Elucidating the dynamic role of PTPsigma in synaptic nano-organization and NMDA receptor function

微调突触传递的效率对学习和记忆至关重要，而它的破坏则是 与多种病理有关，包括自闭症谱系障碍、抑郁症和焦虑症。 因此，确定调节突触强度的机制是神经科学的中心目标。既然这样 可塑性通常是由NMDA型谷氨酸受体的激活触发的，了解这种调节 对NMDA受体的研究尤为关键。最近的研究表明，突触强度和NMDA受体 突触内蛋白质的纳米级组织可以直接影响激活。许多 突触蛋白，包括囊泡释放机制和突触后支架和受体，显示出 具有高蛋白质密度的局部区域的异质组织，称为纳米结构域(NDS)。这些 NDS可以在突触上排列，形成一个“纳米柱”，我们的实验室已经证明，它是 动作电位诱发的囊泡融合和最大受体激活。我们实验室的新工作已经确定 突触后细胞黏附分子LRRTM2在脑内AMPA受体定位中的新作用 纳米柱。虽然CAM在突触的形成和发育中扮演着既定的角色，但这些最近的 研究结果表明，在成熟期，细胞间黏附分子可能协调突触的纳米结构和功能 Synapse。然而，突触前组织和囊泡融合部位的沟通机制 对突触后密度内的蛋白质进行比对仍不清楚。在这项提议中，我将 研究突触前CAMPTPσ是否协调纳米柱对齐。Ptpσ对以下方面很重要 突触的形成，存在于成熟的突触中，并与前和 位于纳米柱内的突触后机制。PTPσ的丢失对突触前和突触后都有影响 生理，最显著的是NMDA受体介导的反应。以前研究ptpσ的尝试依赖于 长期的操纵，例如击倒和击倒。然而，解释因其本身而变得复杂 在发育过程中突触形成的初始作用。我建议通过以下方式来阐明ptpσ正在进行的功能 通过外源蛋白酶的裂解来剧烈地破坏其裂隙相互作用。这种高度的特异性和敏锐性 这种方法将允许我操纵ptpσ的S裂解相互作用，以分离它们的功能，而不会妥协 它在突触形成中的早期作用。在整个项目中，我将使用超分辨率显微镜， 电生理学、分子生物学和活细胞成像来测试ptpσ裂解相互作用在 维持纳米柱的排列和调节NMDA受体介导的传递。这项工作将提供 关于突触前凸轮关键家族在发育过程中的作用的新见解，并将测试 突触纳米结构和NMDA受体功能协调的新候选机制。这个 根据该奖学金获得的培训将提供深入和多样化的方法论和专业培训 这项发展将为我在生物医学大学担任学术研究员的职业生涯做好准备。

Fine tuning the efficiency of synaptic transmission is essential for learning and memory, while its disruption is associated with diverse pathologies including autism spectrum disorder, depression, and anxiety disorders. Thus, identifying mechanisms that regulate synaptic strength is a central goal in neuroscience. Since such plasticity is frequently triggered by activation of NMDA-type glutamate receptors, understanding the regulation of NMDA receptors is particularly critical. Recent studies indicate synaptic strength and NMDA receptor activation can be directly affected by the nanometer-scale organization of proteins within the synapse. Many synaptic proteins, including vesicle release machinery and postsynaptic scaffolds and receptors, display a heterogeneous organization with local regions of high protein density, known as nanodomains (NDs). These NDs can be aligned across the synapse to form a “nanocolumn”, which our lab has demonstrated is the site of action potential-evoked vesicle fusion and maximal receptor activation. New work from our lab has established a novel role for the postsynaptic cell-adhesion molecule (CAM) LRRTM2 in positioning AMPA receptors within the nanocolumn. While CAMs have well-established roles in synapse formation and development, these recent findings highlight the possibility that CAMs may coordinate synaptic nanostructure and function in the mature synapse. However, the mechanism by which presynaptic organization and vesicle fusion sites are communicated to proteins within the postsynaptic density to enable alignment to occur remains unknown. In this proposal I will investigate whether the presynaptic CAM PTPσ coordinates nanocolumn alignment. PTPσ is important for synapse formation, is present in the mature synapse, and forms indirect interactions with both pre- and postsynaptic machinery located within the nanocolumn. Loss of PTPσ impacts both pre- and postsynaptic physiology, most notably NMDA receptor-mediated responses. Previous attempts to study PTPσ have relied on chronic manipulations, such as knockouts and knockdowns. However, interpretations are complicated by its initial role in synapse formation during development. I propose to elucidate the ongoing functions of PTPσ by acutely disrupting its cleft interactions via cleavage by an exogenous protease. This highly specific and acute approach will allow me to manipulate PTPσ’s cleft interactions to isolate their functions, without compromising its earlier role in synapse formation. Throughout this project, I will use super-resolution microscopy, electrophysiology, molecular biology, and live-cell imaging to test the role of PTPσ cleft interactions in maintaining nanocolumn alignment and regulating NMDA receptor-mediated transmission. This work will provide novel insight regarding the roles of a critical family of presynaptic CAMs following development and will test a new candidate mechanism for the coordination of synaptic nanostructure and NMDA receptor function. The training obtained under this fellowship will provide deep and diverse training in methodologies and professional development that will prepare me excel in my career as an academic researcher at a biomedical university.