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.