The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death

Abstract

Background: Currently, several amyloid beta (Aβ) antibodies, including the protofibril selective antibody BAN2401, are in clinical trials. The murine version of BAN2401, mAb158, has previously been shown to lower the levels of pathogenic Aβ and prevent Aβ deposition in animal models of Alzheimer's disease (AD). However, the cellular mechanisms of the antibody's action remain unknown. We have recently shown that astrocytes effectively engulf Aβ₄₂ protofibrils, but store rather than degrade the ingested Aβ aggregates. In a co-culture set-up, the incomplete degradation of Aβ₄₂ protofibrils by astrocytes results in increased neuronal cell death, due to the release of extracellular vesicles, containing N-truncated, neurotoxic Aβ.

Methods: The aim of the present study was to investigate if the accumulation of Aβ in astrocytes can be affected by the Aβ protofibril selective antibody mAb158. Co-cultures of astrocytes, neurons, and oligodendrocytes, derived from embryonic mouse cortex, were exposed to Aβ₄₂ protofibrils in the presence or absence of mAb158.

Results: Our results demonstrate that the presence of mAb158 almost abolished Aβ accumulation in astrocytes. Consequently, mAb158 treatment rescued neurons from Aβ-induced cell death.

Conclusion: Based on these findings, we conclude that astrocytes may play a central mechanistic role in anti-Aβ immunotherapy.

Keywords: Alzheimer’s disease; Amyloid-β; Antibody; Astrocyte; Clearance; Neuron.

Fig. 1

mAb158 reduces Aβ accumulation in astrocytes. Exposure of co-cultures to Aβ₄₂-555 protofibrils for 24 h results in large Aβ inclusions in astrocytes (a). Concurrent addition of mAb158 clearly reduces the intracellular Aβ₄₂-555 in astrocytes (b). However, mAb158 added 3 days after the Aβ₄₂-555 protofibril exposure had no effect on Aβ₄₂-555 accumulation (c). Using the Zen software, the 555-intensity and 555-stained area of the inclusions were measured. The 555-intensity/number of living cells (d), 555-intensity/number of Aβ inclusions (e), and total 555-stained area of Aβ-555 inclusions (f) decreased significantly in co-cultures treated with Aβ₄₂-555 protofibrils + mAb158, compared to cultures exposed to Aβ₄₂-555 protofibrils only or cultures treated with mAb158 3 days after Aβ₄₂-555 protofibril exposure. Confocal imaging demonstrates intracellular co-localization of Aβ₄₂-555 protofibrils and mAb158 in astrocyte (g). GFAP (green), DAPI (blue), mAb158 (white), and Aβ₄₂-555 (red). The image is an orthogonal view of a confocal z-stack image. The XZ plane and YZ plane are shown at the top and to the right of the XY image, respectively. Scale bar 20 μm. The experiments were performed in triplicates with independent cell cultures, and 10 images/experiment were analyzed. Statistical analysis using Mann–Whitney U test (***P < 0.001)

Fig. 2

mAb158 rescues neurons from Aβ-induced toxicity. Aβ₄₂-555 protofibril exposure of co-cultures results in neuronal cell death 12 days following the removal of the Aβ₄₂-555 protofibrils (a). Simultaneous addition of mAb158, significantly rescued the neurons (b), but this effect was not seen when the mAb158 was added 3 days after the Aβ₄₂-555 protofibrils (c). The relative number of living neurons was significantly decreased 12 days following Aβ₄₂-555 protofibril exposure (24 h + 12 days, **P < 0.01), but no neurotoxic effect was observed at 24 h + 12 days in cultures exposed to Aβ₄₂-555 protofibrils together with mAb158. When mAb158 were added 3 days after the Aβ₄₂-555 protofibril removal, no protective effect of mAb158 was found (***P < 0.001) (d). βIII-tubulin (green) and DAPI (blue). Scale bar 20 μm. The experiments were performed in triplicates with independent cell cultures, and 10 images/experiment were analyzed using Mann–Whitney U test

Fig. 3

mAb158 reduces intracellular Aβ load, at least partly through an Fcγ receptor-independent pathway. Aβ₄₂ protofibril accumulation in astrocytes (a) is reduced by preincubating Aβ₄₂ protofibrils with mAb158 (b) or with N297D (c). Although, there was a clear reduction in Aβ accumulation with the presence of N297D as compared to Aβ₄₂ protofibril exposed co-cultures, the cultures treated with mAb158 had significantly lower 555-intensity/number of living cells (d), 555-intensity/number of Aβ inclusions (e), and total 555-stained area of Aβ inclusions (f), compared to cultures treated with N297D. GFAP (green), DAPI (blue), and Aβ (red). Scale bar 20 μm. The experiments were performed in triplicates with independent cell cultures, and 10 images/experiment were analyzed. Statistical analysis using Mann–Whitney U test (***P < 0.001)

Fig. 4

Anti-Aβ protofibril selective antibodies reduce the levels of intracellular Aβ. The Aβ levels in total cell lysates were measured using Aβ_1-x and Aβ_x-42 ELISA. Both Aβ_1-x (a) and Aβ_x-42 (b) levels were reduced in cultures treated with Aβ₄₂ protofibrils + mAb158, Aβ₄₂ protofibrils + RmAb158, or Aβ₄₂ protofibrils + N297D, compared to cultures exposed to Aβ₄₂ protofibrils only. All concentrations are expressed in nanomolar (nM) units. Mean values are from duplicates of three independent experiments, from two repeated analyses (**P < 0.01)

Fig. 5

In the presence of mAb158, Aβ is cleared from the cell culture media. Western blot analysis showed a reduction of Aβ in the cell culture media of cultures exposed to Aβ₄₂ protofibrils + mAb158, compared to cultures exposed to Aβ₄₂ protofibril only (a). The reduction was seen both in low-molecular Aβ at ~ 10 kDa (b) and ~ 5 kDa (c) and in the smear of Aβ detected at ~ 38–160 kDa (d). Intensity measurements were performed from three independent cell culture experiments, and statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (*P < 0.05)

Fig. 6

mAb158 F(ab’)₂ fragments reduce Aβ accumulation in astrocytes. To confirm that the effect of mAb158 on Aβ₄₂ protofibril degradation was a Fcγ receptor-independent mechanism, cell cultures were exposed to the F(ab’)₂ fragments of mAb158 together with the Aβ₄₂ protofibrils. Astrocytes in co-cultures exposed to Aβ₄₂ protofibrils accumulated Aβ (a) but not when co-treated with mAb158 (b). In cultures treated with F(ab’)₂ mAb158 + Aβ₄₂ protofibrils, the accumulation of Aβ was reduced, almost to the same level as for mAb158 (c). The 555-intensity/number of living cells (d), 555-intensity/number of Aβ inclusions (e), and total 555-stained area of Aβ inclusions (f) were all significantly reduced in co-cultures treated with Aβ₄₂ protofibril + mAb158 and Aβ₄₂ protofibril + mAb158 F(ab’)₂ fragments, as compared to Aβ₄₂ protofibril exposed co-cultures. GFAP (green), DAPI (blue), and Aβ (red). Scale bar 20 μm. The experiments were performed in triplicates with independent cell cultures, and 10 images/experiment were analyzed using Mann–Whitney U test (***P < 0.001)

Fig. 7

Proteosomal or endosomal-lysosomal inhibitors do not alter the mAb158-mediated effects on Aβ accumulation in astrocytes. Co-cultures were incubated with the proteosomal inhibitor mg-132 or the lysosomal inhibitor bafilomycin for 30 min prior to the Aβ₄₂ protofibril exposure and mAb158 treatment. The inhibitors were also present during the 24-h exposure. Neither of the inhibitors affected the mAb158-mediated reduction of Aβ deposits (a–d). Measurement of 555-intensity/number of living cells (e), 555-intensity/number of Aβ inclusions (f), and total 555-stained area of Aβ inclusions (g) demonstrated that there was a significant reduction in Aβ inclusions in Aβ₄₂ protofibril-exposed cultures when co-treated with mAb158 + bafilomycin or mAb158 + mg-132. This confirms that the inhibitors did not interfere with the action of mAb158. GFAP (green), DAPI (blue), and Aβ (red). Scale bar 20 μm. The experiments were performed in triplicates with independent cell cultures, and 10 images/experiment were analyzed using Mann–Whitney U test (**P < 0.01)