内限制性mGlu 5将抑制 突触可塑性的变化是学习和记忆的基础，也是运动、社交和 焦虑样行为，而阻断细胞表面限制性mGlu 5不会这样做。这将 是第一个报告的位置特异性功能的细胞内与细胞表面定位 体内mGlu 5。因为mGlu 5是越来越多的从体内发出信号的受体之一， 细胞，拟议的实验将提高其他细胞内受体的知识， 好.靶向细胞内与细胞表面定位受体的药物的未来体内研究 有望为FXS，自闭症， 强迫症和其他mGlu 5调节的疾病。