Fig 1: Microglial cells express functional amylin receptors. a Primary cultures of human fetal microglia (HFMs) that are stained with microglial antibody, Iba-1 (green), and DyLight-594-labeled lectin (red). b These primary cultured HFMs were also stained for the two dimeric proteins that are components of the amylin receptor 3 (AMY3) subtype, the calcitonin receptor (CTR), and the receptor-associated membrane protein 3 (RAMP3). c Cultured HFMs were loaded with the fluorescent intracellular calcium dye, Fluo-8L-AM (green), and with lectin (red), an in vivo microglial marker. Arrowheads indicate cells from which Ca2+ signals were recorded. The same cell culture is also stained with lectin (red). The field in c shows that a majority of cells are microglia. d Summary of data on intracellular calcium changes after human amylin (hAmylin) and Aβ1–42 without and with application of the amylin receptor antagonist, AC253 in HFM (*p < 0.05, n = 123 cells in 12 culture wells from four different batch of the culture cells). The candidate traces for intracellular calcium changes are illustrated in e–i. Elevations of Ca2+induced by acute (30 s) application of either hAmylin (1 μM, e) or Aβ1–42 (1 μM, f). The changes in Ca2+ were blocked by AC253 (10 μM, g–i). Traces correspond to cells identified in c with arrowheads. Scale bar = 20 μm
Fig 2: Amylin receptors on microglia in human Alzheimer’s disease brain and BV2 microglial cells. a, b Section from the inferior frontal gyrus of the AD patient (Braak Stage 6) after dual immunostaining both with the antibody to microglial marker Iba-1 (yellow) and calcitonin receptor (CTR) (dark blue). Arrows show microglial cells expressing both Iba-1 and CTR; thin arrows point to microglial cells expressing only Iba-1. c, d Single microglial cells are presented at a higher magnification, with thin arrows pointing to Iba-1 location (yellow) and arrows pointing to the CTR location (dark blue). e Coronal section from the inferior frontal gyrus of the AD patient after double immunohistochemical staining with antibody against CTR and receptor activity modifying protein 3 (RAMP3). Notice the microglia-like profile (mgl) expressing both CTR (blue) and RAMP3 (yellow). f BV2 microglial cells expressed CTR (red). g These BV2 cells were cultured in the presence of Aβ1–42 1 μM for 24 h. The Aβ was taken up into the cells and formed plaque-like structures stained with thioflavin S (green). Scale bars = 10 μm
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