Heat Shock Protein Therapeutics for Stroke

摘要 热休克蛋白，包括热休克蛋白70，是由热休克和许多应激诱导的分子伴侣，包括 并在蛋白质合成过程中辅助蛋白质折叠和蛋白质变性后的再折叠。Hsp70 蛋白质表达在多种模型中具有神经保护作用，这些模型使用了许多不同的过度表达方法。 表情本研究将探讨Hsp 70在以下方面的保护、抗凋亡和抗炎作用：（1） 神经元和胶质细胞的原代培养;和（2）局灶性脑缺血后。这项研究的一个新方面将 使用一种新的方法静脉内施用重组Hsp 70、Hsp 70 C和突变体Hsp 70 C-DEVD蛋白， 开发了称为Fv的抗DNA抗体的单链片段。Fv-Hsp 70结合核苷 在所有细胞上发现补救转运蛋白ENT 2，并且Fv-Hsp 70通过ATP非依赖性ENT 2进入这些细胞 传送器我们的初步研究结果表明：（a）Fv-Hsp 70对体外培养的神经元和胶质细胞有保护作用;（B）FvHsp 70对体外培养的神经元和胶质细胞有保护作用; 减少大脑中动脉闭塞后的梗死体积并改善行为结果 （c）在体内的MCAO;以及在体内的MCAO后在血液白细胞中诱导的促炎和促凋亡基因。 在体内用FvHsp 70处理可减少MCAO。因此，我们提出以下目标。 具体目标#1a：证明Fv-Hsp 70、Fv-Hsp 70 C和突变体Fv-Hsp 70 C-DEVD保护培养的 神经元和星形胶质细胞从氧葡萄糖剥夺（OGD）。具体目标#1b：开始探索反 通过检测Hsp 70、NF B和NF κ B之间的相互作用， 和TNF;并表明Hsp 70阻断了原代脑细胞中NF B的活化。 具体目标#2：证明Fv-Hsp 70构建体减少梗死体积并改善行为功能。 大鼠局灶性脑缺血模型的结果。Fv-Hsp 70、Fv-Hsp 70 C和Fv-Hsp 70 C-DEVD在体外实验中的作用 将相互比较，并与载体、单独的Hsp 70、单独的Fv和Fv-绿色荧光蛋白（GFP）进行比较， 对照具体目标#3a。在MCAO中风后对大鼠血液进行基因组分析， 目的#2并证明MCAO诱导一组损伤性促凋亡和促炎基因， 抑制血液中白细胞中的促生存基因。具体目标#3b：证明用Fv- MCAO后大鼠中的Hsp 70构建体减弱血液白细胞中的有害基因应答， 增加大鼠血液白细胞中抗凋亡基因、抗炎基因和其它促存活基因。 意义：这些研究将提供一个原则的证据，即Hsp 70，静脉注射作为一种Fv- 融合蛋白，进入大脑，改善中风的结果。更一般地，Fv蛋白递送方法 在这里使用的蛋白质可以用于输送任何蛋白质来治疗中风，其他急性脑损伤和脊髓损伤。 脊髓，并可能治疗退行性神经疾病。我们还提出，监测基因表达 与啮齿动物中有效的中风治疗相关的外周血的变化可用于评估 在人类中的潜在治疗反应性。

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.