Misfolded amyloid β-42 induced impairment of the endosomal-lysosomal pathway revealed by real-time optical monitoring

Abstract

Misfolding and aggregation of proteins is strongly linked to several neurodegenerative diseases but how such species bring about their cytotoxic actions remains poorly understood. Here we used specifically-designed optical reporter probes and live fluorescence imaging in primary hippocampal neurons to characterise the mechanism by which prefibrillar, oligomeric forms of the Alzheimer's-associated peptide, Aβ42, exert their detrimental effects. We used a pH-sensitive reporter, Aβ42-CypHer, to track Aβ internalisation in real time, demonstrating that oligomers are rapidly taken up into cells in a dynamin-dependent manner, and trafficked via the endo-lysosomal pathway resulting in accumulation in lysosomes. In contrast, a non-assembling variant of Aβ42 (vAβ42) assayed in the same way is not internalised. Tracking ovalbumin uptake into cells using CypHer or Alexa Fluor tags shows that preincubation with Aβ42 significantly disrupts protein uptake. Our results identify a potential mechanism by which amyloidogenic aggregates directly impair cellular function through disruption of the endosomallysosomal pathway. (150 words)

Nanoscale reorganisation of synaptic proteins in Alzheimer's disease

“…[32] We also measured Aβ1-42 on cell viability in primary hippocampal neurons by using the Ready-probes assay. [33] Compared with the DMSO-treated neurons, neurons incubated with both 5-and 10-μM Aβ1-42 oligomers showed significantly lower viability (Figure S1A-D), consistent with previous studies. [33] We quantified the components of Aβ preparation by Western blot (Figure S1E) and found that the major oligomer was trimer, which accounted for about 5% of the total mass (Figure S1F).…”

“…[33] Compared with the DMSO-treated neurons, neurons incubated with both 5-and 10-μM Aβ1-42 oligomers showed significantly lower viability (Figure S1A-D), consistent with previous studies. [33] We quantified the components of Aβ preparation by Western blot (Figure S1E) and found that the major oligomer was trimer, which accounted for about 5% of the total mass (Figure S1F). Meanwhile, the immunostaining showed that a significant fraction of Aβ was colocalised with PSD-95, suggesting its direct targeting to synapses (Figure S1G-I).…”