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Supplemental Information MICROGLIA CONVERT AGGREGATED AMYLOID- INTO NEUROTOXIC FORMS THROUGH THE SHEDDING OF MICROVESICLES Pooja Joshi, Elena Turola, Ana Ruiz, Alessandra Bergami, Dacia Dalla Libera, Luisa Benussi, Paola Giussani, Giuseppe Magnani, Giancarlo Comi, Giuseppe Legname, Roberta Ghidoni, Roberto Furlan, Michela Matteoli and Claudia Verderio Inventory of Supplemental Information Supplemental Information includes 4 gures and 1 Table. fi 0,0 0,2 0,4 0,6 0,8 1,0 1,2 5~8 nm 100 nm A B 100 nm Figure S1. TEM analysis of A species present in samples of aggregated A 1-42 after overnight incubation with MVs and evaluation of their binding capacity 4M A 1-42 was incubated overnight with MVs and then centrifuged at 10000g for 30 min. A-B Representative TEM images of 5-8 nm wide A 1-42 fibrils retrieved in the pellet fraction (A) and of globular A 1-42 species, present in the supernatant (B). C-E Representative confocal images of neurons exposed to the supernatant (sup) (D) or to the pellet (E) fractions obtained after centrifugation of 488- Ab 1-42 / MVs mixture. Corresponding binding A tub colocalizing area 3 tub A MVs sup pellet D E C 10 m * *
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Supplemental Information MICROGLIA CONVERT AGGREGATED AMYLOID- b INTO NEUROTOXIC FORMS THROUGH THE SHEDDING OF MICROVESICLES - PowerPoint PPT Presentation
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MICROGLIA CONVERT AGGREGATED AMYLOID- INTO NEUROTOXIC FORMS THROUGH THE SHEDDING OF MICROVESICLESPooja Joshi, Elena Turola, Ana Ruiz, Alessandra Bergami, Dacia Dalla Libera, Luisa Benussi, Paola Giussani, Giuseppe Magnani, Giancarlo Comi, Giuseppe Legname, Roberta Ghidoni, Roberto Furlan, Michela Matteoli and Claudia Verderio
Inventory of Supplemental Information Supplemental Information includes 4 figures and 1 Table.
0,0
0,2
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5~8 nm
100 nm
A B
100 nm
Figure S1. TEM analysis of A species present in samples of aggregated A 1-42 after overnight incubation with MVs and evaluation of their binding capacity 4M A 1-42 was incubated overnight with MVs and then centrifuged at 10000g for 30 min. A-B Representative TEM images of 5-8 nm wide A 1-42 fibrils retrieved in the pellet fraction (A) and of globular A 1-42 species, present in the supernatant (B). C-E Representative confocal images of neurons exposed to the supernatant (sup) (D) or to the pellet (E) fractions obtained after centrifugation of 488- Ab 1-42 / MVs mixture. Corresponding binding quantification is shown in C (ANOVA, p<0.001, Holm-Sidak Method p<0.05).
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Figure S3. Western Blot of A species in MVs produced from A-preloaded microglia. Relates to figure 4Western blot analysis of A 1-42 species present in shed MVs (P2 and P3 fractions) and exosomes (P4 fraction) constitutively produced during 24 h by 4X106 microglia pre-exposed to biotinylated A1-42 (4M).The blot was carried out using a 15% Tris-glycine gel and the membrane was probed with streptavidine. Shed MVs and exosomes produced by 8X106 donor microglia were probed in parallel for the EMV markers Tsg101 and the exosomal marker Alix (lower panel). Numbers below each lane indicate the estimated amount of loaded proteins.
MW MVs exos
P2 P3 P4
Alix
P2 P3 P4
34 30 75 (g)
68 60 150 (g)
4 KD
20 KD25 KD
90 KD
50 KD
Alix
Tsg101
Figure S2. Thioflavin T emission spectra of aggregated A 1-42 (solid blue line), incubated overnight with MVs (dashed blue line) or acutely exposed to MVsNo changes were detected in Thioflavin-T spectra upon addition of MVs just before mixing Thioflavin-T with samples of aggregated A 1-42.
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540510495480465 525
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Thioflavin-TAA-MVs
Aβ-MVs(AA)
Aβ 1-42
Aβ 1-40
Aβ 1-38
Aβ 1-39Aβ 1-37Aβ 1-18
Figure S4. Mass spectrometry spectra of MVs from an AD patient. Relates to figure 5Representative SELDI TOF MS spectra of MVs isolated from the CSF of a patient with AD showing the most common Ab peptides captured by immunoproteomic assay employing 6E10 and 4G8 monoclonal antibodies
Gender (F/M) Age (average ± S.D.)HC 10/10 68.0 ± 6.3MCI 27/26 68.8 ± 6.5AD 50/39 64.6 ± 7.1
Table S1. Clinical features of MCI and AD patients
