Heat Shock Protein Therapeutics for Stroke

描述（由申请人提供）：热休克蛋白，包括Hsp70，是由热休克和包括缺血在内的许多应激诱导的伴侣蛋白，在蛋白质合成和蛋白质变性后的再折叠过程中协助蛋白质折叠。使用多种不同的过表达方法，在多种模型中，Hsp70蛋白表达具有神经保护作用。本研究将探讨Hsp70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；(2)局灶性脑缺血后。该研究的一个新方面将是静脉注射重组Hsp70、Hsp70C和突变Hsp70C- devd蛋白，使用新开发的抗dna抗体单链片段（Fv）。Fv-Hsp70结合在所有细胞上发现的核苷回收转运体ENT2，并通过ATP不依赖的ENT2转运体进入这些细胞。我们的初步数据表明：(a) Fv-Hsp70在体外对神经元和神经胶质细胞有保护作用；(b)体内FvHsp70减少脑梗死体积，改善大脑中动脉闭塞（MCAO）后的行为结局；(c)在体内用FvHsp70治疗MCAO后，血液白细胞中诱导的促炎和促凋亡基因减少。因此，我们提出以下目标。具体目的#1a：证明Fv-Hsp70、Fv-Hsp70C和突变型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）。特异性目的#1b：通过检测脑原代细胞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阻断了原代脑细胞中NF:B的激活。具体目标2：证明Fv-Hsp70构建体在局灶性脑缺血大鼠模型中减少梗死面积并改善行为结果。将Fv-Hsp70、Fv- hsp70c和Fv- hsp70c - devd相互比较，并与对照、Hsp70单独对照、Fv单独对照和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。特异性目的#3b：证明在MCAO后大鼠中使用Fv- Hsp70构建物治疗可减弱血液白细胞中的有害基因反应，并将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脑并改善脑卒中预后提供原理证明。更一般地说，这里使用的Fv蛋白递送方法可以用于递送任何蛋白质来治疗中风、其他脑和脊髓的急性损伤，并可能用于治疗退行性神经系统疾病。我们还提出，监测与啮齿类动物中风有效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性。

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.