Aβ42 peptide

Aβ-42 peptide (Bachem, Bubendorf, Switzerland) was dissolved in 100% hexafluoroisopropanol (HFIP) to a final concentration of 1 mM. The solution was aliquoted, HFIP was evaporated and the resulting pellets were stored at −20 °C until needed. These aliquots were stable for at least three months. To generate amyloid aggregates, Aβ-42 peptide was dissolved to a 30 μM concentration in 20 mM sodium phosphate buffer (PBS), pH 7.4, at 25 °C. The samples were sonicated for 15 min and then centrifuged at 14,000× g for 15 min at 4 °C. The clear supernatant was collected, and the peptide concentration was checked by evaluating the absorbance of the resulting solution by means of a double-beam Lambda-20 spectrophotometer (Perkin Elmer Life Sciences, Norwalk, CT, USA) (ε280 = 1490 mol⁻¹ cm⁻¹) and adjusted to a final concentration of 25 μM. Enriched solutions of fibrillar Aβ-42 (Fib) were collected after 72 h of incubation of the supernatant at room temperature (RT).

Aβ₄₂ peptide (American Peptide Company), HiLyte^™ Fluor 488-Aβ₄₂, or HiLyte^™ Fluor 555-Aβ₄₂ (AnaSpec) were used for all Aβ oligomerizations and injections^{27 (link)}. Under a fume hood, 0.1 mg Aβ peptide was dissolved in ice cold 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP, Fluka) then vortex for a few seconds. The solution was dried with a gentle stream of nitrogen to obtain a peptide film at the bottom of the vial. Prior to use, the film was resuspended in anhydrous dimethyl sulfoxide (DMSO) to form a 5 mM solution, sonicated in a water bath for 10 min and diluted in sterile phosphate-buffered saline (PBS) to a final concentration of 100 μM. HiLyte^™ Fluor 488- or 555-Aβ or Aβ₄₂ peptide and E3 or E4 native particles were combined, vortexed, held under oligomer forming conditions (room temperature) for 24 h at a final concentration of 50 μM Aβ and 5 μM APOE and stored at −20 ^oC until use (abbreviated AβE3 or AβE4). The same molar concentration (10:1) of scrambled Aβ (AnaSpec) was dissolved in vehicle, combined with isolated E3 or E4 particles, held under oligomer forming conditions and stored at −20 ^oC until use (abbreviated scrAβE3 or scrAβE4). scrAβE3 or scrAβE4 was utilized as negative controls throughout the subsequent experiments. Furthermore, Aβ₄₂ peptide and scrambled Aβ was incubated with PBS vehicle as negative control for APOE particles.

Aβ42 peptide was purchased from American Peptide Company (Sunnyvale, CA, USA) and was prepared as previously described (Byun et al., 2015). It was dissolved in hexafluoroisopropanol for 72 h at room temperature (RT) and lyophilized. The peptide was then dissolved again in dimethylsulfoxide. Anti‐ZO‐1, anti‐Claudin 5 (Thermo Fisher Scientific, Waltham, MA, USA), anti‐GAPDH (Abcam, Cambridge, MA, USA), anti‐GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti‐CD31 (R&D Systems, Minneapolis, MN, USA), antiphospho CREB (Cell signaling Technology, Danvers, MA, USA), anti‐CREB (Cell signaling Technology), and anti‐Annexin A1 (Invitrogen, Carsbad, CA, USA) were used for Western blot analysis, and Anti‐ZO‐1 (Thermo Fisher Scientific) was used for immunofluorescence images. Y27632, MK801, and l‐glutamate were purchased from Sigma‐Aldrich Co. (St. Louis, MO, USA), Rhosin was purchased from Merck Millipore (Billerica, MA, USA), and human Annexin A1 (ANXA1) recombinant protein was purchased from MyBioSource (San Diego, CA, USA). For experiments, bEnd.3 cells and/or pericytes were treated with Aβ42 (2 μm and/or 5 μm; from 0 to 24 h, differently for each experiment), ANXA1 (1 μg mL⁻¹ and/or 2 μg mL⁻¹; for 24.5 h), Y27632 (30 μm; for 24 h), Rhosin (10 μm; for 24 h), l‐glutamate (30 μm; for 30 min or 24 h), and MK801 (10 μm; for 30 min or 24 h).