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 热激蛋白（包括HSP70）是由热冲击引起的伴侣，许多压力包括 蛋白质合成过程中的缺血并有助于蛋白质折叠，并在蛋白质变性后重新折叠。 HSP70 蛋白质表达在多种模型中具有多种不同方法的神经保护作用 表达。该建议将解决HSP70的受保护，抗凋亡和抗炎作用：（1） 神经元和神经胶质的主要培养； （2）局灶性脑缺血之后。研究的新方面将 使用新近使用新近的重组HSP70，HSP70C和突变体HSP70C-DEVD蛋白使用重组HSP70，使用新的 开发的抗DNA抗体的单链片段称为FV。 FV-HSP70与核苷结合 在所有细胞上发现的打捞转运蛋白ENT2，FV-HSP70通过ATP独立ENT2进入这些细胞 转运蛋白。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； （b）FVHSP70 减少梗塞体积并改善大脑中动脉闭塞后的行为结果 （MCAO）体内； （c）和促炎和促凋亡基因在血清细胞中诱导的基因 MCAO通过体内的FVHSP70处理减少。因此，我们提出以下目标。 特定目标＃1A：证明FV-HSP70，FV-HSP70C和突变体FV-HSP70C-DEVD受保护的培养 来自氧气葡萄糖剥夺（OGD）的神经元和星形胶质细胞。特定目标＃1B：开始探索反 - 通过检查HSP70，NF¿B之间的相互作用，侵蚀性和抗炎机制 来自大脑的原代细胞中的TNF；并表明HSP70阻断了原代脑细胞中的NF¿B激活。 特定目的＃2：证明FV-HSP70构造减少梗塞量并改善行为 局灶性脑缺血大鼠模型的结果。 FV-HSP70，FV-HSP70C和FV-HSP70C-DEVD的影响 将相互比较，与媒介物，仅HSP70，单独使用FV和FV绿色荧光蛋白（GFP） 控件。特定目标＃3A。在MCAO中风后进行大鼠血液的基因组分析 目标＃2并证明MCAO会影响一套破坏性的促凋亡和促炎基因 抑制血液中白细胞的促阳离子基因。特定目的＃3b：证明使用FV-治疗 MCAO后大鼠中的HSP70构造减弱了血清细胞中有害基因的反应，并将 增加抗凋亡性，增加抗炎作用并增加大鼠血清细胞中其他促生物性基因。 意义：这些研究将提供原理证明，HSP70作为FV-静脉内给药 融合蛋白进入大脑并改善中风的结果。更一般地，FV蛋白输送方法 这里使用的可能有助于输送任何蛋白质治疗中风，对大脑和脊柱的其他急性损伤 脐带，可能治疗退化性神经系统疾病。我们还建议监测基因表达 可以使用与啮齿动物中有效卒中治疗相关的外周血的变化来评估 人类的潜在治疗响应能力。