Heat Shock Protein Therapeutics for Stroke

热休克蛋白治疗中风

• 批准号: 7937620
• 负责人: Robert Nishimura
• 金额: $ 53.99万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937620
• 关键词: Acute; Address; Anti-DNA Antibodies; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Apoptotic; Astrocytes; Attenuated; Behavioral; Binding; Biological Markers; Blood; Brain; Caspase; Cells; Cerebral Ischemia; Chimeric Proteins; Client; Coupled; Data; Future; Gene Expression; Gene Expression Profiling; Genes; Genome; Genomics; Glucose; Green Fluorescent Proteins; Heat shock proteins; Heat-Shock Response; Human; In Vitro; Infarction; Inflammation; Inflammatory; Inflammatory Response; Injury; Ischemia; Leukocytes; Measures; Mediating; Methods; Microglia; Middle Cerebral Artery Occlusion; Mitochondria; Modeling; Molecular Chaperones; Mutate; Neuroglia; Neurons; Nucleosides; Outcome; Oxygen; Pathway interactions; Property; Protein Biosynthesis; Protein Denaturation; Proteins; RNA; Rattus; Recombinants; Research Personnel; Rodent; Role; Signal Transduction; Sorting - Cell Movement; Spinal Cord; Stress; Stroke; TNF gene; Therapeutic; Transfection; Transgenes; Viral; acute stroke; apoptotic protease-activating factor 1; brain cell; caspase-3; caspase-9; deprivation; improved; in vivo; inhibitor/antagonist; mutant; nervous system disorder; novel; peripheral blood; protein degradation; protein expression; protein folding; public health relevance; response

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.

描述（由申请人提供）：热休克蛋白（包括HSP70）是热休克引起的伴侣，以及许多应力，包括缺血和蛋白质合成过程中蛋白质折叠，并在蛋白质变性后重新折叠。 Hsp70蛋白表达在多种模型中使用多种不同表达方法具有神经保护作用。该提案将解决HSP70的保护性，抗凋亡和抗炎作用：（1）神经元和神经胶质的主要培养物； （2）局灶性脑缺血之后。该研究的一个新方面将是使用新开发的抗DNA抗体的单链片段静脉内给予重组HSP70，HSP70C和突变的HSP70C-DEVD蛋白，称为FV。 FV-HSP70与在所有细胞上发现的核苷挽救蛋白转运蛋白ENT2结合，而FV-HSP70通过ATP Indepent Indepent Ent2转运蛋白进入这些细胞。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； （b）FVHSP70减少梗塞体积并改善体内大脑中动脉闭塞（MCAO）后的行为预后； （c）MCAO后血清细胞诱导的血清细胞诱导的促炎和促凋亡基因通过体内的FVHSP70治疗降低。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气葡萄糖剥夺（OGD）的影响。特定目的＃1b：开始探索通过检查脑中原代细胞中HSP70，NF：B和TNF之间的相互作用，从而探索保护的抗凋亡和抗炎机制；并表明HSP70阻止了原代脑细胞中的NF：B激活。具体目的＃2：证明FV-HSP70构建体可减少梗塞体积，并改善局灶性脑缺血大鼠模型中的行为结果。 FV-HSP70，FV-HSP70C和FV-HSP70C-DEVD的效果将相互比较，并且单独使用HSP70，单独使用HSP70，单独使用FV和FV绿色荧光蛋白（GFP）对照。特定目标＃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诱导了一组破坏性的促凋亡和促炎基因，同时抑制血液中白细胞中的促生物性基因。具体目的＃3B：证明MCAO之后大鼠中用FV-HSP70构建体的治疗减弱了血清细胞中有害的基因反应，并会增加抗凋亡，增加抗炎和增加抗炎症并增加大鼠血液白细胞中其他亲苏的基因。 意义：这些研究将提供原理证明，即HSP70作为FV-融合蛋白静脉内给药，进入大脑并改善中风的预后。更一般而言，此处使用的FV蛋白输送方法可能有助于输送任何蛋白质以治疗中风，对脑和脊髓的其他急性损伤，以及可能治疗退化性神经系统疾病。我们还建议监测与啮齿动物中有效中风治疗相关的外周血中基因表达的变化，可用于评估人类的潜在治疗反应能力。 公共卫生相关性：该提案将检查热休克蛋白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突变体传递到原代脑细胞和脑。我们将证明FV-HSP70构造可保护神经元和神经胶质免受氧气和葡萄糖剥夺的影响，并且FVHSP70构造可保护大鼠脑免受中风。 FV-HSP70介导的保护部分将部分归因于血液和大脑中炎症的阻塞和促凋亡途径的阻塞。有人提出，FVHSP70可用于治疗人类用中风治疗人类，并且可以将与改善大鼠结局相关的大鼠血液中的基因表达变化用作生物标志物，以预测接受FVHSP70治疗的人类的改善。