Heat Shock Protein Therapeutics for Stroke

描述（由申请人提供）：热休克蛋白（包括Hsp70）是由热休克和许多应激（包括缺血）诱导的分子伴侣，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重折叠。Hsp70蛋白表达在使用许多不同的过表达方法的各种模型中具有神经保护作用。本研究将探讨热休克蛋白70在神经元和神经胶质细胞原代培养及局灶性脑缺血后的保护、抗凋亡和抗炎作用。该研究的一个新方面是使用新开发的称为Fv的抗DNA抗体的单链片段静脉内施用重组Hsp70、Hsp70C和突变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ATP非依赖性ENT2转运蛋白进入这些细胞。我们的初步数据显示：（a）Fv-Hsp 70在体外保护神经元和神经胶质;（B）FvHsp 70在体内减少梗塞体积并改善大脑中动脉闭塞（MCAO）后的行为结果;（c）通过用FvHsp 70在体内治疗，减少了MCAO后在血液白细胞中诱导的促炎和促凋亡基因。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保护培养的神经元和星形胶质细胞免受氧葡萄糖剥夺（OGD）。具体目标1b：开始通过检查来自脑的原代细胞中Hsp70、NF：B和TNF之间的相互作用来探索抗凋亡和抗炎保护机制;并显示Hsp70阻断原代脑细胞中NF：B活化。具体目标#2：证明Fv-Hsp70构建体在大鼠局灶性脑缺血模型中减少梗死体积并改善行为结果。将Fv-Hsp70、Fv-Hsp70C和Fv-Hsp70C-DEVD的作用相互比较，并与媒介物、单独的Hsp70、单独的Fv和Fv-绿色荧光蛋白（GFP）对照比较。具体目标#3a。在目标#2中产生的MCAO中风后对大鼠血液进行基因组分析，并证明MCAO诱导一组破坏性促凋亡和促炎基因，同时抑制血液中白细胞中的促存活基因。具体目标3b：证明在MCAO后大鼠中用Fv-Hsp70构建体治疗减弱了血液白细胞中的有害基因应答，并将增加大鼠血液白细胞中的抗凋亡基因、抗炎基因和其他促存活基因。 重要性：这些研究将提供Hsp70作为Fv-融合蛋白静脉内施用进入脑并改善中风结果的原理证明。更一般地，本文使用的Fv蛋白递送方法可用于递送任何蛋白以治疗中风、对脑和脊髓的其他急性损伤，并且可能治疗退行性神经疾病。我们还建议，监测外周血中与啮齿动物有效治疗中风相关的基因表达变化可用于评估人类潜在的治疗反应。 公共卫生相关性：该提案将研究热休克蛋白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脑。我们将证明，Fv-Hsp70结构保护神经元和神经胶质细胞从氧和葡萄糖剥夺和FvHsp70结构保护大鼠脑中风。Fv-Hsp70介导的保护将部分归因于血液和脑中炎症的阻断和促凋亡途径的阻断。提出FvHsp70可用于治疗患有中风的人类，并且与大鼠中改善的结果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用FvHsp70治疗的人类中改善的结果。

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.