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

代谢型谷氨酸受体mGlu5在许多神经元中起着重要作用 突触形成、突触可塑性和突触效能变化等过程。 不足为奇的是，mGlu5信号受损与突触发生障碍有关，如 脆性X综合征(FXS)、自闭症和强迫症(OCD)。的确， 从基因或药物上阻断mGlu5功能有力地改善了动物模型 然而，探索性临床试验显示了不同程度的成功。 这样的结果并没有使mGlu5作为治疗靶点无效，而是突显了一种 更好地了解受体的功能，包括其细胞和位置特异性。例如, 我们已经证明，60%-90%的mGlu5位于细胞内的膜上，在那里它耦合到 不同的信号系统与其细胞表面的对应系统。重要的是，细胞内的mGu5是 足以建立海马区和纹状体的长期抑郁，这是突触的一种形式 FXS、自闭症和强迫症患者的学习和记忆功能障碍。该计划的目标是 拟议的研究是开发动物模型，使测试两个特定位置 MGlu5信号与候选治疗药物影响细胞内受体的能力 细胞膜与细胞表面。我们的中心假设是，通过发送信号的效果 体内的mGlu5是“位置依赖的”。在目标1中，我们将利用CRISPR技术 要生成两个小鼠品系：一个将在mGlu5上添加一个简短的C-末端标签，该标签将 将受体仅靶向内质网和核膜；另一种将结合一种 MGlu5上的N端短表位，它只针对细胞表面的受体。在AIM 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 在突触模型中，细胞内的mGlu5是关键的，而不是细胞表面定位的mGlu5 学习和记忆。我们假设阻断细胞内受限的mGlu5会抑制 学习和记忆以及运动、社会和精神障碍相关突触可塑性的变化 焦虑样行为，而阻断细胞表面受限的mGlu5不会做到这一点。这将会 首次报道细胞内定位功能与细胞表面定位功能 MGlu5在体内。因为mGlu5是越来越多的从内部发出信号的受体之一 细胞，拟议的实验将加强对其他细胞内受体的了解，如 井。未来针对细胞内受体和细胞表面受体的药物体内研究 有望导致FXS、自闭症、 强迫症和其他mGlu5调节性疾病。

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.