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microRNA-34a-Mediated Down-Regulation of the Microglial-Enriched Triggering Receptor and Phagocytosis-Sensor TREM2 in Age-Related Macular Degeneration

Fig 6

Control C8B4 murine MG cells efficiently phagocytose Aβ42 peptides; miRNA-34a treated MG cells do not; (A) C8B4 MG cells (ATCC CRL-2540) were cultured for 3 days (control a-d, top row); cells were treated with 5 μM of Aβ42 for 24 hr before staining; Aβ42 peptide (American Peptide Company, Sunnyvale, CA, cat # 62-0-80A); Aβ42 peptide was prepared as previously described [33]; briefly, Aβ42 peptides were initially solubilized in hexafluoroisopropanol (HFIP; Fluka Chemical, cat# 52512; Sigma-Aldrich, St. Louis MO), aliquoted, and stored at −20°C as an HFIP film. After vacuum evaporation of HFIP, aliquoted peptide was re-suspended with DMSO to 5 mM and diluted to 5 μM into the cell culture media; (B) cells were treated with a scrambled miRNA-34a sequence (miRNA-34a-sc; 30 nM;, control, a-d, middle row); or (C) with an LNA-stabilized miRNA-34a (30 nM; miRNA-34a stressed; a-d; bottom row); treatments were for 24 hr before incubation with 5 uM of Aβ42 (made up as in [33]) for another 24 hr before assay; MG cells were subsequently stained using a murine amyloid beta MABN10 (red fluorescence λmax~650 nm; anti-Aβ antibody, clone W0-2; Millipore, Bellerica MA), a TREM-2 antibody (M-227): sc-48765 (green fluorescence; λmax~510 nm; Santa Cruz, Santa Cruz CA) or DAPI nuclear stain (blue fluorescence; λmax~470 nm) as in Fig 3; arrows indicate Aβ42 uptake into MG cells; note decreased presence of TREM2 in miRNA-34a treated MG cells (bottom row, panel B) and decrease in ingested Aβ42 peptide within C8B4 cells (C; bottom row, panel d). Taken together these results support a miRNA-34a-mediated impairment of sufficient TREM2 to phagocytose Aβ42 peptide from the extracellular space; note self-aggregation of Aβ42 peptide after 24 hrs and Aβ42 peptide affinity for TREM2 containing cells (leftmost panels) and internalization (rightmost panel; yellow merge; λmax~580 nm); magnification 20x; Aβ peptide quantification was performed using SlideBook 5.0 (Intelligent Imaging Innovations) and ImageJ (NIH) software; under these conditions about 42% of externalized Aβ42 was cleared; additional relevant methods have been described [10,44].

Fig 6