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

阐明 PTPsigma 在突触纳米组织和 NMDA 受体功能中的动态作用

• 批准号: 10606077
• 负责人: Emily M. DeMarco
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606077
• 关键词: AMPA Receptors; Action Potentials; Acute; Affect; Anxiety Disorders; Binding; Brain; Cell Adhesion Molecules; Cells; Chronic; Cognition; Communication; Compensation; Confocal Microscopy; DLG4 gene; DNA; Development; Electrophysiology (science); Engineering; Evoked Potentials; Excitatory Synapse; Experimental Designs; Extracellular Domain; Family; Fellowship; Genetic; Genetic Complementation Test; Glutamate Receptor; Goals; Hippocampus; Image; Knock-out; Lead; Learning; Measures; Mediating; Memory; Mental Depression; Methodology; Microscopy; Modeling; Molecular Biology; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; N-terminal; Nanostructures; Neurons; Neurosciences; Optics; Pathology; Peptide Hydrolases; Physiology; Play; Positioning Attribute; Probability; Process; Protein Family; Protein Tyrosine Phosphatase; Proteins; Receptor Activation; Regulation; Research Personnel; Resistance; Resolution; Role; Scaffolding Protein; Site; Structure; Synapses; Synaptic Cleft; Synaptic Transmission; Techniques; Testing; Training; Universities; Vesicle; Work; autism spectrum disorder; career; density; experimental study; extracellular; improved; insight; knock-down; live cell imaging; member; mutant; nano; nanocolumn; nanoscale; neuropsychiatric disorder; neurotransmitter release; novel; patch clamp; postsynaptic; presynaptic; protein complex; receptor; receptor function; response; scaffold; single molecule; superresolution microscopy; synaptogenesis; transmission process; vesicular release

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.

微调突触传递的效率对学习和记忆至关重要，而它的中断是 与包括自闭症谱系障碍、抑郁症和焦虑症在内的多种病理学相关。 因此，确定调节突触强度的机制是神经科学的中心目标。因为这种 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 NMDA受体的功能尤为重要。最近的研究表明，突触强度和NMDA受体 激活可以直接受到突触内蛋白质的纳米级组织的影响。许多 突触蛋白，包括囊泡释放机制和突触后支架和受体，显示出一种 纳米结构域是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（ND）。这些 神经元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了这是神经元在突触中的位置。 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证实 突触后细胞粘附分子（CAM）LRRTM2在定位AMPA受体中的新作用， 纳米柱虽然CAM在突触形成和发育中具有公认的作用，但最近的研究表明， 研究结果强调了CAMs可能在成熟的神经元中协调突触纳米结构和功能的可能性。 突触然而，突触前组织和囊泡融合位点的沟通机制 与突触后致密物中的蛋白质结合以使对齐发生仍然是未知的。在这份提案中，我将 研究突触前CAM PTPσ是否协调纳米柱对齐。PTPσ对于以下方面很重要： 突触形成，存在于成熟的突触中，并与前突触和后突触形成间接相互作用。 位于纳米柱内的突触后机械。PTPσ的丧失影响突触前和突触后神经元的功能。 生理学，最显著的是NMDA受体介导的反应。以前研究PTPσ的尝试依赖于 慢性操纵，如击倒和击倒。然而，由于其 在发育过程中突触形成的初始作用。我建议通过以下方式阐明PTPσ的持续功能： 通过外源蛋白酶的切割来剧烈破坏其裂缝相互作用。这种高度特异性和急性 一种方法将使我能够操纵PTPσ的裂缝相互作用，以隔离它们的功能，而不会损害 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，以测试PTPσ裂缝相互作用在 维持纳米柱排列和调节NMDA受体介导的传递。这项工作将提供 新的见解关于一个关键的家庭的作用突触前CAM以下的发展，并将测试一个 突触纳米结构和NMDA受体功能协调的新候选机制。的 在该研究金下获得的培训将在方法和专业方面提供深入和多样化的培训。 这将使我在生物医学大学的学术研究生涯中做好准备。