Structural progression of amyloid-β Arctic mutant aggregation in cells revealed by multiparametric imaging

Abstract

The 42-amino-acid β-amyloid (Aβ42) is a critical causative agent in the pathology of Alzheimer's disease. The hereditary Arctic mutation of Aβ42 (E22G) leads to increased intracellular accumulation of β-amyloid in early-onset Alzheimer's disease. However, it remains largely unknown how the Arctic mutant variant leads to aggressive protein aggregation and increased intracellular toxicity. Here, we constructed stable cell lines expressing fluorescent-tagged wildtype (WT) and E22G Aβ42 to study the aggregation kinetics of the Arctic Aβ42 mutant peptide and its heterogeneous structural forms. Arctic-mutant peptides assemble and form fibrils at a much faster rate than WT peptides. We identified five categories of intracellular aggregate—oligomers, single fibrils, fibril bundles, clusters, and aggresomes—that underline the heterogeneity of these Aβ42 aggregates and represent the progression of Aβ42 aggregation within the cell. Fluorescence-lifetime imaging (FLIM) and 3D structural illumination microscopy (SIM) showed that all aggregate species displayed highly compact structures with strong affinity between individual fibrils. We also found that aggregates formed by Arctic mutant Aβ42 were more resistant to intracellular degradation than their WT counterparts. Our findings uncover the structural basis of the progression of Arctic mutant Aβ42 aggregation in the cell.

Walnut‐Derived Peptide PW5 Ameliorates Cognitive Impairments and Alters Gut Microbiota in APP/PS1 Transgenic Mice

“…In briefly, a single point mutation in Aβ42, Glu22 (E) to Gly22 (G), could lead to rapid and aggressive amyloid fibril formation in Flpin‐T‐REx HEK293 cell line. Moreover, the reporter protein mCherry was tagged to Aβ42 as a marker for fluorescence imaging, which allows observation and quantitative study of amyloidogenesis . To observe the intracellular Aβ42 aggregation after WPH treatment, microscopy and flow cytometry were applied to detect the aggregates and fluorescence intensity.…”

“…Cells were cultured in basic culture medium supplemented with 50 µg/mL of hygromycin B (Roche), 5 µg mL –1 of blasticidin S (Invivogen) and 5 m m of l ‐glutamine (Gibco) at 37 °C under humidified air with 5% CO 2 . Aβ42‐mCherry (WT) cell line was used as the control …”

Targeting Aggrephagy for the Treatment of Alzheimer’s Disease

“…Lu et al showed that cells overexpressing APP Arctic mutation (APP693G) resulted in APP amyloidogenic processing and increased Aβ-42 levels. Using super-resolution microscopy, they found that elevated Aβ-42 monomers progressively assembled into degradation-resistant aggresomes in the perinuclear space [57]. In general, ubiquitin binds to Aβ for proteasomal degradation [58].…”

Capturing Aβ42 aggregation in the cell

“…These considerations further enhance the importance of the study by Lu et al (5) and urge a more systematic study of all the various APP mutations on A␤ aggregation in vivo. The technical advancements that allow the investigators to quantify the rate of formation of the various forms of deposits are undoubtedly of great value in this research field.…”

“…The paper by Lu et al (5) provides a welcome demonstration of these advances in their study of the aggregation propensity and stability of the "Arctic" mutant (E22G or E693G) A␤42 aggregates in engineered human embryonic kidney cells. The authors were able, in particular, to characterize the nature and dynamics of the A␤42 peptide assemblies in living cells using fluorescence-lifetime imaging microscopy (FLIM) to monitor the conversion of soluble protein fragments into amyloid fibrils.…”