Acid-sensing channels as novel target for brain ischemia

描述（申请人提供）：脑酸中毒是急性神经系统疾病特别是缺血的常见特征，在神经元损伤的病理中起着重要作用。然而，酸中毒损伤的细胞和分子机制仍然不确定，多因素和模糊。我们有大量的初步数据表明，新描述的酸敏感离子通道（asic）， Degenerin/EnaC超家族成员的激活，以及随后通过这些通道的Ca2+进入，在很大程度上负责酸中毒诱导的，不依赖谷氨酸受体的神经元损伤。在培养的小鼠皮质神经元中，降低pH可激活阿米洛利敏感的ASIC电流。在大多数这些神经元中，asic也可渗透到Ca2+，这些通道的激活诱导细胞内Ca2+浓度的增加（[Ca2+]i）。在电压门控Ca2+通道和谷氨酸受体阻滞剂存在的情况下，用酸性溶液短暂孵育神经元激活asic可诱导时间依赖性细胞损伤。然而，这种酸诱导的损伤可以通过asic阻滞剂和减少细胞外[Ca2+]来抑制。酸处理缺乏功能性asic的COS-7细胞不会引起明显的细胞损伤。与原代培养神经元类似，酸处理可诱导器官型脑切片损伤，且asic阻滞剂可抑制脑切片损伤。初步的体内研究也表明，脑室内注射ASIC1阻断剂可减少梗死体积，敲除ASIC1基因可保护小鼠脑免受缺血性损伤。此外，我们的初步研究表明，缺血治疗和代谢抑制显著增强ASIC电流。asic的增强反过来又增加了酸中毒引起的神经元损伤。我们的总体目标是研究asic在中枢神经系统中的病理作用，并验证asic的激活与随后的Ca2+进入在很大程度上负责酸中毒介导的，不依赖谷氨酸的缺血性脑损伤的假设。特定的目标

DESCRIPTION (provided by applicant): Brain acidosis is a common feature in acute neurological diseases particularly in ischemia, and has been assumed to play an important role in the pathology of neuronal injury. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain uncertain, multifactorial and vague. We have substantial preliminary data demonstrating that activation of newly described acid-sensing ion channels (ASICs), members of Degenerin/EnaC superfamily, and subsequent Ca2+ entry through these channels are largely responsible for acidosis-induced, glutamate receptor-independent neuronal injury. In cultured mouse cortical neurons, lowering pH activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are also permeable to Ca2+, and activation of these channels induces increases in the concentration of intracellular Ca2+([Ca2+]i). Activation of ASICs by brief incubation of neurons with acidic solutions induces time-dependent cell injury in the presence of the blockers for both voltage-gated Ca2+ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing the extracellular [Ca2+]. Acid treatment of COS-7 cells that lack functional ASICs does not induce significant cell injury. Similar to the primary cultured neurons, acid treatment induces injury in organotypic brain slices, and the injury of brain slices is inhibited by the blockers of ASICs. Preliminary in vivo studies also demonstrate that intraventricular injection of ASIC1 blocker reduced the infarction volume, and knockout of the ASIC1 gene protects the mouse brain from ischemic injury. Furthermore, our preliminary studies demonstrate that ischemic treatment and metabolic inhibition dramatically potentiate the ASIC currents. This potentiation of ASICs in turn increases acidosis-induced neuronal injury. Our overall objective is to investigate the pathological role of ASICs in the central nervous system and to test the hypothesis that activation of ASICs with subsequent Ca2+ entry is largely responsible for acidosis-mediated, glutamate-independent ischemic brain injury. Specific Aims Aim 1. Ca2+ -permeability of acid-sensing ion channels in CNS neurons Aim 2. Specific subunit configurations are responsible for acidosis-induced neuronal injury Aim 3. Potentiation of ASIC currents by hypoxia/ischemia Aim 4. Neuroprotective role of ASIC blockers or ASIC gene knockout in an in vivo model of brain ischemia and the organotypic brain slices