Spontaneous aging of mature amyloids alters structural stability, cytotoxicity, and susceptibility to biological clearance.

Accumulation of amyloid fibrils is a key factor in the pathogenesis of progressive diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease, as well as systemic amyloidosis. Although amyloid deposits are known to persist in vivo for years or even decades, it remains unclear how the structure and biological properties of mature fibrils evolve during prolonged post-assembly residence. Here, we addressed this question using a simplified cell-free aqueous model designed to isolate the intrinsic time-dependent behavior of mature amyloid aggregates from the complexity of the biological milieu. Specifically, we investigated the long-term evolution of two polymorphs of lysozyme amyloid fibrils as model systems with distinct clustering propensities that mimic the diversity of amyloid deposits. We challenge the assumption of amyloid stability by demonstrating their spontaneous degradation over 16 months at physiological temperature, a process we define as amyloid "aging". This process is characterized by aggregate declustering, fibril shortening, and progressive depolymerization into monomeric subunits, accompanied by a pronounced reduction in intrinsic toxicity across multiple human cell lines. Notably, "aged" fibrils were disassembled and degraded more efficiently than freshly prepared aggregates by the molecular chaperone α-B-crystallin and immune-associated proteases, including matrix metalloproteinase-9 and cathepsins B and D. However, this enhanced degradation revealed a paradox: accelerated processing of "aged" amyloids did not always reduce cytotoxicity and, in some cases, even exacerbated it, consistent with the generation of biologically active fibril-derived species rather than their complete conversion into non-toxic monomers. We provide the first systematic evidence that mature amyloids are dynamic structures undergoing spontaneous degradation that fundamentally alters their cytotoxicity and susceptibility to biological clearance. Our results introduce amyloid aging as a previously underappreciated dimension of amyloid biology and emphasize the need to account for fibril "age" when evaluating pathogenic potential and developing anti-amyloid therapeutic strategies. Spontaneous "aging" of amyloid fibrils leads to suprastructural remodeling: declustering, fibril shortening, and attenuated cytotoxicity. While "aged" aggregates are degraded more efficiently by immune-associated proteases and α-B-crystallin than "fresh" fibrils, this enhanced degradation does not reduce cytotoxicity and, in some cases, exacerbates it, highlighting the complex interplay between aggregate maturity and biological outcomes.