Macromolecular crowding inhibits degradation of alpha‐synuclein amyloid fibrils induced by cathepsins and MMP9

Abstract

The accumulation of amyloid fibrils in the brain leads to cognitive and motor impairment in incurable neurodegenerative disorders, particularly in Alzheimer's and Parkinson's diseases. Emerging anti‐amyloid immunotherapies rely on recruiting immune proteases to clear these pathological aggregates, yet a stark disconnect remains between the high proteolytic efficiency observed in dilute in vitro assays and the limited efficacy of these treatments in vivo. This discrepancy likely arises because standard models neglect the dense macromolecular crowding characteristic of the cellular milieu. Here, we address this gap by investigating the degradation of alpha‐synuclein amyloids by key immune proteases (cathepsin B, cathepsin D, and matrix metalloproteinase‐9) in the presence of synthetic crowding agents at physiological densities. We found that macromolecular crowding in a crowding agent‐ and dose‐dependent manner inhibits protease‐induced amyloid declusterization, cluster size reduction, fibril fragmentation, and structural disorganization. We propose that the observed inhibition arises from the excluded volume effect, which stabilizes fibril structure and restricts both enzyme mobility and steric access to the fibril surface rather than suppressing catalytic activity. By restricting the extent of enzymatic amyloid degradation, this “crowding shield” modulated the cytotoxic consequences of enzymatic processing in human cell lines, attenuating enzyme‐induced shifts in fibril toxicity in a condition‐dependent manner. These findings identify the crowded cellular environment as a critical stabilizer of pathogenic protein aggregates, explaining their persistence in vivo.