Mechanistic Actions of PDZ Domain Mediated Protein Interactions on Neural Development and Anesthetic-Mediated Neurotoxicity

项目总结： 在人类中，早期多次暴露于需要麻醉的程序是患上 学习障碍和注意力障碍的发展以及较长但不是较短的持续时间 单次暴露期间的麻醉也与不良后果有关。虽然临床前研究 显示全身麻醉持续时间与不良细胞和 功能结果潜在的分子机制仍有待充分阐明。有证据表明 损伤的海马棘发生和突触发生可能参与了 早期接触麻醉剂会导致长期的认知障碍，而突触支架蛋白PSD- 涉及95个PDZ结构域介导的蛋白质-蛋白质相互作用和突触活动。我们之前的研究 已经证明了PDZ结构域介导的蛋白质相互作用被临床上相关的 吸入麻醉剂的浓度。最近，我们发现将出生后7天(PND)7的小鼠暴露于 异氟醚抑制树突棘发育，改变突触可塑性，损害学习和记忆 与PSD-95 PDZ结合区的麻醉剂破坏有关的功能。我们的结果显示， PDZ相互作用和PDZ结构域介导的突触功能的破坏可能在 早期麻醉剂暴露的发病机制--产生长期的认知损害。我们假设 早期吸入麻醉通过破坏PDZ结构域介导的神经发育 导致PSD-95-NMDAR和相关突触复合体解偶联的相互作用导致抑制 几个重要的下游信号通路对树突棘、突触和乔木至关重要 发育，从而产生长期的神经认知功能障碍。为了解决这一假设，我们的目标是 将确定与麻醉诱导的PSD-95 PDZ解偶联有关的信号通路和机制- NMDAR-nNOS突触复合体对脊髓变化和突触成熟对神经回路至关重要 形成(目标1)；我们将在体内验证受干扰的关键下游信号组件 突触复合体，PSD-95-NMDAR-nNOS，并确定恢复它们是否足以防止 麻醉对树突棘和突触发育、长时程增强和记忆的有害影响(目标2)； 评估PDZ结构域中断是否会延迟从NR2B到NR2A的发育转换，影响生长 谷氨酸能神经元树突与关键可塑性相关蛋白的时空表达模式 突触，并确定是否可以使用过度表达和击倒来防止Arbor干扰 方法(目标3)。拟议的研究将确定信号通路和连接机制 麻醉诱导PSD-95、PDZ-NMDAR和相关突触复合体的解偶联作用 导致长期认知障碍的树突棘、突触和乔木发育。

Project Summary: In humans, multiple early exposures to procedures requiring anesthesia is a significant risk factor for development of learning disabilities and disorders of attention and longer, but not shorter, durations of anesthesia during a single exposure are also associated with adverse outcomes. While preclinical studies show a dose–response relationship between the duration of general anesthesia and adverse cellular and functional outcomes the underlying molecular mechanisms remain to be fully elucidated. Evidence indicates that impaired hippocampal spinogenesis and synaptogenesis may be involved in the mechanisms by which early anesthetic exposure produces long-term cognitive impairment, and that synaptic scaffolding protein PSD- 95 PDZ domain-mediated protein-protein interactions and synaptic activities are involved. Our previous studies have demonstrated that PDZ domain-mediated protein interactions are disrupted by clinically relevant concentrations of inhaled anesthetics. Recently, we showed that exposing postnatal day (PND) 7 mice to isoflurane inhibits dendritic spine development, alters synaptic plasticity, and impairs learning and memory function in relation to the anesthetic disruption of PSD-95 PDZ binding domains. Our results showed that the disruption of PDZ interactions and PDZ domain-mediated synaptic function may play important roles in the pathogenesis of early anesthetic exposure-produced long-term cognitive impairment. We hypothesize that early exposure to inhalational anesthesia alters neural development by disrupting PDZ-domain mediated interactions causing uncoupling of PSD-95-NMDAR and associated synaptic complexes resulting in inhibition of several prominent downstream signaling pathways critical to dendritic spine, synapse, and arbor development, thereby producing long-term neurocognitive dysfunction. To address this hypothesis, our aims will identify the signaling pathways and mechanisms that link anesthesia induced uncoupling of PSD-95 PDZ- NMDAR-nNOS synaptic complex to changes in spine and synapse maturation critical to neural circuit formation (Aim 1); we will validate, in vivo, key downstream signaling components effected by disruption of the synaptic complex, PSD-95-NMDAR-nNOS, and determine if restoring them can sufficiently prevent the deleterious effects of anesthesia on dendritic spine and synaptic development, LTP, and memory (Aim 2); assess whether PDZ domain disruption delays the NR2B to NR2A developmental switch, affects growing dendrites and the spatial and temporal expression patterns of critical plasticity related proteins in glutamatergic synapses, and determine if arbor disturbances can be prevented using over-expression and knock-down approaches (Aim 3). The proposed studies will identify the signaling pathways and mechanisms linking anesthesia induced uncoupling of PSD-95 PDZ-NMDAR and associated synaptic complexes to changes in dendritic spine, synapse, and arbor development that lead to long-term cognitive impairment.