Engineering chaperones for extracellular amyloids

描述（由申请方提供）：错误折叠的蛋白质自缔合成毒性组装体是大量神经退行性疾病中的主要病理事件。我们目前的知识表明，防止错误折叠的构象异构体的积累和有毒组件的形成可能具有神经保护作用。一种显著的神经保护分子是热休克蛋白70（Hsp 70）。这种有效的分子伴侣在果蝇和小鼠模型中表现出对几种细胞内淀粉样蛋白的强大神经保护作用，包括Ataxin-1，Ataxin-3和α-突触核蛋白。这些结果表明，激活细胞内Hsp 70的治疗策略可能在神经退行性疾病中具有广泛的应用。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Hsp 70的新策略提供了针对细胞外淀粉样蛋白的独特治疗机会。本申请的主要目标是确定一种新的工程化形式的分泌型Hsp 70（secHsp 70）的疾病修饰能力。为此，我们有初步的数据表明，secHsp 70，而不是野生型Hsp 70，完全阻断果蝇眼睛中的细胞外淀粉样蛋白-β 42（Abeta 42）肽的毒性。值得注意的是，这种强大的保护发生在没有Hsp 70共伴侣Hsp 40的细胞外供应的情况下。因此，我们的中心假设是secHsp 70与Abeta 42的错误折叠构象结合，并阻止神经毒性组装体如寡聚体和原纤维的形成。基本原理是，Hsp 70在细胞外环境中的有意表达可以靶向可能在早期变得活跃或在整个疾病过程中持续活跃的各种A β 42组装体。我们计划通过追求以下具体目标来验证我们的假设：（1）表征secHsp 70在表达人APP和BACE的果蝇中的保护活性。我们的工作假设是secHsp 70将阻止或延迟APP衍生的Abeta 42的细胞外蛋白毒性组装体的形成。(2)评估secHsp 70在阿尔茨海默病小鼠模型中的保护活性。我们将使用体细胞脑转基因技术来测试secHsp 70在tgCRND 8小鼠脑中的保护活性。创新：在果蝇和小鼠中的实验方法的系统组合将是至关重要的，以评估新的secHsp 70融合在各种实验范例中的活性，测试其与正常Hsp 70的潜在协同相互作用，并确定其调节预形成的寡聚体和恢复体内神经毒性的能力。在完成这些目标后，我们预期以下预期结果：首先，我们将定义secHsp 70对APP/BACE果蝇中Abeta 42病理学的神经保护潜力;其次，我们将定义secHsp 70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的影响，因为它们将导致secHsp 70在其他脑淀粉样变性（如朊病毒疾病）以及淀粉样蛋白全身分布的疾病（如2型糖尿病）中的应用。

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.