Heat Shock Protein Therapeutics for Stroke

摘要 热休克蛋白，包括Hsp70，是由热休克和许多应激诱导的伴侣蛋白，包括 在蛋白质合成和蛋白质变性后的再折叠过程中，缺血和辅助蛋白质折叠。热休克蛋白70 在使用多种不同方法的各种模型中，蛋白质的表达具有神经保护作用 表情。这项提案将解决Hsp70在以下方面的保护、抗凋亡和抗炎作用：(1) 原代培养神经元和神经胶质细胞；(2)局灶性脑缺血后。这项研究的一个新方面将是 将重组Hsp70、Hsp70C和突变型Hsp70C-DEVD蛋白通过一种新的 开发了一种被称为Fv的抗DNA抗体的单链片段。Fv-Hsp70与核苷结合 在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 传送器。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 大脑中动脉闭塞后缩小脑梗塞体积和改善行为结局 (C)在下列情况下在血白细胞中诱导的促炎基因和促凋亡基因 体内应用FvHsp70可减少大脑中动脉闭塞。因此，我们提出以下目标。 特异性目标#1a：证明Fv-Hsp70、Fv-Hsp70C和突变体Fv-Hsp70C-DEVD对培养的动物具有保护作用 缺氧缺糖(OGD)引起的神经元和星形胶质细胞。具体目标#1b：开始探索反 热休克蛋白70、核因子B相互作用对细胞凋亡和抗炎保护机制的研究 和肿瘤坏死因子；并表明HSP70阻断了原代脑细胞中的核因子B的激活。 具体目标2：证明FV-Hsp70结构可减少脑梗塞体积并改善行为学 大鼠局灶性脑缺血模型的转归。Fv-Hsp70、Fv-Hsp70C和Fv-Hsp70C-DEVD的作用 将相互比较，并与载体、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卒中后对大鼠血液进行基因组图谱分析 目的#2并证明MCAO诱导了一组破坏性的促凋亡和促炎症基因，而 抑制血液中白细胞中的促生存基因。具体目标#3b：演示使用FV- 大鼠大脑中动脉阻塞后热休克蛋白70的构建可减弱血白细胞中有害基因的反应 增强大鼠血白细胞的抗凋亡、抗炎和其他促生存基因。 意义：这些研究将提供一个原则证明，Hsp70作为Fv静脉注射- 融合蛋白，进入大脑，改善中风的预后。更广泛地说，FV蛋白递送方法 可用于输送任何蛋白质来治疗中风、脑和脊椎的其他急性损伤 脐带，可能还用于治疗退行性神经疾病。我们还建议监测基因的表达 外周血中与有效的中风治疗相关的变化可以用来评估 人类潜在的治疗反应性。

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.