Fluorescence-based techniques for the detection of the oligomeric status of proteins: implication in amyloidogenic diseases.

Intrinsically disordered proteins (IDPs) have captured attention in the last couple of decades due to their functional roles despite a lack of specific structure. Moreover, these proteins are found to be highly aggregation prone depending on the mutational and environmental changes to which they are subjected. The aggregation of such proteins either in the intracellular context or extracellular matrix is associated with several adverse pathophysiological conditions such as Alzheimer's, Parkinson's, and Huntington's diseases, Spinocerebellar ataxia, and Type-II diabetes. Interestingly, it has been noted that the smaller oligomers formed by IDPs are more toxic to cells than their larger aggregates. This necessitates the development of techniques that can detect the smaller oligomers formed by IDPs for diagnosis of such diseases during their early onset. Fluorescence-based spectroscopic and microscopic techniques are highly effective as compared to other techniques for the evaluation of protein oligomerization, organization, and dynamics. In this review, we discuss several fluorescence-based techniques including fluorescence/Förster resonance energy transfer (FRET), homo-FRET, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM), and photobleaching image correlation spectroscopy (pbICS) that are routinely used to identify protein oligomers in extracellular and intracellular matrices.

Cytotoxic Effects of Transthyretin Aggregates in an Epidermoid Cell Line.

OBJECTIVE: Skin amyloid deposits can occur as part of systemic amyloidoses including those involving misfolded- aggregated transthyretins (agTTR). Pathological effects of agTTR on the skin are not well understood. The main objective of the current study was to examine the toxicity of agTTR upon a human keratinocyte cell line. METHODS: Cells were analyzed for indicators of oxidative stress after treatment with normal soluble TTR or the same pre-aggregation concentration of agTTR. Hydrogen peroxide production was analyzed as an indicator for the involvement of reactive oxygen species. RESULTS: Treatment of cells with agTTR significantly increased hydrogen peroxide production (p < 0.05 vs. controls). Glutathione (GSH) and catalase were analyzed as indicators of endogenous cellular antioxidant activity. Treatment of cells with agTTR resulted in significant decreases in both catalase activity and GSH levels (p < 0.05 vs. controls). CONCLUSION: agTTR disrupts redox balance and induces oxidative stress in these epidermoid cells.

Molecular Insights into the Effect of Metals on Amyloid Aggregation.

Amyloid diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and type 2 diabetes (T2D) are characterized by accumulation of misfolded proteins' species, e.g., oligomers and fibrils. The formation of these species occurs via self-assemble of the misfolded proteins in a process which is named "aggregation." It is known that essential divalent metal ions initiate the aggregation of these misfolded proteins, and that specific concentrations of these metal ions may be implicated in the pathology of amyloid diseases. This chapter focuses on the effects of two of the most common divalent metal ions in the brain-Zn2+ and Cu2+, and while Zn2+ ion is known as a metal that is release from the pancreas. Specifically, the spotlight of this chapter illustrates recent computational molecular modelling studies that investigate the effect of the concentrations of metal ions on aggregation of the misfolded proteins amylin, amyloid ß, and α-synuclein. The challenges for computational molecular modeling and future perspectives are discussed.

Boosting the Photodynamic Degradation of Islet Amyloid Polypeptide Aggregates Via a "Bait-Hook-Devastate" Strategy.

Photosensitizers that can generate reactive oxygen species (ROS) upon irradiation have emerged as promising agents for photodynamic degradation of toxic amyloid aggregates that are linked to many amyloidogenic diseases. However, due to the ultrastable ß-sheet structure in amyloid aggregates and inefficient utilization of the generated ROS, it usually requires high stoichiometric concentration of the photosensitizer and/or intensive light irradiation to fully dissociate aggregates. In this work, we have developed a "bait-hook-devastate" strategy to boost the efficiency of the photodynamic degradation of amyloid aggregates. This strategy employs anionic polyacrylic acid as a bait to accumulate cationic human islet amyloid polypeptide (IAPP) aggregates and positively charged photosensitizer TPCI in a confined area through electronic interactions. Multiple characterization studies proved that the utilization rate of ROS generated by TPCI was remarkably improved via this strategy, which amplified the ability of TPCI to dissociate IAPP aggregates. Rapid and complete degradation of IAPP aggregates could be achieved by irradiating the system under very mild conditions for less than 30 min, and the IAPP-mediated cytotoxicity was also largely alleviated, providing a new paradigm to accelerate photodynamic degradation of amyloid aggregates for further practical applications.

Link Between Diabetes and Alzheimer's Disease due to the Shared Amyloid Aggregation and Deposition Involving both Neurodegenerative Changes and Neurovascular Damages.

Diabetes and Alzheimer's disease are two highly prevalent diseases among the aging population and have become major public health concerns in the 21st century, with a significant risk to each other. Both of these diseases are increasingly recognized to be multifactorial conditions. The terms "diabetes type 3" or "brain diabetes" have been proposed in recent years to provide a complete view of the potential common pathogenic mechanisms between these diseases. While insulin resistance or deficiency remains the salient hallmarks of diabetes, cognitive decline and non-cognitive abnormalities such as impairments in visuospatial function, attention, cognitive flexibility, and psychomotor speed are also present. Furthermore, amyloid aggregation and deposition may also be drivers for diabetes pathology. Here, we offer a brief appraisal of social impact and economic burden of these chronic diseases and provide insight into amyloidogenesis through considering recent advances of amyloid-ß aggregates on diabetes pathology and islet amyloid polypeptide on Alzheimer's disease. Exploring the detailed knowledge of molecular interaction between these two amyloidogenic proteins opens new opportunities for therapies and biomarker development.

Pro-oxidative effects of aggregated transthyretin in human Schwannoma cells.

Neurotoxicity mechanisms of amyloidogenic polypeptides such as transthyretin (TTR) are not well understood. Misfolded and aggregated TTRs (agTTR) lead to age-related diseases such as senile systemic amyloidosis and familial amyloid polyneuropathy (FAP). Among other clinical manifestations in TTR amyloidic disease, peripheral nerve tissue, including Schwann cell, degeneration has been observed. In this study, we examined potential toxic effects of agTTR in human Schwannoma cells (sNF94.3 peripheral nerve sheath line). Cells were treated with agTTR (2.4µM pre-aggregation concentration) or, as controls, normal, soluble TTR (2.4µM) or no-TTR treatment, and then analyzed for different pro-oxidant and anti-oxidant markers: hydrogen peroxide (H2O2), catalase (CAT), glutathione (GSH), and more generalized cellular antioxidant capacity. In the latter case, cytosolic fractions were prepared after agTTR (or control) treatments and analyzed in oxidation assays. Relative to treatment with normal soluble TTR, cells treated with agTTR increase their release of H2O2. Residual CAT activity is decreased after agTTR treatment. The Schwannoma cells also exhibit significantly lower levels of GSH after agTTR treatment (p<0.05, relative to controls). More generally, cytosols from agTTR-treated cells exhibited a lower capacity to prevent oxidation relative to those from control cells (TTR-treated, or non-TTR-treated). These results suggest that agTTR (a) stimulates production of reactive oxygen species, (b) leads to lower levels of endogenous antioxidants, and (c) decreases overall cellular antioxidant capacity, in Schwannoma cells.