Project Summary Numerous aging-associated neurodegenerative diseases are rooted in the misfolding of proteins and the generation of toxic protein aggregates, with Alzheimer's disease (AD) being the most prevalent form of dementia characterized by the aggregation of the amyloid beta peptide (Aβ) and hyperphosphorylated tau. Few effective strategies are currently available for most of these diseases. In this proposal, we will test the hypothesis that novel molecular chaperones can be evolved to recognize and target aggregation-prone proteins and ameliorate their associated toxicity, thus providing a new approach to intervene in protein misfolding diseases. Using Aβ as the model substrate, our specific goal is to provide proof-of-principle for a new platform for the directed evolution of molecular chaperones that can protect Aβ sequences from aggregation. The efficacy of the evolved chaperones in ameliorating Aβ toxicity will be tested in vitro and in vivo. The proposed studies will establish a new, facile, and broadly applicable approach to evolve novel molecular chaperones tailored to specific aggregation-prone target proteins and thus broadly impact diverse diseases rooted in protein misfolding. The evolved TMMCs are expected to achieve a specificity that is normally unattainable by other approaches, such as small molecules or general chaperone upregulation, and can supplement the reduced capacity of the cellular chaperone network during aging to protect neuronal cells from toxic protein aggregates.