Intraneuronal accumulation of amyloid-β (Aβ) peptides represent an early pathological feature in Alzheimer’s disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of Aβ(1–40) and Aβ(1–42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreover, SH-SY5Y cells internalise consistently twice the amount of Aβ(1–42) compared to Aβ(1–40); an imaging-based quantification showed that cells treated with 1 µM peptide for 8 h contained 800,000 peptides of Aβ(1–42) and 400,000 of Aβ(1–40). Both variants co-localised to >90% with lysosomes or other acidic compartments. Dynasore and chlorpromazine endocytosis inhibitors were both found to reduce uptake, particularly of Aβ(1–42). Overexpression of the C-terminal of the clathrin-binding domain of AP180, dynamin2 K44A, or Arf6 Q67L did however not reduce uptake of the Aβ variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced Aβ(1–40) and Aβ(1–42) uptake considerably. This study clarifies mechanisms of Aβ(1–40) and Aβ(1–42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal Aβ in AD.

Alzheimer's disease is associated with the aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of Aβ(1-42) self-assembly, in order to shed light on how this pathological reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophysical experiments and theoretical approaches, we show that zwitterionic DOPC lipid vesicles accelerate the Aβ(1-42) fibril growth rate by interacting specifically with the growing fibrils. We probe this interaction with help of a purpose-developed Förster resonance energy transfer assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalise these findings we use mathematical models to fit the aggregation kinetics of Aβ(1-42) and find that lipid vesicles alter specific mechanistic steps in the aggregation reaction; they augment monomer-dependent secondary nucleation at the surface of existing fibrils and facilitate monomer-independent catalytic processes consistent with fibril fragmentation. We further show that DOPC vesicles have no effect on primary nucleation. This finding is consistent with experiments showing that Aβ(1-42) monomers do not directly bind to the lipid bilayer. Taken together, our results show that plain lipid membranes with charge and composition that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of Aβ(1-42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.

Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Aggregation and intracellular deposition of the protein α-synuclein is an underlying characteristic of Parkinson’s disease. α-Synuclein assemblies also undergo cell–cell spreading, facilitating propagation of their cellular pathology. Understanding how cellular interactions and uptake of extracellular α-synuclein assemblies depend on their physical attributes is therefore important. We prepared fragmented fluorescently labeled α-synuclein amyloid fibrils of different average lengths (∼80 nm to >1 μm) and compared their interactions with SH-SY5Y cells. We report that fibrils of all lengths, but not monomers, bind avidly to the cell surface. Their uptake is inversely dependent on their average size, occurs via a heparan sulfate dependent endocytic route, and appears to have a size cutoff of ∼400 nm. The uptake of α-synuclein fibrils, but not monomers, correlates with their cytotoxicity as measured by reduction in metabolic activity, strongly suggesting an intracellular basis for α-synuclein fibril toxicity, likely involving endolysosomes.