Heat Shock Protein Therapeutics for Stroke

热休克蛋白治疗中风

• 批准号: 7937620
• 负责人: Robert Nishimura
• 金额: $ 53.99万
• 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/7937620
• 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; 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; apoptotic protease-activating factor 1; 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; public health relevance; response

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This proposal will examine the neuroprotective properties of the heat shock protein, Hsp70. A newly developed protein delivery method, termed Fv, will be used to deliver Fv-Hsp70 and Hsp70 mutants to primary brain cells and to brain. We will demonstrate that Fv-Hsp70 constructs protect neurons and glia from oxygen and glucose deprivation and that FvHsp70 constructs protect rat brain against stroke. The Fv-Hsp70 mediated protection will be due in part to blockade of inflammation and blockade of pro-apoptotic pathways in both blood and brain. It is proposed that FvHsp70 could be used to treat humans with stroke, and that changes of gene expression in blood of rats that correlate with improved outcome in rats can be used as biomarkers to predict improved outcomes in humans treated with FvHsp70.

描述（由申请人提供）：热休克蛋白，包括Hsp70，是由热休克和包括缺血在内的许多应激诱导的伴侣，并在蛋白质合成期间协助蛋白质折叠以及蛋白质变性后的重新折叠。 Hsp70 蛋白表达在使用多种不同过表达方法的多种模型中具有神经保护作用。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是使用新开发的抗 DNA 抗体（称为 Fv）的单链片段，静脉注射重组 Hsp70、Hsp70C 和突变型 Hsp70C-DEVD 蛋白。 Fv-Hsp70 与所有细胞上发现的核苷补救转运蛋白 ENT2 结合，并且 Fv-Hsp70 通过不依赖于 ATP 的 ENT2 转运蛋白进入这些细胞。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 减少体内大脑中动脉闭塞 (MCAO) 后的梗塞体积并改善行为结果； (c) MCAO后血液白细胞中诱导的促炎和促凋亡基因通过体内FvHsp70处理而减少。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 可以保护培养的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：通过检查脑原代细胞中 Hsp70、NF:B 和 TNF 之间的相互作用，开始探索抗凋亡和抗炎保护机制；并表明 Hsp70 可阻断原代脑细胞中 NF:B 的激活。具体目标#2：证明 Fv-Hsp70 构建体可减少局灶性脑缺血大鼠模型的梗塞体积并改善行为结果。 Fv-Hsp70、Fv-Hsp70C 和 Fv-Hsp70C-DEVD 的效果将相互比较，并与媒介物、单独的 Hsp70、单独的 Fv 和 Fv-绿色荧光蛋白 (GFP) 对照进行比较。具体目标#3a。在目标 #2 中产生 MCAO 中风后，对大鼠血液进行基因组分析，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：证明在 MCAO 后用 Fv-Hsp70 构建体治疗大鼠可减弱血液白细胞中的有害基因反应，并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：这些研究将为 Hsp70 作为 Fv 融合蛋白静脉注射进入大脑并改善中风预后提供原理证明。更一般地说，这里使用的 Fv 蛋白递送方法可用于递送任何蛋白质以治疗中风、其他大脑和脊髓急性损伤，并可能用于治疗退行性神经系统疾病。我们还建议，监测与啮齿动物有效中风治疗相关的外周血基因表达变化可用于评估人类潜在的治疗反应。 公共健康相关性：该提案将检查热休克蛋白 Hsp70 的神经保护特性。一种新开发的蛋白质递送方法，称为 Fv，将用于将 Fv-Hsp70 和 Hsp70 突变体递送至原代脑细胞和大脑。我们将证明 Fv-Hsp70 构建体可以保护神经元和神经胶质细胞免受缺氧和葡萄糖的影响，并且 FvHsp70 构建体可以保护大鼠大脑免受中风。 Fv-Hsp70 介导的保护部分归因于血液和大脑中炎症的阻断和促凋亡途径的阻断。有人提出，FvHsp70 可用于治疗人类中风，并且与大鼠结果改善相关的大鼠血液中基因表达的变化可用作生物标志物，以预测接受 FvHsp70 治疗的人类结果的改善。