Aquaporin 4

To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3–4 h (ref. 6 (link)). The cells were then spun down and washed with PBS and plated in a complete medium containing Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum, 1% non-essential amino acids, 1% vitamins and 1% antibiotic/antimycotic on plastic (non-coated) tissue culture plates. After 6–12 h, the non-adherent cells were rinsed away and fresh medium was replaced every 2–3 days. Cultures were confirmed to be morphologically consistent with pericyte cultures and were PDGFRβ-positive, desmin-positive, glial fibrillar acidic protein-negative, aquaporin 4-negative, microtubule-associated protein 2-negative, NeuN-negative, von Willebrand Factor-negative and ionized calcium-binding adapter molecule 1-negative. Primary pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific) and grown overnight. For Cy3-Aβ40 uptake experiments, pericytes were initially incubated with 1 μM Cy3-Aβ40 in DMEM at 4 °C for 1 h. Unbound Cy3-Aβ40 was removed by several washes with cold DMEM. Cy3-Aβ40 uptake was determined at 37 °C after 30 min incubation with and without 50 μg ml⁻¹ non-immune IgG or specific function-blocking antibodies raised against the extracellular domain of LRP1 (Santa Cruz Biotechnology; sc-16166), LRP2 (a generous gift from Dr Scott Argraves, Medical University of South Carolina), very low-density lipoprotein receptor (R&D Systems; AF2258) and low-density lipoprotein receptor (R&D Systems; AF2255)6 (link). Cy3-Aβ40 (1 μM) internalization and lysosomal colocalization in pericytes were determined 48 h after siRNA silencing of Lrp1, Lrp2, Vldlr, Ldlr, Apoer2 or control siRNA (siCtrl)6 (link). Adenoviral-mediated re-expression of the LRP1 minigene (Ad.mLRP1) or GFP was performed in pericytes with silenced Lrp1. LysoTracker green DND-26 (Invitrogen) was added at 100 nM and incubated for 30 min at the end of the experiment. Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS, briefly incubated with 0.1% Triton X-100 for 20 s and then stained with Alexa Fluor phalloidin conjugates, F-actin (Invitrogen) and Hoechst 33342 (Invitrogen). Slides were scanned using Zeiss 510 confocal microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA). The Cy3-Aβ40 relative intensity and its lysosomal colocalization in pericytes were measured with the NIH Image J software.