Edited by Wolfgang PetiThe aggregation of the tau protein into neurofibrillary tangles is believed to correlate with cognitive decline in several neurodegenerative disorders, including Alzheimer's disease. Recent studies suggest that tau's interactions with the cell membrane could serve as a toxicity pathway and also enhance fibrillation into paired helical filaments (PHFs). Conformational changes associated with tau-membrane interactions are poorly understood, and their characterization could improve our understanding of tau pathogenicity. In this study, we investigated the molecular level structural changes associated with the interaction of the tau hexapeptide PHF6 with model lipid membranes and characterized the effects of these interactions on membrane stability and peptide fibrillation. We used two PHF6 forms, the aggregation-prone PHF6 with N-terminal acetylation (Ac-PHF6) and the nonaggregation prone PHF6 with a standard N terminus (NH 3 ؉ -PHF6). We found that both PHF6 peptides are neurotoxic and exhibit similar membrane-mediated changes, consisting of: 1) favorable interactions with anionic membranes, 2) membrane destabilization through lipid extraction, and 3) membrane-mediated fibrillation. The rate at which these changes occurred was the main difference between the two peptides. NH 3 ؉ -PHF6 displayed slow membrane-mediated fibrillation after 6 days of incubation, whereas Ac-PHF6 adopted a ␤-sheet conformation at the surface of the membrane within hours. Ac-PHF6 interactions with the membrane were also accompanied by membrane invagination and rapid membrane destabilization. Overall, our results reveal that membrane interactions could play a critical role in tau toxicity and fibrillation, and highlight that unraveling these interactions is important for significantly advancing the development of therapeutic strategies to manage tau-associated neurodegenerative diseases.

Misfolding and aggregation of amyloid proteins into fibrillar aggregates is a central pathogenic event in neurodegenerative disorders such as Alzheimer’s (AD) and Parkinson’s diseases (PD). Currently, there is a lack of reliable sensors for detecting the range of protein aggregates involved in disease etiology, particularly the prefibrillar aggregate conformations that are more neurotoxic. In this study, the fluorescent sensing of two novel oligomeric p-phenylene ethynylenes (OPEs), anionic OPE1– and cationic OPE2+, for detecting prefibrillar and fibrillar aggregates of AD-associated amyloid-β (Aβ40 and Aβ42) and PD-associated α-synuclein proteins (wildtype, and single mutants A30P, E35K, and A53T) over their monomeric counterparts, were tested. Furthermore, the performance of OPEs was evaluated and compared to thioflavin T (ThT), the most widely used fibril dye. Our results show that OPE1– and OPE2+ exhibited aggregate-specific binding inducing large fluorescence turn-on and spectral shifts based on a combination of backbone planarization, hydrophobic unquenching, and superluminescent OPE complex formation sensing modes. OPEs exhibited higher selectivity, higher binding affinity, and comparable limits of detection for Aβ40 fibrils compared to ThT. OPE2+ exhibited the largest fluorescence turn-on and highest sensitivity. Significantly, OPEs detected prefibrillar aggregates of Aβ42 and α-synuclein that ThT failed to detect. The superior sensing performance, the nonprotein specific detection, and the ability to selectively detect fibrillar and prefibrillar amyloid protein aggregates point to the potential of OPEs to overcome the limitations of existing probes and promise significant advancement in the detection of the myriad of protein aggregates involved in the early stages of AD and PD.

Photodynamic therapy (PDT) has been explored as a therapeutic strategy to clear toxic amyloid aggregates involved in neurodegenerative disorders such as Alzheimer's disease. A major limitation of PDT is off-target oxidation, which can be lethal for the surrounding cells. We have shown that a novel class of oligo-pphenylene ethynylenes (OPEs) exhibit selective binding and fluorescence turn-on in the presence of prefibrillar and fibrillar aggregates of disease-relevant proteins such as amyloid-β (Aβ) and αsynuclein. Concomitant with fluorescence turn-on, OPE also photosensitizes singlet oxygen under illumination through the generation of a triplet state, pointing to the potential application of OPEs as photosensitizers in PDT. Herein, we investigated the photosensitizing activity of an anionic OPE for the photo-oxidation of Aβ fibrils and compared its efficacy to the well-known but nonselective photosensitizer methylene blue (MB). Our results show that, while MB photo-oxidized both monomeric and fibrillar conformers of Aβ40, OPE oxidized only Aβ40 fibrils, targeting two histidine residues on the fibril surface and a methionine residue located in the fibril core. Oxidized fibrils were shorter and more dispersed but retained the characteristic β-sheet rich fibrillar structure and the ability to seed further fibril growth. Importantly, the oxidized fibrils displayed low toxicity. We have thus discovered a class of novel theranostics for the simultaneous detection and oxidization of amyloid aggregates. Importantly, the selectivity of OPE's photosensitizing activity overcomes the limitation of off-target oxidation of traditional photosensitizers and represents an advancement of PDT as a viable strategy to treat neurodegenerative disorders.

Human cystatin C (HCC) is a commonly known inhibitor of cysteine proteases and also macromolecule used as a marker in the clinical diagnosis of the kidney function. This protein exhibits a strong tendency to misfolding via the domain swapping mechanism and then it is able to form oligomers, fibrils and amyloid deposits that can be built in brain blood vessels. The aim of our study was precise characterisation of morphology of these oligomers obtained in various conditions. Different types of HCC oligomers and aggregates were generated using native cystatin C and its covalently stabilised variant. These HCC oligomer species were generated in different compositions of solution, pH values, ionic strength, and also using various temperature and agitation conditions. The micromorphology of obtained HCC aggregates and oligomers was described on the basis of electron microscopy images and topographic data from the atomic force microscopy. In parallel the process of oligomer formation was characterised using 1H NMR diffusometry. In the next step, these HCC oligomers were selected as targets for nanosensing studies. At this stage, preliminary construction studies of a nanosensor selective for detection of HCC variants were initiated. For this research stage the initial immobilisation protocol of antibodies, selective to HCC oligomers, on the gold nanoparticles was tested.

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