Engineering chaperones for extracellular amyloids

描述(由申请人提供)：错误折叠的蛋白质自身结合成有毒组件是大量神经退行性疾病的主要病理事件。我们目前的知识表明，防止错误折叠的构象积累和有毒组件的形成可能具有神经保护作用。一种值得注意的神经保护分子是热休克蛋白70(Hsp70)。这种有效的分子伴侣对果蝇和小鼠模型中的几种细胞内淀粉样蛋白表现出强大的神经保护作用，包括Aaxin-1，Aaxin-3和α-Synuclein。这些结果表明，激活细胞内HSP70的治疗策略可能在神经退行性疾病中有广泛的应用。然而，在细胞外空间中不存在这样的质量控制机制；因此，分泌HSP70的新策略为治疗细胞外淀粉样蛋白提供了一个独特的治疗机会。这项应用的主要目标是确定一种新的、工程形式的分泌型Hsp70(SecHsp70)的疾病修改能力。为此，我们有初步数据表明，secHsp70，而不是野生型Hsp70，可以完全阻断细胞外淀粉样β42(Abeta42)多肽对果蝇眼睛的毒性。值得注意的是，这种强大的保护作用是在没有细胞外供应Hsp70辅助伴侣Hsp40的情况下发生的。因此，我们的中心假设是secHsp70与错误折叠的Abeta42构象结合，防止形成神经毒性组件，如低聚物和原纤维。其基本原理是，Hsp70在细胞外环境中的有意表达可能针对各种Abeta42组件，这些组件可能在疾病早期变得活跃或在整个疾病过程中持续活跃。我们计划通过追求以下具体目标来验证我们的假设：(1)鉴定SecHsp70在表达人APP和BACE的果蝇中的保护活性。我们的工作假设是secHsp70将防止或延迟APP衍生的Abeta42的细胞外蛋白毒性组装的形成。(2)评估secHsp70在阿尔茨海默病小鼠模型中的保护作用。我们将利用体脑转基因技术来检测secHsp70在tgCRND8小鼠脑组织中的保护活性。创新：在果蝇和小鼠身上系统地结合实验方法将是至关重要的，以评估新的secHsp70融合在各种实验范式中的活性，测试其与正常Hsp70的潜在协同作用，并确定其调节预先形成的寡聚体和逆转体内神经毒性的能力。在完成这些目标后，我们预计会出现以下预期结果：首先，我们将确定secHsp70在APP/BACE果蝇Abeta42病理上的神经保护潜力；其次，我们将定义secHsp70在阿尔茨海默病小鼠模型中的功能相关性。这些结果预计将产生积极影响，因为它们将导致secHsp70在其他脑淀粉样变性中的应用，如普里恩疾病，以及淀粉样蛋白全身性分布的疾病，如2型糖尿病。

DESCRIPTION (provided by applicant): Self-association of misfolded proteins into toxic assemblies is the main pathological event in a large number of neurodegenerative disorders. Our current knowledge indicates that preventing the accumulation of misfolded conformers and the formation of toxic assemblies may have neuroprotective consequences. One remarkable neuroprotective molecule is the Heat shock protein 70 (Hsp70). This potent molecular chaperone demonstrated robust neuroprotection against several intracellular amyloids in Drosophila and mouse models, including Ataxin-1, Ataxin-3, and alpha-Synuclein. These results suggest that therapeutic strategies that activate intracellular Hsp70 may have broad applications in neurodegenerative diseases. However, no such quality control mechanism exists in the extracellular space; thus, new strategies to secrete Hsp70 present a unique therapeutic opportunity against extracellular amyloids. The major goal of this application is to determine the disease-modifying ability of a new, engineered form of secreted Hsp70 (secHsp70). To that end, we have preliminary data showing that secHsp70, but not wild type Hsp70, completely blocks toxicity of the extracellular amyloid-beta42 (Abeta42) peptide in the Drosophila eye. Notably, this robust protection occurs without extracellular supply of the Hsp70 co-chaperone Hsp40. Thus, our central hypothesis is that secHsp70 binds to misfolded conformations of Abeta42 and prevents the formation of neurotoxic assemblies such as oligomers and protofibers. The rationale is that the deliberate expression of Hsp70 in the extracellular milieu could target various Abeta42 assemblies that potentially become active early or are continuously active throughout the course of disease. We plan to test our hypothesis by pursuing the following specific aims: (1) Characterize the protective activity of secHsp70 in flies expressing human APP and BACE. Our working hypothesis is that secHsp70 will prevent or delay the formation of extracellular proteotoxic assemblies of APP-derived Abeta42. (2) Assess the protective activity of secHsp70 in a mouse model of Alzheimer's disease. We will use the somatic brain transgenesis technique to test the protective activity of secHsp70 in the brain of tgCRND8 mice. Innovation: The systematic combination of experimental approaches in Drosophila and mice will be crucial to assess the activity of the new secHsp70 fusion in a variety of experimental paradigms, test its potential synergistic interaction with normal Hsp70, and determine its ability to regulate pre-formed oligomers and revert neurotoxicity in vivo. Upon completion of these aims, we anticipate the following expected outcomes: First, we will define the neuroprotective potential of secHsp70 on Abeta42 pathology in APP/BACE flies; second, we will define the functional relevance of secHsp70 in a mouse model of Alzheimer's disease. These outcomes are expected to have a positive impact because they will lead to the application of secHsp70 in other cerebral amyloidosis, such as prion diseases, as well as in disorders with systemic distribution of amyloids, such as Type 2 diabetes.