Effects of Charged Cholesterol Derivatives on Aβ40 Amyloid Formation

Understanding of the mechanistic progess of amyloid-β peptide (Aβ) aggregation is critical for elucidating the underlying pathogenesis of Alzheimer’s disease (AD). Herein, we report for the first time the effects of two cholesterol derivatives, negatively charged cholesterol sulfate (cholesterol-SO₄) and positively charged 3β-[<i>N</i>-(dimethyl­amino­ethane)­carbamoyl]-cholesterol (DC-cholesterol), on the fibrillization of Aβ40. Our results demonstrate that both of the nonvesicular forms of cholesterol-SO₄ and DC-cholesterol moderately accelerate the aggregation rate of Aβ40. This effect is similar to that observed for unmodified cholesterol, indicating the importance of hydrophobic interactions in binding of Aβ40 to these steroid molecules. Furthermore, we show that the vesicles formed at higher concentrations of anionic cholesterol-SO₄ facilitate Aβ40 aggregation rate markedly. In contrast, the cationic DC-cholesterol vesicles show the ability to inhibit Aβ40 fibril formation under appropriate experimental conditions. The results suggest that the electrostatic interactions between Aβ40 and the charged vesicles can be of great importance in regulating Aβ40–vesicle interaction. Our results also indicate that the structural properties of the aggregates of the cholesterol derivatives, including the surface charge and the size of the vesicles, are critical in regulating the effects of these vesicles on Aβ40 aggregation kinetics

Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells

Cell-penetrating peptides (CPPs) are promising vectors for the intracellular delivery of a variety of membrane-impermeable bioactive compounds. The mechanisms by which CPPs cross the cell membrane, and the effects that CPPs may have on cell function, still remain to be fully clarified. In this work, we employed confocal Raman microscopy (CRM) and atomic force microscopy (AFM) to study the infiltration and physiological effects of the amphipathic CPP transportan (Tp) on the metastatic melanoma cell line SK-Mel-2. CRM enabled the detection of label-free Tp within the cells. Raman maps of live cells revealed rapid entry (within 5 min) and widespread distribution of the peptide throughout the cytoplasm and the presence of the peptide within the nucleus after ∼20 min. Principal component analysis of the CRM data collected from Tp-treated and untreated cells showed that Tp Raman bands were not positively correlated with lipid Raman bands, indicating that Tp entered the cells via a nonendocytic mechanism. Analysis of intracellularly recovered Tp by mass spectrometry showed that Tp remained intact in SK-Mel-2 cells for up to 24 h. The Raman spectroscopic data also showed that, although Tp was predominantly unstructured (random coil) in aqueous solution, it accumulated to high densities within the cells with mostly β-sheet and α-helical structures. AFM was employed to measure the effect of Tp treatment on cell stiffness. These data showed that Tp induced a significant increase in cell stiffness within the first hour of treatment, which was partially abated after 2 h. It is hypothesized that the increase in cell stiffness was the result of cytoskeletal changes triggered by Tp

Poly(4-styrenesulfonate) as an Inhibitor of Aβ40 Amyloid Fibril Formation

The formation of amyloid, a cross-β-sheet fibrillar aggregate of proteins, is associated with a variety of neurodegenerative diseases. Amyloidogenic proteins such as β-amyloid (Aβ) are known to exist with a large amount of polyelectrolyte macromolecules in vivo. The exact nature of Aβ–polyelectrolyte interactions and their roles in Aβ-aggregation are largely unknown. In this regard, we report the inhibiting effect of an anionic polyelectrolyte poly­(4-styrenesulfonate) (PSS) on the aggregation of Aβ40 peptide. The results demonstrate the strong inhibition potential of PSS on the aggregation of Aβ40 and imply the dominant role of hydrophobicity of the polyelectrolyte in reducing the propensity of Aβ40 amyloid formation. Additional studies with poly­(vinyl sulfate) (PVS) and <i>p</i>-toluenesulfonate (PTS), which share similar charge density with PSS except the former lacking the nonpolar aromatic side chain and the latter the aliphatic hydrocarbon backbone, reveal that the presence of both aliphatic backbone and aromatic side chain group in PSS is essential for its Aβ-aggregation inhibition activity. The interactions involved in the Aβ40–PSS complex were further investigated using molecular dynamics (MD) simulation. Our results provide new insights into the structural interplay between polyelectrolytes and Aβ peptide, facilitating the ultimate understanding of amyloid formation in Alzheimer’s disease. The results should assist in developing novel polyelectrolytes as potential chemical tools to study amyloid aggregation

Positively Charged Chitosan and <i>N</i>‑Trimethyl Chitosan Inhibit Aβ40 Fibrillogenesis

Amyloid fibrils, formed by aggregation of improperly folded or intrinsically disordered proteins, are closely related with the pathology of a wide range of neurodegenerative diseases. Hence, there is a great deal of interest in developing molecules that can bind and inhibit amyloid formation. In this regard, we have investigated the effect of two positively charged polysaccharides, chitosan (CHT) and its quarternary derivative <i>N</i>-trimethyl chitosan chloride (TMC), on the aggregation of Aβ40 peptide. Our aggregation kinetics and atomic force microscopy (AFM) studies show that both CHT and TMC exhibit a concentration-dependent inhibiting activity on Aβ40 fibrillogenesis. Systematic pH-dependent studies demonstrate that the attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues in Aβ40 play a key role in this inhibiting activity. The stronger inhibiting activity of TMC than CHT further suggests the importance of charge density of the polymer chain in interacting with Aβ40 and blocking the fibril formation. The possible interactions between CHT/TMC and Aβ40 are also revealed at the atomic level by molecular docking simulation, showing that the Aβ40 monomer could be primarily stabilized by electrostatic interactions with charged amines of CHT and quaternary amines of TMC, respectively. Binding of CHT/TMC on the central hydrophobic core region of Aβ40 peptide may be responsible for blocking the propagation of the nucleus to form fibrillar structures. These results suggest that incorporation of sugar units such as d-glucosamine and <i>N</i>-trimethyl-d-glucosamine into polymer structural template may serve as a new strategy for designing novel antiamyloid molecules

Gold Nanoparticles as a Probe for Amyloid‑β Oligomer and Amyloid Formation

The process of amyloid-β (Aβ) amyloid formation is pathologically linked to Alzheimer’s disease (AD). The identification of Aβ amyloids and intermediates that are crucial players in the pathology of AD is critical for exploring the underlying mechanism of Aβ aggregation and the diagnosis of the disease. Herein, we performed a gold nanoparticle (AuNP)-based study to detect the formation of Aβ amyloid fibrils and oligomers. Our results demonstrate that the intensity of the surface plasmon resonance (SPR) absorption band of the AuNPs is sensitive to the quantity of Aβ40 amyloids. This allows the SPR assay to be used for detection and semiquantification of Aβ40 amyloids and characterization of the kinetics of Aβ amyloid formation. Furthermore, our study demonstrates that the SPR band intensity of the AuNPs is sensitive to the presence of oligomers of Aβ40, and an Aβ40 mutant which forms more stable oligomers. The kinetics of the stable oligomer formation of the Aβ40 mutant can also be monitored following the SPR band intensity change of AuNPs. Our results indicate that this nanoparticle-based method can be used for mechanistic studies of early protein self-assembly and fibrillogenesis