Mechanism of cellular production and in vivo seeding effects of hexameric β-amyloid assemblies

Abstract

BackgroundThe {beta}-amyloid peptide (A{beta}) plays a key role in Alzheimers disease. After its production by catabolism of the amyloid precursor protein (APP) through the action of presenilin 1 (PS1)- or presenilin 2 (PS2)-dependent {gamma}-secretases, monomeric A{beta} can assemble in oligomers. In a pathological context, this eventually leads to the formation of fibrils, which deposit in senile plaques. Many studies suggest that A{beta} toxicity is related to its soluble oligomeric intermediates. Among these, our interest focuses on hexameric A{beta}, which acts as a nucleus for A{beta} self-assembly. MethodsBiochemical analyses were used to identify hexameric A{beta} in a wide range of models; cell lines, cerebrospinal fluid from cognitively impaired patients and transgenic mice exhibiting human A{beta} pathology (5xFAD). We isolated this assembly and assessed both its effect on primary neuron viability in vitro, and its contribution to amyloid deposition in vivo following intracerebral injection. In both cases, we used wild-type mice (C57BL/6) to mimic an environment where hexameric A{beta} is present alone and 5xFAD mice to incubate hexameric A{beta} in a context where human A{beta} species are pre-existing. Using CRISPR-Cas9, we produced stable knockdown human cell lines for either PS1 or PS2 to elucidate their contribution to the formation of hexameric A{beta}. ResultsIn WT mice, we found that neither in vitro or in vivo exposure to hexameric A{beta} was sufficient to induce cytotoxic effects or amyloid deposition. In 5xFAD mice, we observed a significant increase in neuronal death in vitro following exposure to 5M hexameric A{beta}, as well as a 1.47-fold aggravation of amyloid deposition in vivo. At the cellular level, we found hexameric A{beta} in extracellular vesicles and observed a strong decrease in its excretion when PS2 was knocked down by 60%. ConclusionsOur results indicate the absence of cytotoxic effects of cell-derived hexameric A{beta} by itself, but its capacity to aggravate amyloid deposition by seeding other A{beta} species. We propose an important role for PS2 in the formation of this particular assembly in vesicular entities, in line with previous reports linking the restricted location of PS2 in acidic compartments to the production of more aggregation-prone A{beta}.