Deregulated lipid metabolism in stroke

中风中脂质代谢失调

• 批准号: 7983656
• 负责人: RAO M ADIBHATLA
• 金额: $ 32.48万
• 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/7983656
• 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; Coenzyme A; Complement; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Data; Development; Diglycerides; Enzymes; FDA approved; Fibroblast Growth Factor 2; Figs - dietary; Glucose; Health; Health Care Costs; Healthcare; Homeostasis; Hydrolysis; 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; 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; Testing; Therapeutic; Transient Cerebral Ischemia; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; 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）：溶后神经元可以在中风后进入细胞周期，但死亡而不是增殖。我们的数据表明，在近光质原发性皮质神经元培养物中，氧气 - 葡萄糖剥夺（OGD）/二氧化剂后，细胞周期蛋白依赖性激酶4（CDK4）的调节是细胞周期进入的证据。脑缺血后，细胞周期蛋白的表达改变。抑制中风后的细胞周期将通过减轻小胶质细胞/巨噬细胞的神经元死亡和增殖来提供益处。中风后脂质第二信使调节细胞周期：鞘磷脂（SM）合酶（SMS）将磷胆碱从磷脂酰胆碱（PC）转移到神经酰胺中，形成SM和1,2-二酰甘油（DAG）。神经酰胺和DAG是细胞周期的关键调节剂，改变其形成会影响中风后神经元和非神经元细胞命运。三环甘油-9-基-Xanthogenate（D609）抑制导致神经酰胺积累的SMS。神经酰胺可以通过（a）激活蛋白质磷酸酶1和2a（pp1和pp2a），（b）视网膜磷酸化的细胞周期停滞，（b）视网膜细胞瘤（RB）和CDK2的去磷酸化以及CDK抑制剂P21和P27的上调。假设：D609可能会通过中风后增加神经酰胺水平来阻止细胞周期，减弱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和第3天，（b）通过神经酰胺的积累上调的P21和P27显着降低了脑梗塞，（c）（c）（c）在比例中瞬时中大脑动脉（TMCAO）瞬时中等脑磷酸化。我们的研究强烈支持D609的SMS抑制，从而导致细胞周期停滞。要了解D609机制，以下目的将检验假设：目标1：D609是否抑制SMS，阻止细胞周期并在OGD/Rexygenation后在近光质原发性皮质神经元培养物中提供保护？我们的研究表明，D609在OGD/Rexygyation后原代神经元培养物中上调P27，这表明由于SMS抑制而引起的神经酰胺增加。 AIM 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（无论是在神经元和巨噬细胞培养物中，目标1-2）将确认通过抑制SMS介导的D609的作用。 AIM 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神经保护是由于神经酰胺水平升高，P21的上调和TMCAO后的细胞周期停滞。小胶质细胞/巨噬细胞是中风后迅速上调并导致脑损伤的主要来源。我们预计D609将减少TMCAO后的小胶质细胞/巨噬细胞以及TNF-1和IL-1SS表达的增殖，从而提供益处。使用CRE/LOXP系统有条件地敲除体内SMS沉默和SMS2（神经元特异性）敲除作为替代方案。翻译潜力：TPA在中风患者中使用有限。尽管预测为时过早，但影响中风细胞周期系统的脂质代谢产物尚未进行广泛研究，也没有接受中风临床试验。该建议探讨了D609的治疗潜力以及它如何影响脂质第二信使神经酰胺，从而调节体外和体内中风模型的细胞周期。 公共卫生相关性：中风是全球医疗保健问题，也是残疾的主要原因。在美国； /年的医疗保健费用> 630亿美元。目前，FDA批准的TPA在中风患者中使用非常有限。令人失望的NXY-059中风临床试验强调了对新治疗的必要性。这项研究旨在通过在药理学上改变酶系统来调节脂质代谢产物，该酶系统可能会提供益处，并可能提供开发铅分子的线索。这项研究的远距离推力是制定策略，以最大程度地减少中风的残疾。