An Engineered Multifunctional Fusion Protein Targeting Aβ Oligomers, Microglia and Autophagy Ameliorates Cognitive Deficits and Amyloid Pathology in Alzheimer’s Disease Mice

In Alzheimer's disease (AD), Amyloid-β (Aβ) oligomers function as key neurotoxic agents that underpin the disease's progression. A diverse array of therapeutic entities, including peptides, single-chain variable fragments (scFvs), and small molecules, have demonstrated the ability to interact with Aβ oligomers, thereby suppressing their aggregation and associated neurotoxicity. Despite these advances, such agents frequently struggle to promote the phagocytosis and subsequent breakdown of aggregated Aβ by microglia. Moreover, the dense accumulation of Aβ oligomers may resist enzymatic hydrolysis within the acidic lysosomal lumen, contributing to lysosomal stress and dysfunction. To overcome these problems, we engineered a multifunctional fusion protein, p62-LIR-W20-Tuftsin (W20-LT), consisting of an oligomer-specific scFv, a microglia-targeting Tuftsin peptide, and a p62-LIR peptide to activate autophagy. In vitro assays demonstrated that W20-LT significantly outperformed the parental W20 by promoting the rapid microglial uptake of Aβ oligomers and enhancing their intracellular clearance through an autophagy-associated pathway. In APPswe/PS1dE9 (APP/PS1) mice, a low-dose regimen (0.5 µg, every 3 days) of W20-LT, but not W20, significantly ameliorated cognitive deficits and reduced amyloid pathology. Mechanistically, W20-LT was associated with enhanced autophagy-lysosomal pathway activity, as indicated by increased LC3B-II and reduced p62 levels, together with downregulated CatD and LAMP1 levels, thereby mitigating neuroinflammation. In summary, our findings suggest that W20-LT represents a promising proof-of-concept therapeutic strategy that combines scFv-based Aβ oligomer recognition with enhanced autophagy-associated clearance, thereby mitigating AD pathology.