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

项目概要： 在人类中，多次早期暴露于需要麻醉的手术是一个重要的风险因素， 学习障碍和注意力障碍的发展，以及更长的，但不是更短的， 单次暴露期间的麻醉也与不良结果相关。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。证据表明 受损的海马棘发生和突触发生可能参与了 早期的麻醉剂暴露会产生长期的认知障碍，而突触支架蛋白PSD- 95 PDZ结构域介导的蛋白质-蛋白质相互作用和突触活动。我们以前的研究 已经证明PDZ结构域介导的蛋白质相互作用被临床相关的 吸入麻醉剂的浓度。最近，我们发现出生后第7天（PND）的小鼠暴露于 异氟烷抑制树突棘发育，改变突触可塑性，损害学习和记忆 与PSD-95 PDZ结合结构域的麻醉破坏有关的功能。我们的研究结果表明， PDZ相互作用和PDZ结构域介导的突触功能的破坏可能在PDZ介导的突触功能中起重要作用。 早期麻醉剂麻醉产生长期认知障碍的发病机制。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改变 引起PSD-95-NMDAR和相关突触复合物解偶联的相互作用，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 发展，从而产生长期的神经认知功能障碍。为了解决这个问题，我们的目标是 将确定连接麻醉诱导的PSD-95 PDZ解偶联的信号通路和机制， NMDAR-nNOS突触复合物对脊髓变化和突触成熟的影响对神经回路至关重要 形成（目标1）;我们将验证，在体内，关键的下游信号传导组件的破坏影响， 突触复合物，PSD-95-NMDAR-nNOS，并确定恢复它们是否可以充分防止 麻醉对树突棘和突触发育、LTP和记忆的有害影响（目的2）; 评估PDZ结构域破坏是否延迟NR 2B到NR 2A的发育转换， 树突和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表达和敲低来预防乔木干扰 方法（目标3）。拟议的研究将确定信号通路和机制， 麻醉诱导的PSD-95 PDZ-NMDAR和相关突触复合物的解偶联， 树突棘、突触和乔木发育导致长期认知障碍。

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.