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

星形细胞双孔域 K 通道参与缺血病理学

• 批准号: 7987672
• 负责人: MIN ZHOU
• 金额: $ 38.41万
• 依托单位: ORDWAY RESEARCH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7987672
• 关键词: Accounting; Acidosis; Affect; Antibodies; Apoptotic; Area; Astrocytes; Brain; Carotid Arteries; Cause of Death; Cell Death; Cell Proliferation; Cerebral Ischemia; Cerebrovascular Circulation; Chemosensitization; Electrophysiology (science); Event; Failure; Future; GLAST Protein; Genes; Glial Fibrillary Acidic Protein; Glucose; Gramicidin; Hippocampus (Brain); Hour; Hypoglycemia; Hypoxia; Immunohistochemistry; Individual; Infarction; Ischemia; Lead; Lesion; Light; Mediating; Membrane Potentials; Metabolic; Modeling; Monitor; Necrosis; Neuraxis; Neuronal Injury; Neurons; Neuroprotective Agents; Neurotransmitters; Outcome; Oxygen; Pathology; Peripheral; Pharmaceutical Preparations; Physiological; Potassium Channel; Predisposition; Proliferation Marker; Rattus; Riluzole; Role; Signal Transduction; Simulate; Slice; Staining method; Stains; Stimulus; Stroke; Testing; Therapeutic; Uncertainty; United States; Withdrawal; artery occlusion; base; brain cell; cell type; deprivation; disability; immunocytochemistry; insight; knock-down; member; neuronal survival; neurotransmitter release; novel; post stroke; potassium channel protein TREK-1; public health relevance; resilience; response; therapeutic target; voltage

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.

描述（由申请人提供）：星形胶质细胞是大脑中数量最多的细胞类型，已知可为中枢神经系统（CNS）提供结构、代谢和稳态支持。尽管星形胶质细胞在脑缺血中比神经元能更好地存活，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。电压无关的 K+ 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至关重要。现在我们知道，双孔域 K+ 通道 (K2Ps) K+ 通道的两个成员 TWIK-1 和 TREK-1 属于长期寻找的负责星形胶质细胞被动电导的 K+ 通道。 K2Ps可以受到多种理化和病理刺激的动态调节，包括脑缺血产生的神经元损伤因子（CIPNJFs），例如缺氧、低血糖、酸中毒和神经递质的病理性释放。 K2P表达的病理诱导还导致坏死和凋亡细胞死亡以及细胞增殖，这是发生在缺血性梗塞和半暗带区域的两个突出的病理事件。为了了解星形胶质细胞 K2P 的生理表达如何为星形胶质细胞提供针对早期缺血性损伤的保护，以及长期缺血条件如何诱导反应性星形胶质细胞中 K2P 表达的改变及其对中风后结果的影响，我们假设星形胶质细胞的活性K2P 可以通过 CIPNJF 进行调节，从而保护星形胶质细胞免受早期缺血性损伤，反应性星形胶质细胞中 K2P 表达的改变有助于梗死周围半暗带区域的稳态功能受损。提出了五个具体目标来探索这些完全未知的领域。 1) CIPNJF 对星形胶质细胞膜电位和被动电导的调节。这将在大鼠海马切片中进行，并用短杆菌肽穿孔贴片记录来监测 K2Ps 调节，而不干扰 CIPNJF 介导的细胞内能量衰竭和改变的信号转导； 2) 神经保护剂和 TREK-1 通道调节剂利鲁唑和西帕三嗪调节星形胶质细胞对 CIPNJF 的电生理反应； 3) 通过在器官型海马切片培养物中用 siRNA 选择性沉默星形胶质细胞 K2P，鉴定特定的 K2P-CIPNJF 相互作用机制； 4) 通过共聚焦免疫细胞化学鉴定可逆性颈中动脉闭塞 (rMCAO) 后半影区制备的切片中大鼠海马反应性星形胶质细胞中 K2P 的表达。 5)使用来自大鼠rMCAO半暗区的急性制备的海马切片中的电生理学来识别大鼠局灶性缺血半暗区的反应性星形胶质细胞中的功能性K2P。拟议的研究应该为脑缺血中星形细胞 K2P 的生理作用和病理参与以及这些主要星形细胞 K+ 通道是否可以成为中风治疗策略的潜在靶点提供新的见解。 公共健康相关性：通过测试以下假设：两个新发现的星形胶质细胞双孔域 K+ 通道 TWIK-1 和 TREK-1 的生理表达为脑缺血发作早期的星形胶质细胞提供早期保护，以及 K2P 表达如何反应性星形胶质细胞的改变，可能是中风病理学中星形胶质细胞稳态功能受损的基础，拟议的研究将为星形胶质细胞 K2P 在大脑中的作用提供新的见解。缺血。研究结果还应为基于这些新近认识的且功能重要的星形细胞 K+ 通道的未来中风治疗方案提供启示。