Heat Shock Protein Therapeutics for Stroke

热休克蛋白治疗中风

• 批准号: 8461190
• 负责人: Robert Nishimura
• 金额: $ 48.56万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461190
• 关键词: Acute; Address; Anti-DNA Antibodies; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Apoptotic; Astrocytes; Attenuated; Behavioral; Binding; Biological Markers; Blood; Brain; Caspase; Cells; Cerebral Ischemia; Chimeric Proteins; Client; Coupled; Data; Future; Gene Expression; Gene Expression Profiling; Genes; Genome; Genomics; Glucose; Green Fluorescent Proteins; Heat shock proteins; Heat-Shock Response; Human; In Vitro; Infarction; Inflammation; Inflammatory; Inflammatory Response; Injury; Ischemia; Leukocytes; MMP9 gene; Measures; Mediating; Methods; Microglia; Middle Cerebral Artery Occlusion; Mitochondria; Modeling; Molecular Chaperones; Mutate; Neuroglia; Neurons; Nucleosides; Outcome; Oxygen; Pathway interactions; Property; Protein Biosynthesis; Protein Denaturation; Proteins; RNA; Rattus; Recombinants; Research Personnel; Rodent; Role; Signal Transduction; Sorting - Cell Movement; Spinal Cord; Stress; Stroke; TNF gene; Therapeutic; Transfection; Transgenes; Viral; acute stroke; brain cell; caspase-3; caspase-9; deprivation; improved; in vivo; inhibitor/antagonist; mutant; nervous system disorder; novel; peripheral blood; protein degradation; protein expression; protein folding; response

ABSTRACT Heat shock proteins, including Hsp70, are chaperones induced by heat shock and many stresses including ischemia and assist protein folding during protein synthesis and re-folding after protein denaturation. Hsp70 protein expression is neuroprotective in a variety of models using a number of different methods of over expression. This proposal will address protective, anti-apoptotic and anti-inflammatory roles of Hsp70 in: (1) primary cultures of neurons and glia; and (2) following focal cerebral ischemia. A novel aspect of the study will be to intravenously administer recombinant Hsp70, Hsp70C and mutant Hsp70C-DEVD proteins using a newly developed single-chain fragment of an anti-DNA antibody referred to as Fv. Fv-Hsp70 binds to the nucleoside salvage transporter ENT2 found on all cells and the Fv-Hsp70 enters these cells via the ATP independent ENT2 transporter. Our preliminary data show: (a) that Fv-Hsp70 protects neurons and glia in vitro; (b) FvHsp70 decreases infarct volumes and improves behavioral outcomes following middle cerebral artery occlusions (MCAO) in vivo; (c) and pro-inflammatory and pro-apoptotic genes induced in blood leukocytes following MCAO are decreased by treatment with FvHsp70 in vivo. Therefore, we propose the following aims. Specific Aim #1a: Demonstrate that Fv-Hsp70, Fv-Hsp70C and mutant Fv-Hsp70C-DEVD protect cultured neurons and astrocytes from oxygen glucose deprivation (OGD). Specific Aim #1b: Begin to explore the anti- apoptotic and anti-inflammatory mechanisms of protection by examining the interaction between Hsp70, NF¿B and TNF in primary cells from brain; and show that Hsp70 blocks NF¿B activation in primary brain cells. Specific Aim #2: Demonstrate that the Fv-Hsp70 constructs decrease infarct volumes and improve behavioral outcomes in rat models of focal cerebral ischemia. The effects of Fv-Hsp70, Fv-Hsp70C and Fv-Hsp70C-DEVD will be compared to each other and to vehicle, Hsp70 alone, Fv alone and Fv-Green Fluorescent Protein (GFP) controls. Specific Aim #3a. Perform genomic profiling of rat blood following the MCAO strokes produced in Aim #2 and demonstrate that MCAO induces a damaging set of pro-apoptotic and pro-inflammatory genes while suppressing pro-survival genes in leukocytes in blood. Specific Aim #3b: Demonstrate that treatment with Fv- Hsp70 constructs in rats following MCAO attenuates the deleterious gene response in blood leukocytes and will increase anti-apoptotic, increase anti-inflammatory and increase other pro-survival genes in rat blood leukocytes. Significance: These studies will provide a proof of principle that Hsp70, administered intravenously as a Fv- fusion protein, enters brain and improves outcome from stroke. More generally, the Fv protein delivery method used here could be useful for delivering any protein to treat stroke, other acute injuries to the brain and spinal cord, and possibly to treat degenerative neurological diseases. We also propose that monitoring gene expression changes in peripheral blood that correlate with effective stroke treatments in rodents can be used to assess potential treatment responsiveness in humans.

抽象的 热休克蛋白（包括 Hsp70）是由热休克和许多应激（包括 缺血并在蛋白质合成过程中协助蛋白质折叠以及蛋白质变性后的重新折叠。热休克蛋白70 蛋白质表达在多种模型中具有神经保护作用，使用多种不同的方法 表达。该提案将解决 Hsp70 在以下方面的保护、抗凋亡和抗炎作用：(1) 神经元和神经胶质细胞的原代培养； (2)局灶性脑缺血后。该研究的一个新颖方面将 使用新的方法静脉注射重组 Hsp70、Hsp70C 和突变型 Hsp70C-DEVD 蛋白 开发了一种抗 DNA 抗体的单链片段，称为 Fv。 Fv-Hsp70 与核苷结合 所有细胞上都发现了补救转运蛋白 ENT2，Fv-Hsp70 通过不依赖于 ATP 的 ENT2 进入这些细胞 运输者。我们的初步数据显示：(a) Fv-Hsp70 在体外保护神经元和神经胶质细胞； (b) FvHsp70 减少大脑中动脉闭塞后的梗塞体积并改善行为结果 (MCAO)体内； (c) 以及在血液白细胞中诱导的促炎和促凋亡基因 体内用 FvHsp70 处理可减少 MCAO。因此，我们提出以下目标。 具体目标 #1a：证明 Fv-Hsp70、Fv-Hsp70C 和突变体 Fv-Hsp70C-DEVD 可以保护培养物 神经元和星形胶质细胞免受氧糖剥夺（OGD）的影响。具体目标#1b：开始探索反 通过检查 Hsp70、NF¿B 之间的相互作用来研究细胞凋亡和抗炎保护机制 和脑原代细胞中的 TNF；并表明 Hsp70 可以阻断原代脑细胞中 NF¿B 的激活。 具体目标 #2：证明 Fv-Hsp70 构建体可减少梗塞体积并改善行为 局灶性脑缺血大鼠模型的结果。 Fv-Hsp70、Fv-Hsp70C 和 Fv-Hsp70C-DEVD 的作用 将相互比较并与媒介物、单独的 Hsp70、单独的 Fv 和 Fv-绿色荧光蛋白 (GFP) 进行比较 控制。具体目标#3a。在 MCAO 中风后对大鼠血液进行基因组分析 目标 #2 并证明 MCAO 会诱导一组具有破坏性的促凋亡和促炎症基因，同时 抑制血液中白细胞中的促生存基因。具体目标#3b：证明使用 Fv- 进行治疗 MCAO 后大鼠体内的 Hsp70 构建体减弱了血液白细胞中的有害基因反应，并将 增加大鼠血液白细胞中的抗凋亡、抗炎和其他促生存基因。 意义：这些研究将提供原理证明，即 Hsp70 作为 Fv- 静脉注射 融合蛋白进入大脑并改善中风的结果。更一般地，Fv蛋白递送方法 这里使用的可用于输送任何蛋白质来治疗中风、大脑和脊髓的其他急性损伤 脊髓，并可能治疗退行性神经系统疾病。我们还建议监测基因表达 与啮齿动物有效中风治疗相关的外周血变化可用于评估 人类潜在的治疗反应。