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. 2016 May 12;11(1):38.
doi: 10.1186/s13024-016-0098-z.

Accumulation of amyloid-β by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons

Sofia Söllvander  1 , Elisabeth Nikitidou  1 , Robin Brolin  1 , Linda Söderberg  2 , Dag Sehlin  1 , Lars Lannfelt  1 , Anna Erlandsson  3
Affiliations

Accumulation of amyloid-β by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons

Sofia Söllvander et al. Mol Neurodegener. .

Abstract

Background: Despite the clear physical association between activated astrocytes and amyloid-β (Aβ) plaques, the importance of astrocytes and their therapeutic potential in Alzheimer's disease remain elusive. Soluble Aβ aggregates, such as protofibrils, have been suggested to be responsible for the widespread neuronal cell death in Alzheimer's disease, but the mechanisms behind this remain unclear. Moreover, ineffective degradation is of great interest when it comes to the development and progression of neurodegeneration. Based on our previous results that astrocytes are extremely slow in degrading phagocytosed material, we hypothesized that astrocytes may be an important player in these processes. Hence, the aim of this study was to clarify the role of astrocytes in clearance, spreading and neuronal toxicity of Aβ.

Results: To examine the role of astrocytes in Aβ pathology, we added Aβ protofibrils to a co-culture system of primary neurons and glia. Our data demonstrates that astrocytes rapidly engulf large amounts of Aβ protofibrils, but then store, rather than degrade the ingested material. The incomplete digestion results in a high intracellular load of toxic, partly N-terminally truncated Aβ and severe lysosomal dysfunction. Moreover, secretion of microvesicles containing N-terminally truncated Aβ, induce apoptosis of cortical neurons.

Conclusions: Taken together, our results suggest that astrocytes play a central role in the progression of Alzheimer's disease, by accumulating and spreading toxic Aβ species.

Keywords: Alzheimer’s disease; Degradation; Enlarged vacuole; Glia; Microvesicle; Phagocytosis; Protofibrils.

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Figures

Fig. 1
Fig. 1
Aβ42 protofibril deposits are found in glial cells, but not in neurons. Immunocytochemistry of co-cultures containing astrocytes, neurons and oligodendrocytes demonstrates that astrocytes contain large deposits of Aβ42-555 (a). Neurons lack detectable Aβ42-555 levels (b), but the few oligodendrocytes in the culture also contain Aβ42-555 (c). Confocal imaging confirms intracellular localization of large Aβ42-555 inclusions in astrocytes (d). DAPI (blue), GFAP (green) and Aβ42-555 (red). Scale bars: a–c = 20 μm, d = 10 μm
Fig. 2
Fig. 2
Aβ42 protofibrils accumulate intracellulary in astrocytes over time. Time-lapse images from 30 min to 24 h following Aβ42-555 protofibril administration show that astrocytes (A), but not neurons (*), accumulate Aβ42-555 over time (a). Higher magnification of a highly vascularized astrocyte with ingested Aβ42-555 (b) and a neuron with undetectable levels of intracellular Aβ42-555 (c). Immunohistochemistry of tg-ArcSwe mouse brain sections confirm that Aβ plaques are surrounded by GFAP positive astrocytes, Aβ (red), GFAP (green) and DAPI (blue) (d) and that Aβ are engulfed by astrocytes in vivo (e). Scale bars: a–d = 10 μm
Fig. 3
Fig. 3
Aβ42 are stored in astrocytes for a very long time. To follow degradation of intracellular Aβ42-555 in astrocytes following Aβ42-555 protofibril removal, cells were divided into four categories; cells with no detectable levels of Aβ42-555 (–), cells with only small amounts of Aβ42-555 (+), cells containing medium sized Aβ42-555 deposits (++) and cells with large Aβ42-555 inclusions (+++) (a). Astrocytes degrade Aβ42-555 protofibrils very slowly and much Aβ42 remains in the cells 12 days after Aβ42-555 protofibril removal (b). Neurons contain almost no Aβ42 (c). The few oligodendrocytes in the culture also degrade the Aβ42-555 protofibrils slowly (d). Although the glial cells degrade the Aβ42-555 protofibrils slowly, the total number of 555-stained inclusions (24 h: 10.4 ± 7.7; 24 h + 6 days: 7.9 ± 7.1; 24 h + 12 days: 6.2 ± 7.1) in the culture decline significantly from 24 h to 24 h + 6 days (P = 0.013) and 24 h + 12 days (P < 0.001) (e). In line with these results, the total 555-intensity (24 h: 1.7 × 107 ± 8.5 × 106; 24 h + 6 days: 8.3 × 106 ± 5.1 × 106; 24 h + 12 days: 5.9 × 106 ± 4.3 × 106) (f) and total 555-positive area (24 h: 9.6 ± 2.3 μm2; 24 h + 6 days: 6.7 ± 3.5 μm2; 24 h + 12 days: 6.8 ± 2.5 μm2) (g) also decline significantly over time (P < 0.001 already from 24 h to 24 h + 6 days). Aβ inclusions are marked with white arrow heads. Scale bars: 10 μm. The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed. Mann–Whitney U-test ***P < 0.001
Fig. 4
Fig. 4
Aβ42 accumulates in immature lysosomes. The co-localization of Aβ42 and LAMP-1 positive lysosomes increases from 24 h to 24 h + 12 days (a). LysoTracker staining does not overlap with the Aβ42 protofibril inclusions, demonstrating that the Aβ42 containing lysosomes are immature (b). Aβ42 protofibrils co-localize with LAMP-2 (c) and GFAP (d) in sections of 14-month-old tg-ArcSwe mice. Scale bars: a–d = 20 μm
Fig. 5
Fig. 5
Intracellular stored Aβ42 is truncated in the N-terminus. Aβ ELISAs of cell lysates from 24 h and from 6 to 12 days following Aβ42 protofibril removal (24 h + 6 days and 24 h + 12 days, respectively) demonstrate that a high proportion of the accumulated Aβ is N-terminally truncated. The concentrations of Aβ1-x (a) and Aβ protofibrils (b) decrease continuously from 24 h (2434.0 ± 272.0 and 946.9 ± 36.5, respectively) to 24 h + 6 days (862.8 ± 92.0 and 325.8 ± 25.9, respectively) and 24 h + 12 days (468.2 ± 52.2 and 282.9 ± 13.1, respectively), while the concentrations of Aβx-42 remain higher over time (24 h: 2475.0 ± 134.0, 24 h + 6 days: 226.01 ± 318.3 and 24 h + 12 days: 1531.0 ± 253.1) (c). In the pellets, remaining after the cell lysis procedure, the Aβ1-x concentrations decline over time (24 h: 1840.0 ± 289.5, 24 h + 6 days: 938.3 ± 159.7, 24 h + 12 days: 483.3 ± 225.3) (d), while the Aβx-42 concentrations are higher at 24 h (2422.0 ± 387.0) and more stable over time, 24 h + 6 days (1493.0 ± 132.7) and 24 h + 12 days (1488.0 ± 312.6) (e). All concentrations are expressed in picomolar (pM) units. Mean values are from duplicates of three independent experiments
Fig. 6
Fig. 6
Aβ42 protofibril treatment results in formation of enlarged endosomes. Time-lapse experiments demonstrate that Aβ42 protofibril treatment induces the formation of enlarged, dynamic vacuoles (*) in the astrocytes (a). The enlarged vacuoles fuse (white arrow heads) with adjacent vacuoles; forming giant vacuoles with a diameter of 30–50 μm (b). Immunocytochemistry show that the vacuoles express the early endosome marker Rab5 and to a lesser extent the late endosome marker Rab7 (c). Inset in left corner of image c shows higher magnification of relevant structure. Scale bars: a–b = 10 μm, c = 20 μm
Fig. 7
Fig. 7
Aβ42 protofibril treatment induces secondary neuronal cell death. The number of astrocytes in Aβ42 protofibril exposed cultures is significantly increased from 24 h (1.0 ± 0.1) to 24 h + 12 days (1.1 ± 0.1, P = 0.011) (a). Similar to astrocytes, the number of oligodendrocytes increases from 24 h (0.7 ± 0.3) to 24 h + 12 days (1.7 ± 1.5, P < 0.001) in Aβ42 protofibril treated cultures (b). Aβ42 protofibril treatment has no direct effect on neurons, but the neuronal cell number significantly decreases (from both 24 h, 0.9 ± 0.4, P = 0.0061 and 24 h + 6 days, 1.0 ± 0.4, P = 0.0083) in the treated cultures 12 days (0.7 ± 0.5) after Aβ removal (c). The relative number of viable cells in Aβ42 protofibril exposed cultures is normalized to viable cells in unexposed cultures. The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed. Mann–Whitney U-test ***P < 0.001
Fig. 8
Fig. 8
Neuronal cell death is induced by secreted microvesicles. Illustration of the experimental setup (a). Electron microscopy images of Aβ42 protofibril exposed co-cultures demonstrate microvesicle (*) secretion from a single astrocyte (b) and a larger (c) and smaller (d) microvesicle present in the cell culture medium. Western blot analysis show that the isolated microvesicles express Flotillin-1, but that the levels are unchanged in Aβ42 protofibril treated cultures, compared to controls (e). TUNEL assays demonstrate a significant increase (P < 0.001) in apoptotic neurons following treatment with microvesicles from Aβ42 protofibril exposed co-cultures (465.4 ± 150.4), compared to microvesicles from untreated co-cultures (308.3 ± 109.2) (f). The experiments were performed in triplicates with independent cell cultures and 10 images/experiment were analyzed. Microvesicles isolated from medium collected 6 and 12 days after Aβ42 protofibril exposure contain Aβ as revealed in Aβ1-x (396.9 ± 167.2 pM) and Aβx-42 (1239.0 ± 438.7 pM) ELISA. The Aβ1-x concentration is lower than Aβx-42, indicating that there is a truncation of the Aβ42 N-terminus (g). Duplicate samples from 3 independent experiments were analyzed. Mann–Whitney U-test ***P < 0.001. Scale bars: b = 1 μm, c–d = 100 nm
Fig. 9
Fig. 9
Aβ42 protofibril characterization. A chromatogram following Aβ42 protofibril analysis by SEC using a Superdex 75 column. The chromatograms show mV for the absorbance at 214 nm on the y-axis and the retention time in minutes on the x-axis
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