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: 8269917
• 负责人: MIN ZHOU
• 金额: $ 32.69万
• 依托单位: OHIO STATE UNIVERSITY
• 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/8269917
• 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，已知为中枢神经系统（CNS）提供结构，代谢和稳态支持。虽然星形胶质细胞在脑缺血中比神经元能更好地存活，但不同脑细胞之间这种不同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，是长期寻找的K+通道之一，负责星形胶质细胞的被动电导。K2Ps可以受到多种生理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），如缺氧、低血糖、酸中毒和神经递质的病理释放。病理诱导的K2P表达也有助于坏死、凋亡细胞死亡和细胞增殖，这是发生在缺血性梗死和半暗区两个突出的病理事件。为了了解星形胶质细胞K2Ps的生理表达如何保护星形胶质细胞免受早期缺血损伤，以及长期缺血条件如何诱导反应性星形胶质细胞中K2P表达的改变及其对卒中后预后的影响，我们假设星形胶质细胞K2Ps的活性可以通过CIPNJFs调节，从而保护星形胶质细胞免受早期缺血损伤。反应性星形胶质细胞中K2P表达的改变导致梗死周围半暗区稳态功能受损。提出了探索这些完全未知领域的五个具体目标。1) CIPNJFs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，用革兰霉素穿孔贴片记录，以监测K2Ps的调节，而不干扰CIPNJFs介导的细胞内能量衰竭和信号转导的改变；2)神经保护剂、TREK-1通道调节剂利鲁唑和西帕替嗪对星形胶质细胞对CIPNJFs的电生理反应的调节；3)在器官型海马片培养中，通过sirna选择性沉默星形细胞K2Ps，确定特定的K2P-CIPNJF相互作用机制；4)用共聚焦免疫细胞化学方法检测可逆性颈动脉中闭塞（rMCAO）后半暗区大鼠海马反应性星形胶质细胞中K2P的表达。5)利用电生理学方法在大鼠rMCAO半暗区急性制备海马切片中鉴定局灶性缺血半暗区反应性星形胶质细胞的功能性K2P。这些研究将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的星形细胞K+通道是否可能成为脑卒中治疗策略的潜在靶点提供新的见解。