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Cited by 106Alzheimer's disease research and treatmentsExtracellular vesicles in diseaseNeuroinflammation and Neurodegeneration Mechanisms

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Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer’s disease

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Gabrielli, Martina, Prada, Ilaria, Joshi, Pooja, Falcicchia, Chiara, D’Arrigo, Giulia, Rutigliano, Grazia, Battocchio, Elisabetta, Zenatelli, Rossella, Tozzi, Francesca, Radeghieri, Annalisa, Arancio, Ottavio, Origlia, Nicola, Verderio, Claudia·2022·Brain
DOI 10.1093/brain/awac083PMC PMC9420022
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Abstract

Synaptic dysfunction is an early mechanism in Alzheimer’s disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-β released by microglia in association with large extracellular vesicles (Aβ-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex–dentate gyrus circuitry. One hour after Aβ-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24 h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aβ naturally secreted by CHO7PA2 cells, while neither Aβ42 alone nor inflammatory extracellular vesicles devoid of Aβ were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aβ-EV–neuron interaction, we show that Aβ-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aβ-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal–hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer’s disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.

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Figures

Figure 1 Morphological features and Aβ42 content of Aβ-EVs in the 10 000g pellet. (A) Western blot analysis for the EV markers flotillin I, alix and annexin-A2, the Golgi and mitochondria markers GS28 and TOM20, the cytosolic marker GAPDH and for Aβ (6E10) of EVs in the 10 000g pellet from 10 × 106 microglia and relative donor cells (2 µg cell lysate). Normalization to total proteins is shown in Supplementary Fig. 1. (B) Analysis of the purity of EV preparations from Aβ42-treated microglia and control cells using the CONAN assay; AI% is the relative aggregation index of AuNPs, normREF is a sample of monodispersed AuNPs, negREF (HPLC grade water+AuNPs+PBS solution) is the negative control threshold and posREF is the positive control (PBS solution+AuNPs). (C) Size distribution of large Aβ-EVs enriched fraction analysed by TRPS. The fraction of Aβ-EVs of diameter >200 nm represents ∼59% of the 10 000g pellet. (D) Charge measurements of large ctrl-EVs and Aβ-EVs (10 000g pellet) by TRPS. Ctrl-EVs display an averaged surface charge of −10.75 mV, while Aβ-EVs −22.57 mV (t-test, P < 0.001). (E) Aβ42 content in intact Aβ-EVs or Aβ-EVs lysed with 0.57% Triton X-100 (10 000g pellet), as detected by ELISA. Values are normalized to intact Aβ-EVs (t-test, P = 0.002, n = 3). (F) Aβ42 content in large Aβ-EVs enriched fraction (10 000g pellet) produced by microglia exposed for 20 h to Aβ42 with or without Bafilomycin A1 (Baf) during the last 15 h of treatment, as detected by ELISA in the presence of 0.57% Triton X-100. Values are normalized to the condition without Baf (Mann–Whitney Rank Sum Test, P < 0.001, n = 7). (G) Aβ-EVs in the 10 000g pellet from microglia stimulated as in F. Values (EV numbers) are normalized to the condition without Baf. Mann–Whitney Rank Sum Test, P = 0.908, n = 3. (H) Representative cryo-electron microscopy micrograph of ctrl- and Aβ-EVs in the 10 000g pellet. Arrows point to multilamellar EVs; black arrowheads to EVs with rough surface; white arrowhead to tubular vesicles. Scale bars: 100 nm. Box plots show the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers).
Figure 1 Morphological features and Aβ42 content of Aβ-EVs in the 10 000g pellet. (A) Western blot analysis for the EV markers flotillin I, alix and annexin-A2, the Golgi and mitochondria markers GS28 and TOM20, the cytosolic marker GAPDH and for Aβ (6E10) of EVs in the 10 000g pellet from 10 × 106 microglia and relative donor cells (2 µg cell lysate). Normalization to total proteins is shown in Supplementary Fig. 1. (B) Analysis of the purity of EV preparations from Aβ42-treated microglia and control cells using the CONAN assay; AI% is the relative aggregation index of AuNPs, normREF is a sample of monodispersed AuNPs, negREF (HPLC grade water+AuNPs+PBS solution) is the negative control threshold and posREF is the positive control (PBS solution+AuNPs). (C) Size distribution of large Aβ-EVs enriched fraction analysed by TRPS. The fraction of Aβ-EVs of diameter >200 nm represents ∼59% of the 10 000g pellet. (D) Charge measurements of large ctrl-EVs and Aβ-EVs (10 000g pellet) by TRPS. Ctrl-EVs display an averaged surface charge of −10.75 mV, while Aβ-EVs −22.57 mV (t-test, P < 0.001). (E) Aβ42 content in intact Aβ-EVs or Aβ-EVs lysed with 0.57% Triton X-100 (10 000g pellet), as detected by ELISA. Values are normalized to intact Aβ-EVs (t-test, P = 0.002, n = 3). (F) Aβ42 content in large Aβ-EVs enriched fraction (10 000g pellet) produced by microglia exposed for 20 h to Aβ42 with or without Bafilomycin A1 (Baf) during the last 15 h of treatment, as detected by ELISA in the presence of 0.57% Triton X-100. Values are normalized to the condition without Baf (Mann–Whitney Rank Sum Test, P < 0.001, n = 7). (G) Aβ-EVs in the 10 000g pellet from microglia stimulated as in F. Values (EV numbers) are normalized to the condition without Baf. Mann–Whitney Rank Sum Test, P = 0.908, n = 3. (H) Representative cryo-electron microscopy micrograph of ctrl- and Aβ-EVs in the 10 000g pellet. Arrows point to multilamellar EVs; black arrowheads to EVs with rough surface; white arrowhead to tubular vesicles. Scale bars: 100 nm. Box plots show the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers).CC BY-NC · PubMed Central
Figure 2 Large Aβ-EVs alter dendritic spine morphology and synaptic plasticity in cultured neurons. (A) Schematic representation of EV delivery by optical tweezers to RFP-expressing dendrite, preceded and followed by time-lapse imaging of RFP-positive dendritic spines. A z-stack of RFP-positive dendrites was first acquired with a spinning disk microscope, then a low amount of EVs was added to the cell medium and one EV was captured (trapped) above the neurons by the infrared laser tweezers and placed in contact with the imaged dendrite (bright field). After 30 s the laser was switched off, EV adhesion was checked and confocal images were collected at the indicated time points. (B) Representative confocal images taken before and 30 min after contact of ctrl-EVs (centre) or Aβ-EVs (right) following the procedure described in A, showing dendritic spine changes in proximity (top) and far from the EV contact site (bottom). Red and orange circles indicate the site of EV contact. White arrows point to newly generated protrusions. Red arrows point to enlarged spines. Orange arrows point to thinned spines. On the left, dendritic spine images at 0 and 30 min after vehicle addition. Scale bar: 10 µm. (C and D) Temporal analysis of dendritic spine density around the contact site (<7 µm, C) and far from the contact site (>60 µm, D; n = 6 dendrites/condition, 12 experiments). Values are normalized to the pre-adhesion condition (two-way repeated measures ANOVA, followed by the Holm–Sidak method; close to the contact site: P = 0.013 ctrl-EVs versus vehicle; P < 0.001 Aβ-EVs 30 and 40 min versus 0; far from the contact site: P = 0.937). (E–H) Temporal analysis of the density of immature (thin) and mature (mushroom and stubby) dendritic spines around the contact site (E and G) and far from the contact site (F and H) after adhesion of Aβ-EVs or ctrl-EVs or in vehicle-treated neurons (immature spines at the contact site: P < 0.01 Aβ-EVs versus ctrl-EVs and versus vehicle; P < 0.001 Aβ-EVs 20, 30, 40 min versus 0; immature spines far from the contact site: P = 0.656; mature spines at the contact site: P < 0.001 Aβ-EVs versus ctrl-EVs; P = 0.015 Aβ-EVs versus vehicle; P < 0.01 ctrl-EVs versus vehicle; P < 0.001 ctrl-EVs 20, 30, 40 min versus 0; mature spines far from the contact site, ns). (I) Representative images showing Shank-2/Bassoon double-positive puncta in vehicle-treated neurons, neurons exposed to ctrl-EVs or Aβ-EVs. Scale bar: 1 µm. (J) The box plot shows the corresponding fraction of juxtaposed pre- and post-synaptic puncta relative to Bassoon positive synaptic puncta (Kruskal–Wallis one-way ANOVA on Ranks, followed by Dunn’s method, P < 0.05 Aβ-EVs versus vehicle; n = 3 experiments). Box plot shows the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers). (K) Representative traces of mEPSCs recorded from control neurons (vehicle) and neurons exposed to Aβ-EVs or ctrl-EVs for 1 h, before and after induction of synaptic plasticity. Vertical scale bar: 5 pA; horizontal scale bar: 1 s. (L) Temporal plot of mEPSC frequency changes showing that glycine (Gly, 200 µM 3 min in 0 Mg++, preceded by 1 min 0 Mg++) induced a long-lasting increase in mEPSC frequency in both vehicle- and ctrl-EV-treated neurons but not in neurons exposed to Aβ-EVs for 1 h (two-way repeated measures ANOVA, followed by the Holm–Sidak method; 2.931 ± 0.808 ‘vehicle’ fold change from baseline, P = 0.002; 2.409 ± 0.549 ‘ctrl-EVs’ fold change from baseline, P = 0.027; 0.942 ± 0.156 ‘Aβ-EVs’ fold change from baseline, P = 0.902; vehicle versus Aβ-EVs, P = 0.012; ctrl-EVs versus Aβ-EVs post Gly, P = 0.009; vehicle, n = 6 cells; ctrl-EVs, n = 5; Aβ-EVs, n = 8; 7 experiments). Data are expressed as mean ± SEM.
Figure 2 Large Aβ-EVs alter dendritic spine morphology and synaptic plasticity in cultured neurons. (A) Schematic representation of EV delivery by optical tweezers to RFP-expressing dendrite, preceded and followed by time-lapse imaging of RFP-positive dendritic spines. A z-stack of RFP-positive dendrites was first acquired with a spinning disk microscope, then a low amount of EVs was added to the cell medium and one EV was captured (trapped) above the neurons by the infrared laser tweezers and placed in contact with the imaged dendrite (bright field). After 30 s the laser was switched off, EV adhesion was checked and confocal images were collected at the indicated time points. (B) Representative confocal images taken before and 30 min after contact of ctrl-EVs (centre) or Aβ-EVs (right) following the procedure described in A, showing dendritic spine changes in proximity (top) and far from the EV contact site (bottom). Red and orange circles indicate the site of EV contact. White arrows point to newly generated protrusions. Red arrows point to enlarged spines. Orange arrows point to thinned spines. On the left, dendritic spine images at 0 and 30 min after vehicle addition. Scale bar: 10 µm. (C and D) Temporal analysis of dendritic spine density around the contact site (<7 µm, C) and far from the contact site (>60 µm, D; n = 6 dendrites/condition, 12 experiments). Values are normalized to the pre-adhesion condition (two-way repeated measures ANOVA, followed by the Holm–Sidak method; close to the contact site: P = 0.013 ctrl-EVs versus vehicle; P < 0.001 Aβ-EVs 30 and 40 min versus 0; far from the contact site: P = 0.937). (E–H) Temporal analysis of the density of immature (thin) and mature (mushroom and stubby) dendritic spines around the contact site (E and G) and far from the contact site (F and H) after adhesion of Aβ-EVs or ctrl-EVs or in vehicle-treated neurons (immature spines at the contact site: P < 0.01 Aβ-EVs versus ctrl-EVs and versus vehicle; P < 0.001 Aβ-EVs 20, 30, 40 min versus 0; immature spines far from the contact site: P = 0.656; mature spines at the contact site: P < 0.001 Aβ-EVs versus ctrl-EVs; P = 0.015 Aβ-EVs versus vehicle; P < 0.01 ctrl-EVs versus vehicle; P < 0.001 ctrl-EVs 20, 30, 40 min versus 0; mature spines far from the contact site, ns). (I) Representative images showing Shank-2/Bassoon double-positive puncta in vehicle-treated neurons, neurons exposed to ctrl-EVs or Aβ-EVs. Scale bar: 1 µm. (J) The box plot shows the corresponding fraction of juxtaposed pre- and post-synaptic puncta relative to Bassoon positive synaptic puncta (Kruskal–Wallis one-way ANOVA on Ranks, followed by Dunn’s method, P < 0.05 Aβ-EVs versus vehicle; n = 3 experiments). Box plot shows the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers). (K) Representative traces of mEPSCs recorded from control neurons (vehicle) and neurons exposed to Aβ-EVs or ctrl-EVs for 1 h, before and after induction of synaptic plasticity. Vertical scale bar: 5 pA; horizontal scale bar: 1 s. (L) Temporal plot of mEPSC frequency changes showing that glycine (Gly, 200 µM 3 min in 0 Mg++, preceded by 1 min 0 Mg++) induced a long-lasting increase in mEPSC frequency in both vehicle- and ctrl-EV-treated neurons but not in neurons exposed to Aβ-EVs for 1 h (two-way repeated measures ANOVA, followed by the Holm–Sidak method; 2.931 ± 0.808 ‘vehicle’ fold change from baseline, P = 0.002; 2.409 ± 0.549 ‘ctrl-EVs’ fold change from baseline, P = 0.027; 0.942 ± 0.156 ‘Aβ-EVs’ fold change from baseline, P = 0.902; vehicle versus Aβ-EVs, P = 0.012; ctrl-EVs versus Aβ-EVs post Gly, P = 0.009; vehicle, n = 6 cells; ctrl-EVs, n = 5; Aβ-EVs, n = 8; 7 experiments). Data are expressed as mean ± SEM.CC BY-NC · PubMed Central
Figure 3 Large Aβ-EV motion at the axon surface. (A) Schematic representation of large EV delivery to axons using optical tweezers. Axons were selected based on their morphology after RFP transfection. A single EV was trapped by the laser tweezers in bright field and placed in contact with the axon. The trapping laser was switched off 30 s after contact and EV–axon interaction was monitored in bright field time-lapse for 40 min. (B) Sequence of phase contrast images of a large EV moving anterogradely along the axon towards the growth cone. (B′) Corresponding fluorescence image of the axon in B. The top right blurred area indicates the region of the growth cone outside phase contrast images. (C) Trajectory of the EV in B superimposed to the phase contrast image. (D) Trajectory of a static EV superimposed to the phase contrast image. (E) Percentage of large ctrl-EVs and Aβ-EVs that adhered to axons (chi-square test, P = 0.768, n = 68 ctrl-EVs, n = 105 Aβ-EVs, 33 experiments). (F) Percentage of large ctrl-EVs and Aβ-EVs that displayed motility on axons (chi-square, P = 0.002, n = 29 ctrl-EVs, n = 34 Aβ-EVs, 31 experiments). (G) Average speed of large Aβ-EVs and ctrl-EVs (Mann–Whitney test, P = 0.011, n = 8 ctrl-EVs, n = 18 Aβ-EVs, 19 experiments). (H and I) Pathlength (H) and maximum distance from the contact point in both directions (I) reached by large ctrl-EVs and Aβ-EVs in 10 min (Mann–Whitney, P = 0.033, for both; n = 8 ctrl-EVs, n = 13 Aβ-EVs, 19 experiments). (J) Anterograde and retrograde motion of large Aβ-EVs and ctrl-EVs (n = 15). (K) Percentage of large Aβ-EVs and Aβ-EVs pre-coated with annexin-V (coated Aβ-EVs, c-Aβ-EVs) that adhered to axons (chi-square, P = 0.014, n = 105 Aβ-EVs, n = 37 c-Aβ-EVs, 24 experiments). (L) Percentage of large Aβ-EVs and c-Aβ-EVs that displayed motility (chi-square, P = 0.002; n = 34 Aβ-EVs, n = 25 c-Aβ-EVs, 23 experiments). (M) Average speed of large Aβ-EVs and c-Aβ-EVs (t-test, P = 0.142, n = 18 Aβ-EVs, n = 11 c-Aβ-EVs, 16 experiments). Scale bars: 10 µm. Percentage values are raw percentages over total EV tested. Box plots show the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers).
Figure 3 Large Aβ-EV motion at the axon surface. (A) Schematic representation of large EV delivery to axons using optical tweezers. Axons were selected based on their morphology after RFP transfection. A single EV was trapped by the laser tweezers in bright field and placed in contact with the axon. The trapping laser was switched off 30 s after contact and EV–axon interaction was monitored in bright field time-lapse for 40 min. (B) Sequence of phase contrast images of a large EV moving anterogradely along the axon towards the growth cone. (B′) Corresponding fluorescence image of the axon in B. The top right blurred area indicates the region of the growth cone outside phase contrast images. (C) Trajectory of the EV in B superimposed to the phase contrast image. (D) Trajectory of a static EV superimposed to the phase contrast image. (E) Percentage of large ctrl-EVs and Aβ-EVs that adhered to axons (chi-square test, P = 0.768, n = 68 ctrl-EVs, n = 105 Aβ-EVs, 33 experiments). (F) Percentage of large ctrl-EVs and Aβ-EVs that displayed motility on axons (chi-square, P = 0.002, n = 29 ctrl-EVs, n = 34 Aβ-EVs, 31 experiments). (G) Average speed of large Aβ-EVs and ctrl-EVs (Mann–Whitney test, P = 0.011, n = 8 ctrl-EVs, n = 18 Aβ-EVs, 19 experiments). (H and I) Pathlength (H) and maximum distance from the contact point in both directions (I) reached by large ctrl-EVs and Aβ-EVs in 10 min (Mann–Whitney, P = 0.033, for both; n = 8 ctrl-EVs, n = 13 Aβ-EVs, 19 experiments). (J) Anterograde and retrograde motion of large Aβ-EVs and ctrl-EVs (n = 15). (K) Percentage of large Aβ-EVs and Aβ-EVs pre-coated with annexin-V (coated Aβ-EVs, c-Aβ-EVs) that adhered to axons (chi-square, P = 0.014, n = 105 Aβ-EVs, n = 37 c-Aβ-EVs, 24 experiments). (L) Percentage of large Aβ-EVs and c-Aβ-EVs that displayed motility (chi-square, P = 0.002; n = 34 Aβ-EVs, n = 25 c-Aβ-EVs, 23 experiments). (M) Average speed of large Aβ-EVs and c-Aβ-EVs (t-test, P = 0.142, n = 18 Aβ-EVs, n = 11 c-Aβ-EVs, 16 experiments). Scale bars: 10 µm. Percentage values are raw percentages over total EV tested. Box plots show the median (central line) and mean (X), upper and lower quartile (box limits), maximum and minimum values (whiskers).CC BY-NC · PubMed Central
Figure 4 Large Aβ-EVs propagate LTP impairment in the EC–DG circuit. (A) Input–output curves showing the relative amplitude (% maximal Ampl.) as a function of stimulus intensity [Stim. Int., measured in volts (V)] in vehicle-treated slices and slices exposed to 1 × 108/ml Aβ-EVs, ctrl-EVs or vehicle for 1 h. (B) LTP field potential recordings in slices incubated with Aβ-EVs (same amount as in A, yellow triangles), ctrl-EVs (dark blue circles) or vehicle alone with no EVs (light blue diamonds). LTP was reliably elicited in slices incubated with ctrl-EVs [two-way repeated measures ANOVA, followed by the Holm–Sidak method, ctrl-EVs 131 ± 4% of baseline amplitude 40 min after HFS (n = 7 slices, 4 mice); vehicle 134 ± 7% (n = 6 slices, 4 mice); P = 0.473 ctrl-EVs versus vehicle], while was not elicited in Aβ-EVs treated EC slices (93 ± 3% of baseline after HFS, P = 0.154 versus baseline; P < 0.001 versus vehicle and ctrl-EVs; n = 6 slices; 4 mice). (C) Experimental protocol for LTP measurements in EC–DG slices after large EVs or Aβ42 injection. Large Aβ-EVs, ctrl-EVs or i-EVs (0.25 × 108 EVs/μl, 1 μl), Aβ42 (1 μl, 100 nM) or vehicle were injected into the mouse lateral EC. Mice were sacrificed 1 h and 24 h after the injection and horizontal slices containing both the EC and the hippocampus have been cut. LTP was recorded from the EC superficial layer II (stimulus and recording pipette in this layer) and at the synapse between the PP (stimulus) and the DG (recording). (D and E) LTP plots from the EC superficial layer II (D) and the PP–DG synapse (E) in cortico-hippocampal slices after injection of large Aβ-EVs in the lateral EC or large ctrl-EVs in the contralateral EC. Field recordings in EC superficial layer II revealed suppression of LTP 1 h after the injection of Aβ-EVs in the ipsilateral EC (D, yellow triangles), while a stable LTP was recorded in the contralateral EC injected with ctrl-EVs (D, dark blue circles; ctrl-EVs 129 ± 6% versus Aβ-EVs 99 ± 5%, P < 0.001, n = 8 slices, 4 mice each). Twenty-four hours after the injection of Aβ-EVs, LTP was still impaired in EC (D, orange squares; Aβ-EVs 101 ± 2%, P < 0.001 versus ctrl EVs, n = 7 slices, 4 mice). In field recordings from the DG after PP stimulation (E), LTP was normal 1 h after the injection of Aβ-EVs in the EC (E, yellow triangles) and comparable to that obtained in the contralateral hippocampus injected in the EC with ctrl-EVs (E, dark blue circles; Aβ-EVs 159 ± 5% versus ctrl-EVs 147 ± 12%, P = 0.195, n = 8 slices, 4 mice each). In contrast, LTP was blocked 24 h after Aβ-EV injection in the ipsilateral EC (E, orange squares; Aβ-EVs 105 ± 8% P < 0.001 versus ctrl EVs and Aβ-EVs at 1 h, n = 7 slices, 4 mice). (F and G) Effect of the stereotaxic injection of oligomeric Aβ42 in EC on LTP expression in cortico-hippocampal slices. Field recordings in EC revealed that LTP expression is affected 1 h after the injection of Aβ42 in the EC (F, light blue diamonds; 93 ± 7%, P = 0.187 versus baseline, n = 7 slices, 4 mice) but it recovers 24 h after Aβ42 injection (F, dark blue diamonds). Slice recordings from the DG after PP stimulation revealed that hippocampal LTP is normally expressed 1 h after the injection of Aβ42 in the EC (G, light blue diamonds; 176 ± 4%, n = 7 slices, 4 mice) and comparable to that obtained in hippocampal slices 24 h after the injection of Aβ42 in the EC (G, dark blue diamonds; 176 ± 2%, P = 0.039 versus 1 h, n = 7 slices, 4 mice). (H and I) Effect of the stereotaxic injection of i-EVs in the EC on LTP expression in EC–hippocampal slices. Field recordings in EC revealed that LTP expression is affected already 1 h after i-EV injection (H, dark grey diamonds; i-EVs 87 ± 4% of baseline amplitude after HFS versus ctrl-EVs 130 ± 6%, P < 0.001, n = 8 slices, 4 mice each) and remained impaired 24 h after (H, light grey diamonds; 96 ± 6% of baseline amplitude, n = 6 slices, 4 mice; P < 0.001 versus ctrl-EVs and P = 0.120 versus i-EVs at 1 h). Hippocampal LTP was normally expressed 24 h after the injection of i-EVs in the EC (I, light grey diamonds) and comparable to that obtained in hippocampal slices after the injection of ctrl-EVs in the EC (I, dark blue circles; mean LTP was 157 ± 11% of baseline fEPSP slope, P = 0.469 versus ctrl-EVs 159 ± 9%, n = 7 slices, 4 mice each). Inserts show representative traces of field potential. Vertical scale bar: 0.5 mV; Horizontal scale bar: 5 ms. Values are mean ± SEM.
Figure 4 Large Aβ-EVs propagate LTP impairment in the EC–DG circuit. (A) Input–output curves showing the relative amplitude (% maximal Ampl.) as a function of stimulus intensity [Stim. Int., measured in volts (V)] in vehicle-treated slices and slices exposed to 1 × 108/ml Aβ-EVs, ctrl-EVs or vehicle for 1 h. (B) LTP field potential recordings in slices incubated with Aβ-EVs (same amount as in A, yellow triangles), ctrl-EVs (dark blue circles) or vehicle alone with no EVs (light blue diamonds). LTP was reliably elicited in slices incubated with ctrl-EVs [two-way repeated measures ANOVA, followed by the Holm–Sidak method, ctrl-EVs 131 ± 4% of baseline amplitude 40 min after HFS (n = 7 slices, 4 mice); vehicle 134 ± 7% (n = 6 slices, 4 mice); P = 0.473 ctrl-EVs versus vehicle], while was not elicited in Aβ-EVs treated EC slices (93 ± 3% of baseline after HFS, P = 0.154 versus baseline; P < 0.001 versus vehicle and ctrl-EVs; n = 6 slices; 4 mice). (C) Experimental protocol for LTP measurements in EC–DG slices after large EVs or Aβ42 injection. Large Aβ-EVs, ctrl-EVs or i-EVs (0.25 × 108 EVs/μl, 1 μl), Aβ42 (1 μl, 100 nM) or vehicle were injected into the mouse lateral EC. Mice were sacrificed 1 h and 24 h after the injection and horizontal slices containing both the EC and the hippocampus have been cut. LTP was recorded from the EC superficial layer II (stimulus and recording pipette in this layer) and at the synapse between the PP (stimulus) and the DG (recording). (D and E) LTP plots from the EC superficial layer II (D) and the PP–DG synapse (E) in cortico-hippocampal slices after injection of large Aβ-EVs in the lateral EC or large ctrl-EVs in the contralateral EC. Field recordings in EC superficial layer II revealed suppression of LTP 1 h after the injection of Aβ-EVs in the ipsilateral EC (D, yellow triangles), while a stable LTP was recorded in the contralateral EC injected with ctrl-EVs (D, dark blue circles; ctrl-EVs 129 ± 6% versus Aβ-EVs 99 ± 5%, P < 0.001, n = 8 slices, 4 mice each). Twenty-four hours after the injection of Aβ-EVs, LTP was still impaired in EC (D, orange squares; Aβ-EVs 101 ± 2%, P < 0.001 versus ctrl EVs, n = 7 slices, 4 mice). In field recordings from the DG after PP stimulation (E), LTP was normal 1 h after the injection of Aβ-EVs in the EC (E, yellow triangles) and comparable to that obtained in the contralateral hippocampus injected in the EC with ctrl-EVs (E, dark blue circles; Aβ-EVs 159 ± 5% versus ctrl-EVs 147 ± 12%, P = 0.195, n = 8 slices, 4 mice each). In contrast, LTP was blocked 24 h after Aβ-EV injection in the ipsilateral EC (E, orange squares; Aβ-EVs 105 ± 8% P < 0.001 versus ctrl EVs and Aβ-EVs at 1 h, n = 7 slices, 4 mice). (F and G) Effect of the stereotaxic injection of oligomeric Aβ42 in EC on LTP expression in cortico-hippocampal slices. Field recordings in EC revealed that LTP expression is affected 1 h after the injection of Aβ42 in the EC (F, light blue diamonds; 93 ± 7%, P = 0.187 versus baseline, n = 7 slices, 4 mice) but it recovers 24 h after Aβ42 injection (F, dark blue diamonds). Slice recordings from the DG after PP stimulation revealed that hippocampal LTP is normally expressed 1 h after the injection of Aβ42 in the EC (G, light blue diamonds; 176 ± 4%, n = 7 slices, 4 mice) and comparable to that obtained in hippocampal slices 24 h after the injection of Aβ42 in the EC (G, dark blue diamonds; 176 ± 2%, P = 0.039 versus 1 h, n = 7 slices, 4 mice). (H and I) Effect of the stereotaxic injection of i-EVs in the EC on LTP expression in EC–hippocampal slices. Field recordings in EC revealed that LTP expression is affected already 1 h after i-EV injection (H, dark grey diamonds; i-EVs 87 ± 4% of baseline amplitude after HFS versus ctrl-EVs 130 ± 6%, P < 0.001, n = 8 slices, 4 mice each) and remained impaired 24 h after (H, light grey diamonds; 96 ± 6% of baseline amplitude, n = 6 slices, 4 mice; P < 0.001 versus ctrl-EVs and P = 0.120 versus i-EVs at 1 h). Hippocampal LTP was normally expressed 24 h after the injection of i-EVs in the EC (I, light grey diamonds) and comparable to that obtained in hippocampal slices after the injection of ctrl-EVs in the EC (I, dark blue circles; mean LTP was 157 ± 11% of baseline fEPSP slope, P = 0.469 versus ctrl-EVs 159 ± 9%, n = 7 slices, 4 mice each). Inserts show representative traces of field potential. Vertical scale bar: 0.5 mV; Horizontal scale bar: 5 ms. Values are mean ± SEM.CC BY-NC · PubMed Central
Figure 5 Aβ-EVs decrease mEPSC amplitude without affecting their frequency. (A) Experimental protocol for whole-cell patch-clamp recordings in EC–DG slices after Aβ-EV (0.25 × 108 EVs/μl, 1 μl) or vehicle injection. Recording electrodes are shown. (B and C) Representative traces of mEPSCs recorded from pyramidal cells of EC superficial layer II 1 h after injection of Aβ-EVs or vehicle in the EC (B) and corresponding plots of mEPSC frequency and amplitude (C; mEPSC frequency, t-test, P = 0.900; mEPSC amplitude, Mann–Whitney rank sum test, P ≤ 0.001; vehicle, n = 13 cells; Aβ-EVs, n = 13 cells; 7 mice each). (D and E) Representative traces of mEPSCs recorded from granular cells of the DG 24 h after injection of Aβ-EVs or vehicle in the EC (D). The plots show corresponding mEPSC frequency and amplitude (E; mEPSC frequency, t-test, P = 0.655; mEPSC amplitude, t-test, P ≤ 0.001; vehicle, n = 12 cells; Aβ-EVs, n = 13 cells; 6 mice each). Vertical scale bar: 5 pA; Horizontal scale bar: 1 s. Box plots show the median (central line) and mean (X) values, upper and lower quartile (box limits), maximum and minimum values (whiskers).
Figure 5 Aβ-EVs decrease mEPSC amplitude without affecting their frequency. (A) Experimental protocol for whole-cell patch-clamp recordings in EC–DG slices after Aβ-EV (0.25 × 108 EVs/μl, 1 μl) or vehicle injection. Recording electrodes are shown. (B and C) Representative traces of mEPSCs recorded from pyramidal cells of EC superficial layer II 1 h after injection of Aβ-EVs or vehicle in the EC (B) and corresponding plots of mEPSC frequency and amplitude (C; mEPSC frequency, t-test, P = 0.900; mEPSC amplitude, Mann–Whitney rank sum test, P ≤ 0.001; vehicle, n = 13 cells; Aβ-EVs, n = 13 cells; 7 mice each). (D and E) Representative traces of mEPSCs recorded from granular cells of the DG 24 h after injection of Aβ-EVs or vehicle in the EC (D). The plots show corresponding mEPSC frequency and amplitude (E; mEPSC frequency, t-test, P = 0.655; mEPSC amplitude, t-test, P ≤ 0.001; vehicle, n = 12 cells; Aβ-EVs, n = 13 cells; 6 mice each). Vertical scale bar: 5 pA; Horizontal scale bar: 1 s. Box plots show the median (central line) and mean (X) values, upper and lower quartile (box limits), maximum and minimum values (whiskers).CC BY-NC · PubMed Central
Figure 6 CHO-EVs propagate LTP impairment in the EC–DG circuit. (A) Living mouse microglia were exposed to CHO7PA2 cell supernatant containing nanomolar concentrations of Aβ for 20 h and stained with IB4-Alexa568 to label the cell surface before being fixed and counterstained with anti-Aβ antibody 6E10. Scale bar: 10 µm. (B and C) Effect on LTP expression of the stereotaxic injection in the EC of EVs released by microglia exposed for 20 h to CHO7PA2 cell supernatant (CHO-EVs, 0.25 × 108 EVs/μl, 1 μl), compared to the same amount of ctrl-EVs. LTP plots are relative to recordings from EC and PP–DG, 1 h and 24 h after the injection, respectively (two-way repeated measures ANOVA, followed by the Holm–Sidak method, 1 h EC: 85.48 ± 13.09%, P = 0.234 versus baseline; P = 0.023 versus ctrl-EVs; n = 4 slices CHO-EVs; n = 6 slices ctrl-EVs; 24 h PP–DG: 97.29 ± 10.86%, P = 0.802 versus baseline; P < 0.01 versus ctrl-EVs; n = 4 slices CHO-EVs; n = 6 slices ctrl-EVs). Vertical scale bar: 0.5 mV; horizontal scale bar: 5 ms. Values are mean ± SEM.
Figure 6 CHO-EVs propagate LTP impairment in the EC–DG circuit. (A) Living mouse microglia were exposed to CHO7PA2 cell supernatant containing nanomolar concentrations of Aβ for 20 h and stained with IB4-Alexa568 to label the cell surface before being fixed and counterstained with anti-Aβ antibody 6E10. Scale bar: 10 µm. (B and C) Effect on LTP expression of the stereotaxic injection in the EC of EVs released by microglia exposed for 20 h to CHO7PA2 cell supernatant (CHO-EVs, 0.25 × 108 EVs/μl, 1 μl), compared to the same amount of ctrl-EVs. LTP plots are relative to recordings from EC and PP–DG, 1 h and 24 h after the injection, respectively (two-way repeated measures ANOVA, followed by the Holm–Sidak method, 1 h EC: 85.48 ± 13.09%, P = 0.234 versus baseline; P = 0.023 versus ctrl-EVs; n = 4 slices CHO-EVs; n = 6 slices ctrl-EVs; 24 h PP–DG: 97.29 ± 10.86%, P = 0.802 versus baseline; P < 0.01 versus ctrl-EVs; n = 4 slices CHO-EVs; n = 6 slices ctrl-EVs). Vertical scale bar: 0.5 mV; horizontal scale bar: 5 ms. Values are mean ± SEM.CC BY-NC · PubMed Central
Figure 7 Aβ-EVs coated with annexin-V do not propagate LTP impairment in the EC–DG circuit. (A and B) Effect of the stereotaxic injection of Aβ-EVs (0.11 × 108 EVs/μl, 1 μl) or coated Aβ-EVs (c-Aβ-EVs; 0.11 × 108 EVs/μl, 1 μl) in the EC on LTP expression in EC and PP–DG, 1 h and 24 h after the injection, respectively. c-Aβ-EVs impaired LTP in EC 1 h after the injection (A) (two-way repeated measures ANOVA, followed by the Holm–Sidak method, 97.95 ± 11.19%, P = 0.820 versus baseline; P = 0.152 versus Aβ-EVs; n = 8 slices ctrl-EVs; n = 6 slices Aβ-EVs; n = 7 slices c-Aβ-EVs; four mice), while allowing its expression in the DG 24 h later (B) (137.80 ± 5.64%, P = 0.008 versus baseline; P < 0.001 versus Aβ-EVs; n = 8 slices ctrl-EVs; n = 8 slices Aβ-EVs; n = 6 slices c-Aβ-EVs; 5 mice). Inserts show a representative trace of field potential. Vertical scale bar: 0.5 mV; horizontal scale bar: 5 ms. Values are mean ± SEM.
Figure 7 Aβ-EVs coated with annexin-V do not propagate LTP impairment in the EC–DG circuit. (A and B) Effect of the stereotaxic injection of Aβ-EVs (0.11 × 108 EVs/μl, 1 μl) or coated Aβ-EVs (c-Aβ-EVs; 0.11 × 108 EVs/μl, 1 μl) in the EC on LTP expression in EC and PP–DG, 1 h and 24 h after the injection, respectively. c-Aβ-EVs impaired LTP in EC 1 h after the injection (A) (two-way repeated measures ANOVA, followed by the Holm–Sidak method, 97.95 ± 11.19%, P = 0.820 versus baseline; P = 0.152 versus Aβ-EVs; n = 8 slices ctrl-EVs; n = 6 slices Aβ-EVs; n = 7 slices c-Aβ-EVs; four mice), while allowing its expression in the DG 24 h later (B) (137.80 ± 5.64%, P = 0.008 versus baseline; P < 0.001 versus Aβ-EVs; n = 8 slices ctrl-EVs; n = 8 slices Aβ-EVs; n = 6 slices c-Aβ-EVs; 5 mice). Inserts show a representative trace of field potential. Vertical scale bar: 0.5 mV; horizontal scale bar: 5 ms. Values are mean ± SEM.CC BY-NC · PubMed Central
Figure 8 Model for synaptic dysfunction propagation mediated by large Aβ-EVs in Alzheimer’s disease. We propose the following model to explain Aβ-EV implication in the onset and propagation of synaptic dysfunction. In the early stages of Alzheimer’s disease, Aβ starts to accumulate in specific areas of the brain, where it is internalized by microglia (1) and re-secreted in toxic form in association with EVs (2): the higher the Aβ cell load, the higher the Aβ content (as indicated by Bafilomycin experiments showed in this paper). Aβ-EVs induce synaptic alterations at the site of adhesion (3) and, by moving along axonal projection (4), can reach connected neurons (5). While small EVs are internalized by neurons and travel inside neuronal axons to trans-synaptically transfer their cargo (so far reported for small EVs released by primary neurons or isolated from Alzheimer patients’ brains),80–82 large EVs, likely too big to be transported intracellularly, move at the axonal surface towards synaptically connected cells.
Figure 8 Model for synaptic dysfunction propagation mediated by large Aβ-EVs in Alzheimer’s disease. We propose the following model to explain Aβ-EV implication in the onset and propagation of synaptic dysfunction. In the early stages of Alzheimer’s disease, Aβ starts to accumulate in specific areas of the brain, where it is internalized by microglia (1) and re-secreted in toxic form in association with EVs (2): the higher the Aβ cell load, the higher the Aβ content (as indicated by Bafilomycin experiments showed in this paper). Aβ-EVs induce synaptic alterations at the site of adhesion (3) and, by moving along axonal projection (4), can reach connected neurons (5). While small EVs are internalized by neurons and travel inside neuronal axons to trans-synaptically transfer their cargo (so far reported for small EVs released by primary neurons or isolated from Alzheimer patients’ brains),80–82 large EVs, likely too big to be transported intracellularly, move at the axonal surface towards synaptically connected cells.CC BY-NC · PubMed Central

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Metadata

Authors
Gabrielli, M., Prada, I., Joshi, P., Falcicchia, C., D’Arrigo, G., Rutigliano, G., Battocchio, E., Zenatelli, R., Tozzi, F., Radeghieri, A., Arancio, O., Origlia, N., & Verderio, C.
Affiliations
  • CNR Institute of Neuroscience , Vedano al Lambro, MB 20854 , Italy
  • Neuroscience Institute (IT)
  • Scuola Superiore Sant'Anna (IT)
  • University of Milano-Bicocca (IT)
  • University of Brescia (IT)
  • Scuola Normale Superiore (IT)
  • Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (IT)
  • Columbia University (US)
Corresponding author
  • Origlia, Nicola
  • Verderio, Claudia
ORCID
Venue
Brain
Publisher
Oxford University Press (OUP)
Published
2022-03-03
Volume
145
Issue
8
Pages
2849-2868
ISSN
  • 0006-8950 (ISSN-L)
  • 1460-2156
DOI
10.1093/brain/awac083
PMC
PMC9420022
OpenAlex
W4214920994
Cited by
106
References
105
Citations by year
2026: 22 · 2025: 29 · 2024: 27 · 2023: 22 · 2022: 6
FWCI
12.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Brain (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • BOA (University of Milano-Bicocca) (submittedVersion, cc-by-nc)
  • Institutional Research Information System (Università degli Studi di Brescia) (submittedVersion, cc-by-nc)
  • Scuola Normale Superiore di Pisa (submittedVersion, cc-by-nc)
Funders
  • American National Institutes of Health (1R56AG056108-01)
  • Alzheimer’s Association Research Fellowship (2018-AARF-588984)
  • Italian Multiple Sclerosis Foundation
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
Alzheimer's disease, microglia, large extracellular vesicles, amyloid-beta, synaptic dysfunction, long-term potentiation, dendritic spine morphology, synaptic plasticity, vesicle motility, annexin V, amyloid-beta 42, disease propagation
MeSH terms
Extracellular Vesicles, Alzheimer Disease, Animals, Hippocampus, Amyloid beta-Peptides, Microglia, Long-Term Potentiation, Mice

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