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Laser Emission of Thioflavin T Uncovers Protein Aggregation in Amyloid Nucleation Phase.

ABSTRACT: There is currently no definitive test for early detection of neurodegeneration which is linked with protein aggregation. Finding methods capable of detecting intermediate states of protein aggregates, named oligomers, is critical for the early stage diagnosis of over 30 neurodegenerative diseases including Alzheimer's or Parkinson's. Currently, fluorescence-based imaging using Thioflavin T (ThT) dye is the gold standard for detecting protein aggregation. It is used to detect aggregation in vitro and in various tissues, including the cerebrospinal fluid (CSF), whereby the disease-related protein recombinant is seeded with the patient's fluid. The major drawback of ThT is its lack of sensitivity to oligomeric forms of protein aggregates. Here, we overcome this limitation by transferring a ThT-oligomer mixture into solid state thin films and detecting fluorescence of ThT amplified in the process of stimulated emission. By monitoring the amplified spontaneous emission (ASE) we achieved a remarkable recognition sensitivity to prefibrillar oligomeric forms of insulin and lysozyme aggregates in vitro, to Aβ42 oligomers in the human protein recombinants seeded with CSF and to Aβ42 oligomers doped into brain tissue. Seeding with Alzheimer patient's CSF containing Aβ42 and Tau aggregates revealed that only Aβ42 oligomers allowed generating ASE. Thus, we demonstrated that, in contrast to the current state-of-the-art, ASE of ThT, a commonly used histological dye, can be used to detect and differentiate amyloid oligomers and evaluate the risk levels of neurodegenerative diseases to potential patients before the clinical symptoms occur.

SUBMITTER: Hanczyc P

PROVIDER: S-EPMC8451393 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aging is the most important risk factor for neurodegenerative diseases associated with pathological protein aggregation such as Alzheimer's disease. Although aging is an important player, it remains unknown which molecular changes are relevant for disease initiation. Recently, it has become apparent that widespread protein aggregation is a common feature of aging. Indeed, several studies demonstrate that 100s of proteins become highly insoluble with age, in the absence of obvious disease processes. Yet it remains unclear how these misfolded proteins aggregating with age affect neurodegenerative diseases. Importantly, several of these aggregation-prone proteins are found as minor components in disease-associated hallmark aggregates such as amyloid-? plaques or neurofibrillary tangles. This co-localization raises the possibility that age-dependent protein aggregation directly contributes to pathological aggregation. Here, we show for the first time that highly insoluble proteins from aged Caenorhabditis elegans or aged mouse brains, but not from young individuals, can initiate amyloid-? aggregation in vitro. We tested the seeding potential at four different ages across the adult lifespan of C. elegans. Significantly, protein aggregates formed during the early stages of aging did not act as seeds for amyloid-? aggregation. Instead, we found that changes in protein aggregation occurring during middle-age initiated amyloid-? aggregation. Mass spectrometry analysis revealed several late-aggregating proteins that were previously identified as minor components of amyloid-? plaques and neurofibrillary tangles such as 14-3-3, Ubiquitin-like modifier-activating enzyme 1 and Lamin A/C, highlighting these as strong candidates for cross-seeding. Overall, we demonstrate that widespread protein misfolding and aggregation with age could be critical for the initiation of pathogenesis, and thus should be targeted by therapeutic strategies to alleviate neurodegenerative diseases.

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Laser Emission of Thioflavin T Uncovers Protein Aggregation in Amyloid Nucleation Phase.

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