Involvement of astrocytic two-pore domain K+ channels in ischemic pathology

描述(申请人提供)：星形胶质细胞是大脑中数量最多的细胞类型，已知为中枢神经系统(CNS)提供结构、代谢和内环境平衡支持。虽然星形胶质细胞在脑缺血中可以比神经元更好地存活，但不同脑细胞之间存在如此不同的易感性的机制尚不清楚。电压非依赖性钾通道电导或被动电导的主要表达是成熟星形胶质细胞的标志，对于星形胶质细胞对中枢神经系统的稳态支持是必不可少的。现在我们知道，两孔结构域K通道(K2Ps)中的两个成员Twik-1和Trek-1是长期寻找的K通道之一，可以解释星形胶质细胞的被动电导。K2Ps可以受到多种生理、化学和病理刺激的动态调节，包括cerebral-ischemia-produced-neuronal-injury-factors，如缺氧、低血糖、酸中毒和神经递质的病理性释放。病理性地诱导K2P的表达也导致了细胞的坏死和凋亡以及细胞的增殖，这是发生在缺血梗死区和半影区的两个重要的病理事件。为了了解星形胶质细胞K2Ps的生理性表达如何为星形胶质细胞提供早期缺血损伤的保护，以及长期缺血条件如何诱导反应性星形胶质细胞K2P表达的变化及其对卒中后结果的影响，我们假设CIPNJF可以通过调节星形胶质细胞K2Ps的活性来保护星形胶质细胞免受早期缺血损伤，而反应性星形胶质细胞中K2P表达的变化有助于梗死灶周围半暗带区域内平衡功能的受损。为了探索这些完全未知的领域，提出了五个具体目标。1)CIPNJF对星形胶质细胞膜电位和被动电导的调节作用。此项工作将在大鼠海马片上进行，并使用gricidin穿孔贴片记录来监测K2Ps的调制，而不干扰CIPNJFS介导的细胞内能量衰竭和改变的信号转导；2)神经保护剂以及Trek-1通道调节剂利鲁唑和西帕三嗪对CIPNJFS诱导的星形胶质细胞电生理反应的调节；3)在器官型海马片培养中，通过选择性沉默星形胶质细胞K2Ps与siRNA的相互作用，鉴定K2P-CIPNJF的特定相互作用机制；4)用免疫细胞化学方法鉴定颈动脉中动脉阻塞后可逆性脑内半影区(RMCAO)脑片中K2P的表达。5)用电生理学方法鉴定大鼠局灶性脑缺血半暗带区反应性星形胶质细胞中K2P的功能。这些研究将为探讨星形细胞K2P在脑缺血中的生理作用和病理参与，以及这些主要的星形细胞K通道是否可能成为卒中治疗策略的潜在靶点提供新的见解。 公共卫生相关性：通过检验两个新发现的星形胶质细胞两孔结构域K通道Twik-1和Trek-1的生理表达可在脑缺血发作早期对星形胶质细胞提供早期保护的假设，以及K2P表达如何在反应性星形胶质细胞中改变并可能在卒中病理中影响星形胶质细胞的动态平衡功能，拟议的研究将为星形胶质细胞K2Ps在脑缺血中的作用提供新的见解。这一结果也应该为基于这些新认识的和具有重要功能的星形细胞K通道的未来卒中治疗选择提供线索。

DESCRIPTION (provided by applicant): Astrocytes are the most numerous cell types in the brain and are known to provide structural, metabolic and homeostatic support to the central nervous system (CNS). Although astrocytes can better survive than neurons in cerebral ischemia, the mechanisms accounting for such a different susceptibility among different brain cells are not clear. Predominant expression of a voltage-independent K+ channel conductance, or passive conductance, is a hallmark of mature astrocytes and essential for the homeostatic support of astrocytes to the CNS. Now we know that two members of the two-pore domain K+ channels (K2Ps) K+ channels, TWIK-1 and TREK-1, are among the long-sought for K+ channels accounting for astrocyte passive conductance. K2Ps can be dynamically modulated by a variety of physiochemical and pathological stimuli, including cerebral-ischemia-produced-neuronal-injury-factors (CIPNJFs), such as hypoxia, hypoglycemia, acidosis and pathological release of neurotransmitters. Pathological induction of K2P expression also contributes to the necrotic and apoptotic cell death and cell proliferation that are the two prominent pathological events occurring in the ischemic infarct and penumbra regions. To understand how the physiological expression of astrocyte K2Ps offers protection to astrocytes against early ischemic insults, and how the long-term ischemic conditions induce altered K2P expression in reactive astrocytes and its consequence on the post-stroke outcomes, we hypothesize that the activity of astrocytic K2Ps can be modulated by CIPNJFs in a manner protecting astrocytes against early ischemic insults, and the altered expression of K2P in reactive astrocytes contributes to the compromised homeostatic function in the peri- infarct penumbra region. Five specific aims are proposed to explore these completely unknown areas. 1) Modulation of astrocyte membrane potential and passive conductance by CIPNJFs. This will be done in rat hippocampal slices with gramicidin perforated patch recording to monitor K2Ps modulation without interfering with the CIPNJFs mediated intracellular energy failure and altered signal transduction; 2) Modulation of electrophysiological response of astrocytes to CIPNJFs by neuroprotectant and TREK-1 channel modulator riluzole and sipatrigine; 3) Identify specific K2P-CIPNJF interaction mechanisms by selective silencing of astrocytic K2Ps with siRNAs in organotypic hippocampal slice cultures; 4) Identify K2P expression in rat hippocampal reactive astrocytes in slices prepared from the penumbra region after reversible middle carotid artery occlusion (rMCAO) by confocal immunocytochemistry. 5) Identify functional K2P in reactive astrocytes in rat focal ischemia penumbra region using electrophysiology in acutely prepared hippocampal slices from the rat rMCAO penumbra region. The proposed studies should provide novel insights into the physiological roles and pathological involvement of astrocytic K2P in cerebral ischemia and whether these predominant astrocytic K+ channels could be potential targets for stroke therapeutic strategy. PUBLIC HEALTH RELEVANCE: By testing a hypothesis that physiological expression of the two newly identified astrocyte two-pore domain K+ channels, TWIK-1 and TREK-1, offers early protection to astrocytes early on in a cerebral ischemia attack, and how the K2P expression alters in the reactive astrocytes and can underlie compromised homeostatic functions of astrocytes in stroke pathology, the proposed studies will provide novel insights into the roles of astrocyte K2Ps in cerebral ischemia. The results should also shed light on future stroke treatment options based on these newly appreciated and functionally significant astrocytic K+ channels.