How do neurons in the brain decide to refine their synaptic connections in vivo?

大脑中的神经元如何决定在体内完善其突触连接？

• 批准号: 9383862
• 负责人: Hisashi Umemori
• 金额: $ 68.63万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-16 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9383862
• 关键词: Alpha Cell; Autistic Disorder; Axon; Brain; CD47 gene; Cell Adhesion Molecules; Cerebral hemisphere; Complement; Cues; Data; Defect; Development; Disease; Dominant-Negative Mutation; Electrophysiology (science); Electroporation; Emotional; Etiology; Functional disorder; Gene Expression; Genetic; Goals; Hippocampus (Brain); Image; JAK2 gene; Knock-out; Knockout Mice; Limbic System; Maintenance; Mediating; Memory; Mental disorders; Microglia; Molecular; Mus; Neurons; Pathway interactions; Phagocytosis; Phosphotransferases; Physiological; Play; Population; Presynaptic Receptors; Process; Protein Tyrosine Kinase; Regulation; Role; Schizophrenia; Signal Transduction; Signaling Molecule; Social Behavior; Synapses; System; Testing; Time; Work; cingulate cortex; design; gene complementation; in utero; in vivo; insight; mutant; nervous system disorder; neural circuit; neuropsychiatric disorder; neurotransmitter release; novel; optogenetics; overexpression; postsynaptic neurons; prevent; relating to nervous system; response; transgene expression

Formation of functional neural circuits is critical for proper functioning of the brain. To establish the most efficient synaptic circuits, synaptic connections must be refined by neural activity during development. In this proposal, we will determine the molecules and manner by which functional circuits are established by neural activity, focusing on the limbic system (including the hippocampus and cingulate cortex), which is implicated in emotional processing, memory formation and social behavior. Using a mouse genetic system in which restricted populations of hippocampal neurons can be conditionally inactivated, we found that hippocampal axons are refined through activity-dependent competition, where active neuronal connections stay (maintained) while inactive ones leave (eliminated). We further found that a cell adhesion molecule SIRP from postsynaptic neurons stabilizes active synapses through its presynaptic receptor CD47, serving as a "Stay" signal. To identify the signaling molecules that play critical roles in inactive axon elimination ("Go" signal), we generated a new system in which neural activity and gene expression can be conditionally controlled in vivo, using in utero electroporation. When neurotransmitter release is blocked in a subset of neurons in the cingulate cortex, their callosal projections (the major connections between the cerebral hemispheres) are eliminated during development. Using this system, we screened for signaling molecules that are upregulated in inactive neurons right before their axons start to leave and identified the Ca2+-dependent tyrosine kinase Pyk2. Inactive axons were not eliminated when a kinase-dead mutant of Pyk2 was expressed, indicating that Pyk2 activity is necessary for inactive axons to leave. We further identified that a Pyk2-interacting kinase, JAK2, is also necessary for inactive axon elimination. Consistently, Pyk2 and JAK2 are activated in inactive neurons. Finally, we found that overexpression of Pyk2 or JAK2 induces axonal elimination even when axons are active. We propose that the Pyk2-JAK2 pathway is the "Go" signal and serves as the determinant of axon refinement. To further characterize this pathway and to understand how the "Go" and "Stay" pathways regulate activity- dependent axon/synapse refinement, we propose to: Aim 1: Investigate the role of Pyk2 and JAK2 for axon/synapse refinement in physiological conditions. Aim 2: Analyze the electrophysiological consequences of Pyk2/JAK2 inactivation during synapse refinement using conditional KO mice. Aim 3: Examine whether the Pyk2-JAK2 pathway provides cues for microglial clearance of inactive axons in vivo. Aim 4: Investigate the interaction between the Stay (SIRP-CD47) and Go (Pyk2-JAK2) pathways in axon/synapse refinement in vivo. Our project will molecularly delineate how neurons decide to establish functional synaptic connections in the mammalian brain. Pyk2 and JAK2 are associated with various neuropsychiatric disorders. Many forms of mental illness including autism and schizophrenia are associated with abnormal alterations in the limbic circuitry. Thus, our studies should also yield novel insights into the etiology and treatment of such disorders.

功能性神经回路的形成对于大脑的正常运作至关重要。 有效的突触回路，在发育过程中必须通过神经活动来完善突触连接。 建议，我们将确定神经元建立功能电路的分子和方式 活动，重点关注边缘系统（包括海马体和扣带皮层），这与 使用小鼠遗传系统进行情绪处理、记忆形成和社会行为。 有限数量的海马神经元可以有条件地失活，我们发现海马神经元 轴突通过依赖于活动的竞争而得到完善，其中活跃的神经连接保持（维持） 而不活跃的则离开（消除）。 突触后神经元通过其突触前受体 CD47 稳定活跃的突触，充当“停留”角色 为了识别在非活性轴突消除（“Go”信号）中发挥关键作用的信号分子，我们 产生了一个新系统，可以在体内有条件地控制神经活动和基因表达， 当扣带皮层神经元的神经递质释放被阻断时使用子宫内电穿孔。 皮质，其胼胝体投射（大脑半球之间的主要连接）被消除 在开发过程中，我们使用该系统筛选了在非活性状态下上调的信号分子。 在轴突开始离开之前对神经元进行研究，并发现 Ca2+ 依赖性酪氨酸激酶 Pyk2 处于非活性状态。 当 Pyk2 的激酶死亡突变体表达时，轴突没有被消除，表明 Pyk2 活性 我们进一步发现，Pyk2 相互作用的激酶 JAK2 也是非活动轴突离开所必需的。 Pyk2 和 JAK2 在不活动的神经元中被激活，这是消除不活动的轴突所必需的。 我们发现，即使轴突活跃，Pyk2 或 JAK2 的过度表达也会诱导轴突消除。 提出 Pyk2-JAK2 通路是“Go”信号并作为轴突细化的决定因素。 进一步表征该通路并了解“Go”和“Stay”通路如何调节活动- 依赖轴突/突触细化，我们建议： 目标 1：研究 Pyk2 和 JAK2 的作用 目标 2：分析生理条件下的轴突/突触细化。 使用条件 KO 小鼠进行突触细化期间 Pyk2/JAK2 失活 目标 3：检查是否 Pyk2-JAK2 途径为小胶质细胞清除体内不活动的轴突提供线索。 Stay (SIRPα-CD47) 和 Go (Pyk2-JAK2) 通路在轴突/突触细化中的相互作用 我们的项目将从分子角度描述神经元如何决定在体内建立功能性突触连接。 Pyk2 和 JAK2 与多种形式的神经精神疾病有关。 包括自闭症和精神分裂症在内的精神疾病与边缘系统的异常改变有关 因此，我们的研究也应该对此类疾病的病因和治疗产生新的见解。