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 figures and 1 Table. 5~8 nm 100 nm AB 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 4  M 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). A  tub colocalizing area  3 tub A   MVs sup pellet D E C 10  m * *

Figure S3. Western Blot of A  species in MVs produced from A  -preloaded microglia. Relates to figure 4 Western 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 4X10 6 microglia pre-exposed to biotinylated A  1-42 (4  M).The blot was carried out using a 15% Tris-glycine gel and the membrane was probed with streptavidine. Shed MVs and exosomes produced by 8X10 6 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 P (  g) (  g) 4 KD 20 KD 25 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 MVs No changes were detected in Thioflavin-T spectra upon addition of MVs just before mixing Thioflavin-T with samples of aggregated A  Thioflavin-T AA A  -MVs Aβ-MVs(AA)

Aβ 1-42 Aβ 1-40 Aβ 1-38 Aβ 1-39 Aβ 1-37 Aβ 1-18 Figure S4. Mass spectrometry spectra of MVs from an AD patient. Relates to figure 5 Representative 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.) HC10/ ± 6.3 MCI27/ ± 6.5 AD50/ ± 7.1 Table S1. Clinical features of MCI and AD patients