Aggregation shifts amyloid-β peptides from synaptogenic to synaptotoxic
Alberto Siddu, Silvia Natale, Connie H. Wong, Hamidreza Shaye, Thomas C. Südhof
Alberto Siddu, Silvia Natale, Connie H. Wong, Hamidreza Shaye, Thomas C. Südhof
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Research Article Cell biology Neuroscience

Aggregation shifts amyloid-β peptides from synaptogenic to synaptotoxic

Abstract

Whether amyloid-β (Aβ) peptides are synaptogenic or synaptotoxic remains a pivotal open question in Alzheimer’s disease research. Here, we chronically treated human neurons with precisely controlled concentrations of chemically defined synthetic Aβ40, Aβ42, and Aβ42arctic peptides that exhibit distinct aggregation propensities. Remarkably, chronic exposure of human neurons to free Aβ40 at higher concentrations or to free Aβ42 at lower concentrations potently promoted synapse formation. In contrast, aggregated Aβ42 or Aβ42arctic at higher concentrations were neurotoxic and synaptotoxic. The synaptotoxic effects of Aβ peptides manifested as an initial contraction of the synaptic vesicle cluster followed by synapse loss. Aβ40 and Aβ42 peptides with scrambled or inverted sequences were inactive. Thus, our experiments reveal that Aβ peptides exhibit an aggregation-dependent functional dichotomy that renders them either synaptogenic or synaptotoxic, thereby providing insight into how Aβ peptides straddle a thin line between physiological synapse organization and pathological synapse disruption. Among others, our data suggest that Alzheimer’s disease therapies might aim to shift the balance of Aβ peptides from the aggregated to the free state instead of suppressing all Aβ peptides.

Authors

Alberto Siddu, Silvia Natale, Connie H. Wong, Hamidreza Shaye, Thomas C. Südhof

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Figure 1

Chemically defined Aβ42 and Aβ42arctic peptides, but not Aβ40 peptides, display robust toxicity in human neurons.

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Chemically defined Aβ42 and Aβ42arctic peptides, but not Aβ40 peptides, ...
(A) Experimental strategy. The experimental design results in chronic exposure of human neurons to Aβ peptides at defined concentrations for 39 days. Note that the Aβ peptides were not exposed to aggregating conditions prior to culture additions. Once added, however, at least Aβ42 and Aβ42arctic are likely subject to dynamic aggregation. As controls, Aβ40 and Aβ42 peptides with scrambled or reverse sequences were used. (B) Measurements of the toxicity of various Aβ peptides at the indicated concentrations using the MTT assay for cell damage (21). Treatments with DMSO (10%) were included as a highly toxic positive control. The effects of scrambled or reverse-sequence control peptides are shown on the right. (C and D) Measurements of the toxicity of Aβ peptides at the indicated concentrations using the NLS-TdTomato assay that scores the leakage of NLS-TdTomato containing a nuclear localization signal from the nucleus into the cytoplasm. NLS-TdTomato only leaks into the cytoplasm when a cell’s energy state is compromised and ATP levels decline. (C) Representative images; scale bar: 10 μm. (D) Summary graphs of the density of neurons containing cytoplasmic NLS-TdTomato). (E) Quantification of the neuronal cell density measured as the density of NLS-TdTomato-positive nuclei to demonstrate that only high concentrations of Aβ42 and Aβ42arctic cause significant cell death that is modest at the relatively low Aβ42 and Aβ42arctic concentrations used. All neurons were analyzed at DIV45. All numerical data are means ± SEM; numbers of independent experiments are reported in the bars (n = 3). Statistical significance was assessed as prespecified by 1-way ANOVA with post hoc corrections, comparing the mean of each group with control (Ctrl), with **P < 0.01, and ****P < 0.0001. Nonsignificant comparisons are not indicated. Additional representative images, analyses of the data as pseudo replicates, validation of the NLS-TdTomato assay, and confirmation of the results with a third cell toxicity assay in addition to the MTT assay and the NLS-TdTomato assay are shown in Supplemental Figure 1. *P < 0.05; **P < 0.01; ***P <.001; ****P <.0001.