Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles - PubMed (original) (raw)

doi: 10.1038/cdd.2013.180. Epub 2013 Dec 13.

E Turola 1, A Ruiz 2, A Bergami 3, D D Libera 3, L Benussi 4, P Giussani 2, G Magnani 3, G Comi 3, G Legname 5, R Ghidoni 4, R Furlan 3, M Matteoli 6, C Verderio 7

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Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles

P Joshi et al. Cell Death Differ. 2014 Apr.

Abstract

Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are the most toxic forms of Aβ. Preventing soluble Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aβ species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic soluble Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.

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Figures

Figure 1

Figure 1

Microglia-derived MVs promote A_β_ neurotoxicity. Fourteen DIV hippocampal neurons were exposed for 1 h to A_β_ 1–42 or scrambled A_β_ 1–42 (4 μ_M) pre-incubated with MVs (1 μ_g/100 μ_l) overnight in neuronal medium. (a) Overlays of DIC and fluorescence microscopic images of neurons stained for calcein and propidium iodide (PI), after 24 h exposure to A_β 1–42/MVs mixture or under control conditions. (b) Percentage of calcein−/PI+ neurons (dead cells) in cultures exposed to A_β 1–42, scrambled A_β 1–42, MVs or A_β_ 1–42/scrambled A_β_ 1–42 incubated overnight with MVs. AA-MVs refer to freshly isolated MVs added to A_β_ 1–42, just before neuron challenge (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05). (c) Basal [Ca2+]i was measured in single neurons loaded with the ratiometric calcium dye Fura-2 and expressed as F340/380 fluorescence. Representative pseudocolor images of 9DIV control neurons and neurons treated with A_β_1–42/MVs mixture for 1 h. The color scale is shown on the left. (d) Quantification of basal [Ca2+]i in neurons exposed to A_β_ 1–42, MVs or A_β_ 1–42 in combination with MVs. At least 100 neurons/condition were examined. Values are normalized to control (the Kruskal–Wallis ANOVA, _P_=0.002; Dunn's test for comparison among groups, *P<0.05). (e) Quantification of early apoptotic damage, revealed by Annexin-V binding, normalized to SNAP-25 immunoreactive area, in neurons treated as in d. (the Kruskal–Wallis ANOVA, P_=0.001; Dunn's test for comparison among groups, *P<0.05). (f) Confocal microscopic images of 14DIV neurons untreated or pretreated with A_β 1–42 in combination with MVs and stained for β_-3 tubulin, the vesicular glutamate transporter vGlut-1 and the postsynaptic marker PSD-95. Nuclei are stained with Hoechst. Note, fragmentation of neuronal processes and loss of excitatory synapses in neurons exposed to A_β 1–42/ MVs mixture. Density of excitatory synaptic puncta is quantified in g (data follow normal distribution, Student's t_–test, **P<0.001). (h–j) Control cultures and cells treated with A_β 1–42/MVs mixture analyzed for basal [Ca2+]i (h, the Kruskal–Wallis ANOVA, _P_=0.001; Dunn's test for comparison among groups, *P<0.05), early apoptotic damage (i, the Kruskal–Wallis ANOVA, _P_=0.001; Dunn's test for comparison among groups, *P<0.05) and calcein/PI staining (j, the Kruskal–Wallis ANOVA P<0.001; Dunn's test for comparison among groups, *P<0.05), either in the presence or in the absence of the glutamate receptor antagonists APV and CNQX

Figure 2

Figure 2

Shed MVs promote formation of soluble forms of A_β_ 1–42. (a) Basal [Ca2+]i in neurons exposed for 1 h either to A_β_ 1–42/MVs mixture or soluble (sup)/insoluble (pellet) fractions (left panel, the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05). Values are normalized to control. Right panel shows the percentage of calcein−/PI+ neurons under the same conditions (the Kruskal–Wallis ANOVA, P_=0.002; Dunn's test for comparison among groups, *P<0.05). (b) Basal [Ca2+]i in neurons exposed for 1 h to MVs derived from resting, M1 or M2 microglia pre-incubated with extracellular A_β 1–42. Values are normalized to control (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05). (c) Basal [Ca2+]i in neurons exposed to A_β_ 1–42 and/or MVs in the presence or in the absence of neutralizing antibodies for IL-1_β_ and TNF_α_. Values are normalized to control. (d) Representative ThT fluorescence emission spectra of samples containing A_β_1–42 fibrils (dashed red lines) or aggregated A_β_ 1–42 (dashed blue lines) exposed to MVs. (e) Time course of fibrilization of A_β_ 1–42 in the presence (dashed line) or in the absence (solid line) of MVs. (f) Negative staining electron microscopic image of aggregated A_β_ 1–42, incubated overnight in neuronal medium. (g) Representative confocal images of Hylite-488-A_β_ 1–42 (488-A_β_ 1–42) fibrils untreated or treated overnight with MVs and exposed for 1 h to neurons. Neurons are stained in blue for MAP-2 after fixation. Cumulative distribution of fibril size from control (solid line) and MV-treated (dashed line) 488-A_β_ 1–42 fibril preparations is shown on the right. (h) ‘Floating assay' by ultracentrifugation reveals an increase of soluble A_β_ 1–42 species in association with MVs. After centrifugation for 1 h at 100 000 × g, MVs are expected in the top fraction, whereas A_β_ 1–42 aggregates are expected in the pellet. As ELISA indicates, a higher fraction of A_β_ 1–42 species is transported from the bottom to the top of the gradient in samples incubated overnight with MVs. Acute addition of MVs (AA-MVs) does not cause statistically significant changes in A_β_ 1–42 distribution (the Kruskal–Wallis ANOVA, P<0.001; Tukey's test for comparison among groups, *P<0.05). (i) ThT fluorescence emission spectra of aggregated A_β_ 1–42 (blue lines) or A_β_ 1–42 fibrils (red lines), untreated (solid lines) or pretreated (dashed lines) with shed MVs. Spectra of aggregated A_β_ 1–42 or A_β_ 1–42 fibrils exposed to MVs lipids (dotted lines) are also shown. (j) Basal [Ca2+]i of neurons exposed for 1 h to A_β_ 1–42 pretreated with intact MVs, small unilamellar vesicles of MV lipids (MV lipids) or artificial liposomes. Note that vesicles made by lipids extracted from shed MVs but not artificial liposomes significantly enhance basal [Ca2+]i

Figure 3

Figure 3

Binding of newly generated soluble 488-A_β_-1–42 to neurons is competed by PrPC. (a) Representative confocal images of 14DIV neurons exposed to 488-A_β_ 1–42 alone or in combination with MVs, with or without pretreatment with PrP or with the anti-A_β_ antibodies A11 and 6E10. (b) Corresponding quantification of 488-A_β_ 1–42 binding to cultured neurons expressed as colocalizing area between 488-A_β_ and β tubulin, relative to total β tubulin (see Materials and Methods) (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test comparison among groups, *P<0.05). (c and d) Basal [Ca2+]i (c) and percentage of calcein−/PI+ neurons (d) in 9–14 DIV cultures exposed to different combinations of A_β_ 1–42, MVs, A11 plus 6E10 antibodies, full-length or truncated (tPrPC) PrPC (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05). (e–g) Bio-detection of soluble A_β_ 1–42 by fura-2-loaded sensor neurons, expressing functional NMDA receptors. Representative traces of [Ca2+]i changes recorded in neurons upon exposure to KRH containing A_β_ 1–42 alone (4 _μ_M) or MVs alone (1 _μ_g/100 μ_l) or their combination (e). [Ca2+]i responses induced by A_β 1–42/MVs mixture are strongly inhibited by the NMDA receptor antagonist APV (f), as quantified in g. Values represent peak [Ca2+]i increases (ΔF340/380 fluorescence) from about 30 neurons/condition (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05)

Figure 4

Figure 4

Soluble A_β_ forms are released in association with shed MVs from microglia activated with A_β_ 1–42. (a) Living rat microglia were exposed to human A_β_ 1–42 for 12–48 h and stained with IB4-FITC to label the cell surface before being fixed and counterstained with 6E10 antibody, which recognizes human but not rat amyloids. Top left panel shows representative xy plane maximum projection of microglia, revealing several 6E10 immunoreactive puncta inside the cells, some of which are double positive for surface IB4-FITC. Bottom left panel showing single stack of the selected cell, shown at higher magnification, reveals a clear association of internalized A_β_ 1–42 to the cell surface, further revealed by the z axis scan. Note, an increase in the size of internalized A_β_ 1–42 after incubation for 48 h (top right panel). Examples of EMVs, double positive for 6E10 and IB4-FITC are shown in bottom right panels. (b) Western blot analysis of A_β_ 1–42 species present in shed MVs (P2 and P3 fractions) and exosomes (P4 fraction) released upon 30 min ATP stimulation by 4 × 106 microglia pre-exposed to biotinylated A_β_ 1–42 (4 μ_M). Blots were carried out using a 15% Tris-glycine gel and membranes were probed with streptavidin. Shed MVs and exosomes produced by 8 × 106 donor microglia were probed in parallel for the EMV markers Tsg101 and the exosomal marker Alix (lower panels). Numbers below each lane indicate the estimated amount of loaded proteins. (c) Shed MVs and exosomes produced by 1 × 106 rat microglia pre-exposed to human A_β 1–42 were analyzed by a SELDI-TOF MS immunoproteomic assay employing anti-human A_β_ antibodies (4G8 and 6E10) on PS20 chip array to capture A_β_ 1–42 and carboxy-terminally truncated A_β_ isoforms. The following representative spectra of samples in NP40 1% lysis buffer are shown (from top to bottom): 4 μ_M A_β_1–42 peptide incubated overnight in KRH; MVs from control microglia, not exposed to A_β_1–42; MVs from A_β_1–42 preloaded microglia (A_β_-MVs); exosomes from control microglia (exos); exosomes from A_β_1–42 preloaded microglia (A_β_-exos). (d) Basal [Ca2+]i recorded from neurons exposed to MVs produced from microglia either resting or pretreated for 48 h with A_β 1–42 (A_β_-MVs), in the presence or in the absence of anti-A_β_ antibodies (A11+6E10) (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05). See also Supplementary Figure S1

Figure 5

Figure 5

CSF MVs isolated from AD patients. (a) Quantitative flow cytometry analysis of IB4+ MVs in CSF collected from MCI patients (n_=53), AD patients (n_=89) and age- and gender-matched controls (HC; n_=20) (the Mann–Whitney test, P<0.0001 AD versus HC; P<0.0329 MCI versus HC). (b) Correlation between IB4+ MVs and total tau protein in the CSF of MCI and AD patients, (rho=0.46, P<0.0001, Spearman's correlation). (c) Representative confocal images of cultured neurons, exposed to aggregated 488-A_β 1–42 untreated or pretreated overnight with MVs from AD patients and stained for MAP-2 after fixation (red). A_β species bind to MAP-2 dendrites. Note the decrease in the number of large fluorescent A_β clusters in neurons exposed to 488-A_β_ 1–42 in combination with AD MVs. (d) Quantification of 488-A_β_ 1–42 aggregates (larger than 5 μ_m) per field (data follow normal distribution, Student's t_–test, **P<0.001). (e) Quantification of 488-A_β 1–42 binding to cultured neurons, expressed as colocalizing area between 488-A_β and β tubulin, relative to total β tubulin (see materials and Methods; data follow normal distribution, Student's t_–test, **P<0.001). (f) Representative fluorescence microscopy images of 14DIV neurons triple stained for calcein, PI and Hoechst 24 h after exposure to AD MVs or maintained in control conditions. (g) Quantification of the percentage of calcein−/PI+ neurons (dead cells) in cultures exposed to AD MVs or MVs isolated from patients with multiple sclerosis (the Kruskal–Wallis ANOVA, P<0.001; Dunn's test for comparison among groups, *P<0.05). (h) Percentage of dead neurons in cultures exposed to MVs isolated from the CSF of AD patients in the presence of anti-A_β antibodies A11 and 6E10 (ANOVA, P<0.001, the Holm–Sidak method, P<0.05). See also Supplementary Figure S2 and Supplementary Table S1

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