Deregulated lipid metabolism in stroke

中风中脂质代谢失调

• 批准号: 8269920
• 负责人: ROBERT J DEMPSEY
• 金额: $ 31.83万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269920
• 关键词: 1,2-diacylglycerol; Abbreviations; Accounting; Address; Affect; Astrocytes; Attenuated; Brain Injuries; Cell Cycle; Cell Cycle Arrest; Cell Cycle Inhibition; Cell Cycle Proteins; Cell Cycle Regulation; Cell Proliferation; Cells; Ceramides; Cerebral Infarction; Cerebral Ischemia; Cessation of life; Clinical; Clinical Trials; Complement; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Data; Development; Diglycerides; Enzymes; FDA approved; Fibroblast Growth Factor 2; Glucose; Health; Health Care Costs; Healthcare; Homeostasis; In Vitro; Inbred SHR Rats; Infarction; Interleukin-1; Knock-out; Lead; Lecithin; Lipids; Macrophage Activation; Mediating; Metabolism; Microglia; Middle Cerebral Artery Occlusion; Mitotic; Modeling; Neuroglia; Neurons; Oxygen; Palmitoyl Coenzyme A; Pathway interactions; Patients; Phase; Phospholipase C; Phosphorylation; Phosphorylcholine; Proliferating; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Rattus; Recovery; Recovery of Function; Regulation; Reperfusion Therapy; Research; Retinoblastoma; Scheme; Second Messenger Systems; Serine; Small Interfering RNA; Source; Sphingomyelinase; Sphingomyelins; Stroke; System; TNF gene; Testing; Therapeutic; Transient Cerebral Ischemia; Tumor Necrosis Factor-alpha; Up-Regulation; acidic sphingomyelinase; deprivation; disability; in vivo; inhibitor/antagonist; lipid metabolism; macrophage; neuronal survival; neuroprotection; oncoprotein p21; premature; prevent; protein kinase C zeta; public health relevance; research study; response; second messenger; serine palmitoyltransferase; sphingomyelin synthase; thermozymocidin; xanthate D609

DESCRIPTION (provided by applicant): Post-mitotic neurons can enter into the cell cycle after stroke but die instead of proliferating. Our data showed up- regulation of cyclin-dependent kinase 4 (Cdk4) after oxygen-glucose deprivation (OGD)/reoxygenation in near-pure primary cortical neuronal cultures, evidence of cell cycle entry. After cerebral ischemia, expressions of cell cycle proteins are altered. Inhibiting the cell cycle after stroke will provide benefit by attenuating neuronal death and proliferation of microglia/macrophages. Cell cycle regulation by lipid second messengers after stroke: Sphingomyelin (SM) synthase (SMS) transfers phosphocholine from phosphatidylcholine (PC) to ceramide to form SM and 1,2- diacylglycerol (DAG). Ceramide and DAG are key regulators of the cell cycle and altering their formation affects both neuronal and non-neuronal cell fate after stroke. Tricyclodecan-9-yl-xanthogenate (D609) inhibits SMS leading to ceramide accumulation. Ceramide can induce cell cycle arrest by (a) activating protein phosphatases 1 and 2A (PP1 and PP2A), (b) dephosphorylation of retinoblastoma (Rb) and Cdk2 and (c) up-regulation of Cdk inhibitors p21 and p27. Hypothesis: D609 may block the cell cycle, attenuating neuronal death and non-neuronal cell proliferation by increasing ceramide levels after stroke. The effect of D609 on ceramide de novo synthesis pathway will also be examined. In support of our hypothesis, D609 (a) significantly reduced cerebral infarction at reperfusion days 1 and 3, (b) up-regulated p21 and p27 through ceramide accumulation, and (c) attenuated Rb phosphorylation after transient middle cerebral artery occlusion (tMCAO) in rat. Our studies strongly support SMS inhibition by D609 leading to cell cycle arrest. To understand D609 mechanism, the following aims will test the hypothesis: Aim 1: Does D609 inhibit SMS, blocking the cell cycle and providing protection after OGD/reoxygenation in near-pure primary cortical neuronal cultures? Our studies showed that D609 up-regulated p27 in primary neuronal cultures after OGD/reoxygenation, suggesting increased ceramide due to SMS inhibition. Aim 2: How does SMS regulate cell cycle proteins and proliferation of RAW 264.7 macrophages? In vitro silencing of SMS (both in neuronal and macrophage cultures, Aims 1-2) will confirm the actions of D609 mediated through inhibition of SMS. Aim 3: How does D609 regulate SM metabolism, expression of cell cycle proteins, and microglia/macrophage proliferation in rat tMCAO? Our data suggest that D609 neuroprotection is due to increased ceramide levels, up-regulation of p21 and cell cycle arrest after tMCAO. Microglia/macrophages are the primary source of TNF-1 and IL-1 that are rapidly up-regulated after stroke and contribute to brain injury. We anticipate that D609 will reduce proliferation of microglia/macrophages as well as TNF-1 and IL-1ss expression after tMCAO, providing benefit. In vivo SMS silencing and SMS2 conditional (neuron-specific) knockout using cre/loxP system are proposed as alternatives. Translational potential: tPA has limited use in stroke patients. Although it is premature to predict, lipid metabolites that affect the cell cycle system in stroke have not been extensively studied and have not undergone stroke clinical trials. This proposal explores the therapeutic potential of D609 and how it affects lipid second messenger ceramide that regulates the cell cycle both in vitro and in vivo stroke models. PUBLIC HEALTH RELEVANCE: Stroke is a worldwide health care concern and a leading cause of disability. In the USA; healthcare costs are >$63 billion/year. Currently FDA approved tPA has a very limited use in stroke patients. The disappointing NXY-059 stroke clinical trials emphasized the need for new treatments. This research seeks to modulate a lipid metabolite by pharmacologically altering an enzyme system(s) that may offer benefit and may provide clues to develop lead molecules. The long-range thrust of this research is to develop strategies to minimize disabilities due to stroke.

描述（由申请人提供）：有丝分裂后神经元可以在中风后进入细胞周期，但死亡而不是增殖。我们的数据显示，在接近纯的原代皮质神经元培养物中，氧糖剥夺（OGD）/复氧后，细胞周期蛋白依赖性激酶4（Cdk4）上调，这是细胞周期进入的证据。脑缺血后，细胞周期蛋白的表达发生改变。中风后抑制细胞周期将通过减少神经元死亡和小胶质细胞/巨噬细胞的增殖来提供益处。中风后脂质第二信使对细胞周期的调节：鞘磷脂 (SM) 合酶 (SMS) 将磷酸胆碱从磷脂酰胆碱 (PC) 转移到神经酰胺，形成 SM 和 1,2-二酰基甘油 (DAG)。神经酰胺和 DAG 是细胞周期的关键调节因子，改变它们的形成会影响中风后神经元和非神经元细胞的命运。 Tricyclodecan-9-yl-xantogenate (D609) 抑制 SMS 导致神经酰胺积累。神经酰胺可通过 (a) 激活蛋白磷酸酶 1 和 2A（PP1 和 PP2A）、(b) 视网膜母细胞瘤 (Rb) 和 Cdk2 去磷酸化以及 (c) 上调 Cdk 抑制剂 p21 和 p27 来诱导细胞周期停滞。假设：D609 可能通过增加中风后的神经酰胺水平来阻断细胞周期，从而减少神经元死亡和非神经元细胞增殖。还将检查 D609 对神经酰胺从头合成途径的影响。为了支持我们的假设，D609 (a) 在再灌注第 1 天和第 3 天显着减少脑梗塞，(b) 通过神经酰胺积累上调 p21 和 p27，以及 (c) 在短暂性大脑中动脉闭塞 (tMCAO) 后减弱 Rb 磷酸化在大鼠中。我们的研究强烈支持 D609 抑制 SMS 导致细胞周期停滞。为了了解 D609 机制，以下目标将检验该假设： 目标 1：D609 是否会抑制 SMS、阻断细胞周期并在近乎纯的原代皮质神经元培养物中的 OGD/复氧后提供保护？我们的研究表明，OGD/复氧后，D609 上调原代神经元培养物中的 p27，表明由于 SMS 抑制而导致神经酰胺增加。目标 2：SMS 如何调节细胞周期蛋白和 RAW 264.7 巨噬细胞的增殖？ SMS 的体外沉默（在神经元和巨噬细胞培养物中，目标 1-2）将证实 D609 通过抑制 SMS 介导的作用。目标 3：D609 如何调节大鼠 tMCAO 中的 SM 代谢、细胞周期蛋白的表达以及小胶质细胞/巨噬细胞增殖？我们的数据表明，D609 的神经保护作用是由于 tMCAO 后神经酰胺水平升高、p21 上调和细胞周期停滞所致。小胶质细胞/巨噬细胞是 TNF-1 和 IL-1 的主要来源，它们在中风后迅速上调并导致脑损伤。我们预计 D609 将减少 tMCAO 后小胶质细胞/巨噬细胞的增殖以及 TNF-1 和 IL-1ss 的表达，从而带来益处。建议使用 cre/loxP 系统进行体内 SMS 沉默和 SMS2 条件（神经元特异性）敲除作为替代方案。转化潜力：tPA 在中风患者中的应用有限。尽管现在预测还为时过早，但影响中风细胞周期系统的脂质代谢物尚未得到广泛研究，也没有进行中风临床试验。该提案探讨了 D609 的治疗潜力以及它如何影响调节体外和体内中风模型细胞周期的脂质第二信使神经酰胺。 公共卫生相关性：中风是一个全球性的医疗保健问题，也是导致残疾的主要原因。在美国；医疗保健费用每年超过 630 亿美元。目前 FDA 批准的 tPA 在中风患者中的用途非常有限。令人失望的 NXY-059 中风临床试验强调需要新的治疗方法。这项研究试图通过药理学改变酶系统来调节脂质代谢物，这可能会带来益处，并可能为开发先导分子提供线索。这项研究的长期目标是制定策略，尽量减少中风造成的残疾。