An electrophysiological and proteomic analysis of the effects of the superoxide dismutase mimetic, MnTMPyP, on synaptic signalling post ischemia in isolated rat hippocampal slices.

Metabolic stress and the increased production of reactive oxygen species (ROS) are two main contributors to neuronal damage and synaptic plasticity in acute ischemic stroke (AIS). The superoxide scavenger MnTMPyP has been previously reported to have a neuroprotective effect in organotypic hippocampal slices and to modulate synaptic transmission after in vitro hypoxia and oxygen glucose deprivation (OGD). However, the mechanisms involved in the effect of this scavenger remain elusive. In this study we evaluated the concentration dependent effect of MnTMPyP on synaptic transmission during ischemia and post-ischemic synaptic potentiation. We also investigated the complex molecular changes supporting cellular adaptation to metabolic stress and how these are modulated by MnTMPyP. Electrophysiological data showed that MnTMPyP causes a concentration dependent decrease in baseline synaptic transmission and impairment of synaptic potentiation. Proteomic analysis performed on MnTMPyP treated tissue indicated an impairment in vesicular trafficking mechanisms including alteration of Secretory Carrier Membrane Protein 3 (SCAMP3) and RAB proteins and downregulation of mitochondrial-mediated apoptosis. Alterations of vesicular trafficking may lead to reduced probability of neurotransmitter release and AMPA receptor activity, resulting in the observed modulatory effect of MnTMPyP. In ischemia and glucose deprivation, protein enrichment analysis highlighted impairments in cell proliferation and differentiation, such as TGFβ1 and CDKN1B signalling, in addition to downregulation of actin signalling, cell stress and mitochondrial-mediated apoptosis. Taken together our results indicate modulation of neuronal sensitivity to the ischemic insult, and a complex role for MnTMPyP on synaptic transmission and plasticity and provide molecular insights into the mechanisms mediating the effects of MnTMPyP during ischemia

代谢应激与活性氧（reactive oxygen species, ROS）生成增多，是急性缺血性脑卒中（acute ischemic stroke, AIS）引发神经元损伤、影响突触可塑性的两大主要致病因素。超氧化物清除剂MnTMPyP此前已被报道可在器官型海马脑片中发挥神经保护作用，并能在体外缺氧氧糖剥夺（oxygen glucose deprivation, OGD）模型中调控突触传递，但其介导该效应的具体分子机制仍尚不明确。本研究评估了MnTMPyP在缺血过程及缺血后突触增强效应中，浓度依赖性地对突触传递的影响；同时探究了支持细胞适应代谢应激的复杂分子变化，以及MnTMPyP如何调控这些适应性过程。电生理实验数据显示，MnTMPyP可浓度依赖性地降低基线突触传递水平，并损伤突触增强效应。对经MnTMPyP处理的脑组织进行蛋白质组学分析后发现，该清除剂可损害囊泡运输相关机制，包括改变分泌载体膜蛋白3（Secretory Carrier Membrane Protein 3, SCAMP3）与RAB蛋白的表达水平，并下调线粒体介导的细胞凋亡通路。囊泡运输异常可能会降低神经递质释放概率与AMPA（α-氨基-3-羟基-5-甲基-4-异恶唑丙酸）受体活性，这与实验中观察到的MnTMPyP调控效应相契合。在缺血与糖剥夺模型中，蛋白质富集分析结果显示，除肌动蛋白信号通路、细胞应激及线粒体介导的细胞凋亡通路出现下调外，细胞增殖与分化相关通路（如转化生长因子β1（TGFβ1）与细胞周期蛋白依赖性激酶抑制剂1B（CDKN1B）信号通路）也存在功能损伤。综上，本研究结果表明MnTMPyP可调控神经元对缺血损伤的敏感性，其在突触传递与可塑性中发挥复杂调控作用，并为阐明缺血过程中MnTMPyP发挥作用的分子机制提供了新的理论依据。