Hibernation--a New Approach To Stroke Therapy

冬眠——中风治疗的新方法

• 批准号: 9358538
• 负责人: John Hallenbeck
• 金额: $ 141.11万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9358538
• 关键词: Animals; Area; Arousal; Biological Clocks; Blood flow; Body Temperature; Brain; Brain Injuries; Brain Ischemia; Brain region; Cell Culture System; Cell Culture Techniques; Cell Death; Cell Survival; Cell membrane; Cell physiology; Cells; Cellular biology; Choroid; Clinical Trials; Cloning; Complementary DNA; Cytoprotection; Data; Depressed mood; Detection; Development; Dominant-Negative Mutation; Eating; Enzymes; Ependymal Cell; Epithelial Cells; FOS gene; Fourth ventricle structure; Glucose; Goals; Hibernation; Hippocampus (Brain); Histocytochemistry; Homeostasis; Human; Hypothalamic structure; Hypoxia; In Situ Hybridization; In Vitro; Lead; Libraries; Mammalian Cell; Measures; Medial; Messenger RNA; Metabolic; MicroRNAs; Modeling; Molecular; Nature; Neuraxis; Neurons; Organ; Oxygen; Phase; Phosphorylation; Phosphotransferases; Polyribosomes; Post-Translational Protein Processing; Pre-Clinical Model; Preoptic Areas; Protein Biosynthesis; Protein Dephosphorylation; Protein Synthesis Inhibition; Protein phosphatase; Proteins; Rattus; Regulation; Research; Research Project Grants; Resistance; Role; Signal Transduction; Slice; Small Interfering RNA; Spermophilus; Squirrel; Stress; Stroke; Structure of choroid plexus; Structure of nucleus infundibularis hypothalami; Temperature; Testing; Thalamic Nuclei; Third ventricle structure; Time; Tissues; Transfection; Transgenic Mice; Translating; Ubiquitin; Ubiquitin-Conjugating Enzymes; Work; awake; deprivation; gamma-Glutamyl Hydrolase; high throughput screening; hypoperfusion; in vivo; in vivo Model; knock-down; lateral ventricle; metabolic depression; natural hypothermia; neuroblastoma cell; novel strategies; overexpression; phase change; prevent; research study; screening; small molecule; stroke therapy; stroke treatment; suprachiasmatic nucleus

Postischemic progression of brain damage is extremely multifactorial. We are analyzing a state in which nature has solved this problematic biocomplexity. Hibernation with its metabolic, hematologic and cell membrane adjustments permits animals to withstand extremely low blood flow in the brain for protracted periods with no cellular loss. Efforts to isolate and identify the mechanisms that regulate the controlled metabolic depression and tolerance of profound brain hypoperfusion that forms the essence of natural hibernation have been conducted. In hippocampal slices, hibernation confers robust resistance to hypoxia and glucose deprivation as compared to slices from non-hibernating ground squirrels and rats at 37 degrees C, 20 degrees C and 7 degrees C. These findings indicate that hibernation involves tolerance to an in vitro form of ischemic stress that is not strictly dependent on temperature. Protein synthesis (PS) in hippocampal slices is greatly depressed at the same incubation temperatures. PS in vivo was below the limit of autoradiographic detection in brain sections, and in brain extracts was determined to be 0.04% of the average rate from active squirrels. Further, it was threefold reduced in cell-free extracts from hibernating brain at 37 degrees C, eliminating hypothermia as the only cause for protein synthesis inhibition. PS suppression involved blocks of both initiation and elongation and its onset coincided with the entrance phase of the hibernation bout. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2a are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a threefold increase in ribosomal mean transit times in cell-free extracts from hibernators. Phosphorylation of eEF2 is increased, eEF2 kinase activity is increased, and protein phosphatase 2A activity is decreased during hibernation which contributes to the elongation block. No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate "trickle" blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state. Dephosphorylation of Akt/PKB has been found to occur in multiple tissues during hibernation. We find that depending on cellular contex in mammalian cells, cytoprotection can be associated with strong Akt activation, modulated Akt activation or modulated Akt inhibition. By cloning c-fos cDNA from the 13-lined ground squirrel (Spermophilus tridecemlineatus) and using squirrel c-fos mRNA probe for in situ hybridization histochemistry, we systematically analyzed and identified specific brain regions that were activated during six different phases of the hibernation bout. During entrance into torpor, we detected activation of the ventrolateral subdivision of the medial preoptic area ('thermoregulatory center'), and the reticular thalamic nucleus, which is known to inhibit the somatomotor cortex. During torpor, c-fos expression in the cortex was suppressed while the reticular thalamic nucleus remained uniformly active. Throughout torpor the suprachiasmatic nucleus ('biological clock') showed increasing activity, likely participating in phase-change regulation of the hibernation bout. Interestingly, during torpor very strong c-fos activation was seen in the epithelial cells of the choroid plexus and in tanycytes at the third ventricle, both peaking near the beginning of arousal. In arousal, activity of the suprachiasmatic and reticular thalamic nuclei and choroid epithelial cells diminished, while ependymal cells in the lateral and fourth ventricles showed stronger activity. Increasing body temperature during arousal was driven by the activation of neurons in the medial part of the preoptic area. In interbout awake animals, we demonstrated the activation of hypothalamic neurons located in the arcuate nucleus and the dorsolateral hypothalamus, areas involved in food intake. Our observations indicate that the hibernation bout is closely regulated and orchestrated by specific regions of the central nervous system. The small ubiquitin-like modifier (SUMO) leads to widespread effects in cell biology by post-translational modification of many proteins; it functions to preserve homeostasis under stress. We have found massive global SUMOylation of proteins in body tissues during hibernation torpor. We have also found that SUMOylation is essential for cell survival and that maintained or augmented SUMOylation is robustly cytoprotective in cell culture systems. The expression level of ubiquitin conjugating enzyme-9 (Ubc9) protein, the single SUMO-conjugating enzyme, was well correlated with the SUMOylation levels in the squirrels during hibernation bouts. In addition, the overexpression of Ubc9 by transfection enhanced the tolerance of SHSY5Y human neuroblastoma cells to oxygen/glucose deprivation (OGD), while the overexpression of a dominant negative mutant of Ubc9 sensitized the cells to OGD. Further experiments in primary neuronal cultures have shown that overexpression of SUMO1 and SUMO2 are cytoprotective and that knock down of SUMO1 with siRNA increases cell death during OGD. Transgenic mice that overexpress Ubc9, the E2 specific conjugase for SUMOylatiion of proteins, are showing that modest increases of Ubc9 increase global SUMOylation levels and confer a corresponding level of resistance to brain ischemia. We have established that the level of global SUMOylation is directly proportional to the level of cytoprotection in preclinical models of stroke. We have also examined the effects of SUMO-1 and SUMO-2/3 SUMOylated proteins on ischemic tolerance in our cell culture models. We have obtained clear data supporting a role for SUMOylation in ischemic tolerance. Current work adresses the study of molecular mechanisms that can control the level of global SUMOylation. We seek to boost global SUMOylation to levels seen in hibernating animals to harness the extraordinary cytoprotection inherent in this form of post-translational modification. To this end, we have identified microRNAs as regulators of both global SUMOylation and the post-translational modification levels with other ubiquitin-like modifiers. We have developed several high throughput assays for screening of compound libraries. We have identified a group of compounds with a robust capacity to globally SUMOylate proteins and are testing these compounds by means of in vitro and in vivo models of brain ischemia. We have identified molecular entities that can increase gobal SUMOylation and cytoprotect cultured neurons exposed to in vitro oxygen-glucose deprivation. We are also investigating cytoprotective changes in cell state related to such signaling.

脑损伤后脑损伤的进展极为多。我们正在分析一种自然解决这种有问题的生物复杂性的状态。冬眠及其代谢，血液学和细胞膜调节允许动物在旷日持久的较长时期内承受大脑中极低的血液流动，而没有细胞损失。已经努力隔离和确定调节被控制的代谢抑郁症的机制和对构成自然冬眠本质的深刻脑部灌注的耐受性。在海马切片中，冬眠赋予了对缺氧和葡萄糖剥夺的耐药性，与在37度C，20度C和7度处的非阳光地下松鼠和大鼠切片相比，这些发现表明，冬眠涉及耐心的压力并不是严格依赖的温度。海马切片中的蛋白质合成（PS）在相同的孵育温度下大大降低。体内PS含量低于脑部的放射自显影检测极限，并且在脑提取物中确定为活性松鼠平均速率的0.04％。此外，在37摄氏度的冬眠大脑中无细胞提取物中降低了三倍，从而消除了体温过低，这是蛋白质合成抑制的唯一原因。 PS抑制涉及启动和伸长的块及其发作与冬眠回合的入口阶段相吻合。单体峰增加，多核糖体谱分解和eIF2A的磷酸化大大增加与冬眠中的起始阻滞一致。通过冬眠器中无细胞提取物中的核糖体平均过渡时间增加了三倍，证明了伸长块。 EEF2的磷酸化增加，EEF2激酶活性增加，在冬眠期间蛋白质磷酸酶2a活性降低，这有助于伸长块。未检测到核糖体功能或mRNA水平的异常。这些发现暗示对PS的抑制是细胞功能关闭的关闭的组成部分，允许冬眠的地松鼠耐受“ trick流”血流，减少底物和氧气可用性。对控制这些现象的因素的进一步研究可能导致鉴定调节该状态的分子机制。在冬眠期间，已经发现AKT/PKB的去磷酸化发生在多个组织中。我们发现，根据哺乳动物细胞中的细胞contex，细胞保护可能与强AKT激活，调节AKT激活或调节AKT抑制有关。 通过从13层的地松鼠（Spermophilus tridecemlineatus）中克隆C-FOS cDNA，并使用松鼠C-FOS mRNA探针进行原位杂交组织化学，我们可以系统地分析并鉴定出在冬眠效果的六个不同阶段中激活的特定大脑区域。在进入Torpor的过程中，我们检测到内侧前区域（“温度调节中心”）和网状丘脑核的腹外侧细分的激活，已知会抑制体形运动皮质。在TORPOR期间，抑制了皮层中的C-FOS表达，而网状丘脑核保持均匀活跃。在整个过程中，上核核（“生物钟”）表现出增加的活性，可能参与冬眠回合的相位变化调节。有趣的是，在脉络丛的上皮细胞和第三个心室的tanycytes中，观察到非常强的C-FOS激活，都在唤醒开始附近达到峰值。在唤醒中，上骨和网状丘脑核和脉络膜上皮细胞的活性减少，而外侧和第四脑室中的doment室细胞显示出更强的活性。唤醒过程中体温的升高是由前跨区域内侧部分的神经元激活驱动的。在Interbout Awake动物中，我们证明了位于弧形核和背外侧下丘脑的下丘脑神经元的激活，该下丘脑涉及食物摄入量。我们的观察结果表明，中枢神经系统的特定区域密切调节冬眠回合。小型泛素样修饰剂（SUMO）通过许多蛋白质的翻译后修饰导致细胞生物学的广泛影响。它的功能可以在压力下保持体内平衡。我们已经发现冬眠torpor期间人体组织中蛋白质的全球属性大量。我们还发现，sumoylation对于细胞存活至关重要，并且在细胞培养系统中维持或增强的sumoylation具有牢固的细胞保护作用。泛素共轭酶-9（UBC9）蛋白的表达水平与单个SUMO偶联酶的表达水平与冬眠爆发过程中松鼠的Sumoylation水平良好相关。 此外，通过转染对UBC9的过表达增强了SHSY5Y人类神经细胞对氧气/葡萄糖剥夺（OGD）的耐受性，而UBC9显性负突变体的过表达使细胞对OGD敏感。 在原发性神经元培养物中的进一步实验表明，SUMO1和SUMO2的过表达是细胞保护的，并且SUMO1用siRNA击倒SUMO1会增加OGD期间的细胞死亡。过表达UBC9的转基因小鼠，蛋白质的E2特异性结合酶，表明UBC9的适度增加增加了全局Sumoylation水平，并赋予对脑缺血的相应抗性水平。我们已经确定，全局sumoylation的水平与中风临床前模型中的细胞保护水平成正比。我们还研究了SUMO-1和SUMO-2/3 Sumoylated蛋白对细胞培养模型中缺血性耐受性的影响。我们已经获得了明确的数据，支持了缺血性耐受性的sumoylation作用。当前的工作适应了可以控制全局sumoylation水平的分子机制的研究。我们试图将全球sumoylation提高到冬眠动物中看到的水平，以利用这种翻译后修饰形式固有的非凡细胞保护作用。为此，我们将microRNA确定为具有其他类似泛素样修饰剂的全局sumoylation和翻译后修饰水平的调节剂。我们已经开发了几种高通量测定，用于筛选复合库。我们已经确定了一组具有强大能力的化合物，可以通过体外和体内脑缺血模型来测试这些化合物。我们已经确定了可以增加gobal sumoylation和细胞保护培养的神经元的分子实体，暴露于体外氧葡萄糖剥夺。我们还正在研究与此类信号相关的细胞状态细胞保护变化。