钠，钙离子和pH值稳态异常是脑部疾病的重要诱因之一。组织酸化激活酸敏感离子通道1a（ASIC1a），介导钠/钙离子内流，引起神经元缺血性酸化死亡。线粒体钙离子超载是细胞死亡过程中的关键步骤，但ASIC1a通透的阳离子影响线粒体钙离子平衡的机理不明。最近Israel Sekler研究组鉴定出新型的线粒体钠钙交换体NCLX，徐天乐研究组报道线粒体ASIC1a（ASICmito）的重要病理功能。NCLX和ASICmito可能协同调节线粒体钠/钙离子变化，影响神经元存活。我们将综合运用化学荧光标记，分子改造，活细胞光学成像和基因操作小鼠等技术，研究ASIC1a-NCLX异常交互调控介导神经元缺血性损伤的机制。本研究试图揭示一种全新的质膜-线粒体之间的钠/钙离子信号通讯模式，阐述其对细胞死亡的意义，为治疗脑缺血损伤提供新的药物靶点和理论指导。

Aberrant homeostasis of pH, Ca(2+) and Na(+) are leading causes of morbidity in brain disorders. Because of its dual role as a pH sensor and a cationic (primarily Na(+) but also Ca(2+)) channel, ASIC1a is a key player in ischemic neuronal death. The mitochondria are the major hub of cellular death signaling, often triggered by mitochondrial Ca(2+) overload. Yet, how ASIC1a-mediated Na(+) or Ca(2+) signals communicate with mitochondria is unknown, primarily because the molecular identity of the mitochondrial Ca(2+) shuttling components, the Ca(2+) uniporter and the Na(+)/Ca(2+) exchanger remained elusive for many years. The Israel group identified NCLX as the long sought transporter conducting mitochondrial Na(+)/Ca(2+) exchange. The Chinese group is a leader in ASICs physiology and also identified the presence of ASIC1a on the mitochondria. Because NCLX couples between mitochondrial Na(+) and Ca(2+) transport, its communication with ASIC1a channels may provide the missing link to ASIC1a-induced ischemic neuronal death. Our collaborative study will address the hypothesis, supported by our joint preliminary results, that mitochondrial Na(+) load and/or depolarization trigged by ASIC1a leads to inhibition or even reversal of NCLX and thereby to mitochondrial Ca(2+) overload. Toward this goal, we will employ our established.molecular or pharmacological tools to control NCLX and ASIC1a expression and activity. By fluorescently monitoring cytosolic and mitochondrial Na(+) and Ca(2+) levels, while molecularly controlling ASIC1a and NCLX expression, we will first determine if the combined activity of NCLX and ASIC1a leads to mitochondrial Na(+) or Ca(2+) overload and thereby controls neuronal fate. By molecularly controlling MCU expression, we will further compare the contribution of the mitochondrial Ca(2+) uniporter to that of NCLX. A major discovery gained during our joint preliminary experiments is that a specific regulatory domain of NCLX prevented the inhibition/reversal of NCLX. Thus, by molecular modulation of this domain we will seek to enhance the mitochondrial resistance against Ca(2+) overload triggered by ASIC1a and thereby prevent neuronal loss triggered by the activation of the membrane or mitochondrial forms of ASIC1a channels.. The results of this study will identify a novel and fundamental communication pathway linking Na(+) and Ca(2+) signaling between cell membrane and mitochondria, and will show how its aberration leads to neuronal death. Results of this study will also lead to identification of novel molecular target of general importance linked to mitochondrial Ca(2+) overload.

pH值稳态异常是神经系统疾病的重要诱因之一。在缺血缺氧条件下，组织酸化导致细胞外H+浓度增加，激活酸敏感离子通道1a (acid-sensing ion channels, ASIC1a)，介导钠/钙离子内流，导致缺血性神经元死亡。课题组前期工作发现，酸敏感离子通道1a（acid-sensing ion channels, ASIC1a）除了在神经元质膜大量表达，还存在于线粒体内膜，并通过调节线粒体通透性转换孔（mitochondrial permeability transition pore, MPTP）的开放参与氧化应激诱导的细胞死亡（Wang et al., Cell Death Differ 2013）。这一现象提示ASIC1a非通道功能的存在。在本项目中，我们以ASIC1a非通道功能为出发点，以线粒体为研究中心，阐明了ASIC1a非通道功能通过磷酸化受体相互作用蛋白激酶1（Receptor-interacting serine/threonine-protein kinase 1, RIPK1）介导缺血性神经元程序性坏死（necroptosis）的分子机制，明确了胞外酸化通过ASIC1a破坏线粒体离子稳态及代谢功能，证实了线粒体Drp1过度SUMO1化修饰导致线粒体分裂是缺血性中风神经元死亡的原因之一，并且观察到受损神经元和邻近胶质细胞之间存在线粒体转移。线粒体在维持细胞正常功能中发挥重要作用，也是细胞死亡信号通路的中心。在缺血性中风，组织酸化激活ASIC1a非通道信号通路，导致神经元质膜-线粒体离子稳态失衡和代谢受损，神经元可以通过释放受损的线粒体向邻近胶质细胞寻求内源性救助。这一系列工作提出并证实了离子通道“非通道功能”的新观点，拓展了ASIC研究的新领域，深化了对线粒体功能调控的新认识，为缺血性中风和其它神经系统疾病的临床治疗提供了新的药物靶点和治疗策略。