Testing the role of intracellular vs. cell surface mGlu5 in models of synaptic plasticity using CRISPR-modified mice

摘要 神经精神障碍，如自闭症谱系障碍（ASD）、焦虑和抑郁 每年在医疗、经济和社会成本上花费数千亿美元。尽管 尽管取得了进展，但对这些疾病的治疗仍然有限。因此，更安全，更具体的药物 具有很高的临床应用价值。其中一个关键的药物靶点是代谢型谷氨酸 受体mGlu 5拮抗剂，其有力地改善这些疾病的动物模型; 然而，初步临床试验的结果喜忧参半。而不是使mGlu 5无效， 治疗靶点，这些结果突出了更好地理解mGlu 5功能的需要， 包括其亚细胞定位。例如，我们已经表明，70-90%的mGlu 5是 位于细胞内膜上，在那里它与信号系统结合， 其细胞表面对应物。重要的是，细胞内mGu 5足以建立长期的 抑郁症（LTD），一种在ASD、焦虑和抑郁症中功能失调的突触可塑性形式。 萧条这项研究的目的是确定细胞内 mGlu 5通过识别其在细胞表面与细胞内部的功能差异发挥作用， 体外、离体和体内。我们的中心假设是mGlu 5与不同的信号传导相互作用， 这取决于它所处的位置，这导致突触的不同调节。 过程和相关行为。为了验证这一假设，我们开发了一种基因 限制性小鼠系，其中mGlu 5仅存在于细胞内膜上（mGlu 5IM）。我们 将使用这种新衍生的动物系用于以下目的。在目标1中，我们将确定 mGlu 5IM与mGlu 5 WT或mGlu 5 KO对体外信号传导途径具有不同的影响（培养的 神经元）通过测试提出的候选基因和通过使用无偏转录组分析 （RNA-Seq）来鉴定新途径。在目标2中，我们将使用离体切片制备， 确定mGlu 5IM与mGlu 5 WT或mGlu 5 KO变体是否发挥独特的作用和信号传导 通过突触过程中的独特通路，如LTD。在目标3中，我们将确定 变异小鼠是否对感觉运动、焦虑样和抑郁样表现出独特的作用， 行为以及奖励的操作模型。这将是第一次报告的位置- 细胞内mGlu 5在体内的特定功能。由于mGlu 5是越来越多的 从细胞内部发出信号的受体，拟议的实验也将增强 其他细胞内受体的知识。靶向细胞内与细胞内药物的未来研究- 表面定位的受体有望导致更好的药物的开发mGlu 5- 调节的疾病如ASD和抑郁症，

ABSTRACT Neuropsychiatric disorders such as autism spectrum disorder (ASD), anxiety and depression cost hundreds of billions of dollars each year in medical, economic and social costs. Despite progress, treatments for these conditions remain limited. Hence safer, more specific drugs would have high clinical utility. One critical, drug-able target is the metabotropic glutamate receptor, mGlu5, antagonists of which robustly improve animal models of these disorders; however the results of initial clinical trials have been mixed. Rather than invalidating mGlu5 as a therapeutic target, such results highlight the need for a better understanding of mGlu5 function, including its subcellular localization. For example, we have shown that 70-90% of mGlu5 is located on intracellular membranes, where it couples to signaling systems distinct from those of its cell surface counterpart. Importantly, intracellular mGu5 is sufficient for establishing long term depression (LTD), a form of synaptic plasticity that is dysfunctional in ASD, anxiety and depression. The objective of the proposed research is to establish the role that intracellular mGlu5 plays by identifying differences in its function on the cell surface vs. inside the cell, in vitro, ex vivo and in vivo. Our central hypothesis is that mGlu5 interacts with different signaling pathways depending on where it is located and that this results in distinct regulation of synaptic processes and related behaviors. To test this hypothesis we have developed a genetically restricted mouse line in which mGlu5 is present only on intracellular membranes (mGlu5IM). We will use this newly derived animal line in the following aims. In Aim 1, we will determine whether mGlu5IM vs. mGlu5WT or mGlu5KO has different effects on signaling pathways in vitro (cultured neurons) by testing proposed candidate genes and by using unbiased transcriptome profiling (RNA-Seq) to identify new pathways. In Aim 2, we will use ex vivo slice preparations to determine whether mGlu5IM vs. mGlu5WT or mGlu5KO variants play unique roles and signal through distinctive pathways in synaptic processes such as LTD. In Aim 3, we will determine whether the variant mice exhibit unique effects on sensorimotor, anxiety-like and depressive-like behaviors as well as on operant models of reward. This would be the first report of location- specific functions of intracellular mGlu5 in vivo. As mGlu5 is one of a growing number of receptors that signal from inside the cell, the proposed experiments will also enhance knowledge of other intracellular receptors. Future studies targeting drugs to intracellular vs. cell- surface-localized receptors are expected to lead to the development of better drugs for mGlu5- modulated disorders such as ASD and depression,