Heat Shock Protein Therapeutics for Stroke

描述(申请人提供)：热休克蛋白，包括Hsp70，是在热休克和包括缺血在内的多种应激条件下诱导的伴侣蛋白，并在蛋白质合成和蛋白质变性后重新折叠过程中辅助蛋白质折叠。HSP70蛋白的表达在各种模型中具有神经保护作用，使用了许多不同的过度表达方法。这项建议将阐述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；以及(2)局灶性脑缺血后。这项研究的一个新方面将是使用新开发的抗DNA抗体的单链片段Fv静脉注射重组Hsp70、Hsp70C和突变的Hsp70C-DEVD蛋白。Fv-Hsp70与所有细胞上发现的核苷回收转运体ent2结合，并通过不依赖于ATP的ent2转运体进入这些细胞。我们的初步数据显示：(A)Fv-Hsp70在体外对神经元和胶质细胞具有保护作用；(B)FvHsp70在体内可减少大脑中动脉阻塞(MCAO)后的脑梗塞体积，并改善行为结果；(C)在体内，FvHsp70可减少MCAO后血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。具体目的#1a：证明Fv-Hsp70、Fv-Hsp70C和突变体Fv-Hsp70C-DEVD可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：通过检测HSP70、NF：B和肿瘤坏死因子在原代脑细胞中的相互作用，开始探索保护作用的抗凋亡和抗炎机制；并表明HSP70阻断了原代脑细胞中NF：B的激活。具体目标#2：证明FV-HSP70结构可减少大鼠局灶性脑缺血模型的脑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，并与载体、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AIM#2中产生MCAO卒中后，对大鼠血液进行基因组图谱分析，并证明MCAO诱导了一组破坏性的促凋亡和促炎基因，同时抑制了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构建的大鼠可以减轻血白细胞中有害基因的反应，并将增加大鼠血白细胞中的抗凋亡、抗炎和其他促生存基因。意义：这些研究将提供HSP70作为FV融合蛋白静脉注射进入大脑并改善中风预后的原理证据。更广泛地说，这里使用的FV蛋白递送方法可以用于递送任何蛋白质来治疗中风、其他急性脑和脊髓损伤，以及可能用于治疗退行性神经疾病。我们还建议，监测啮齿动物外周血中与有效卒中治疗相关的基因表达变化，可以用于评估人类潜在的治疗反应性。 与公共卫生相关：这项提案将检查热休克蛋白Hsp70的神经保护特性。一种名为FV的新开发的蛋白质递送方法将被用于将FV-Hsp70和Hsp70突变体递送到原代脑细胞和大脑。我们将证明Fv-Hsp70结构保护神经元和胶质细胞免受缺氧和葡萄糖剥夺，以及FvHsp70结构保护大鼠大脑免受中风。Fv-Hsp70介导的保护作用将部分归因于阻断炎症和阻断血液和脑中的促凋亡途径。FvHsp70可用于治疗人类卒中，大鼠血液中与改善大鼠预后相关的基因表达的变化可作为预测FvHsp70治疗人类改善预后的生物标志物。

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.