Fig 2: Features of P2RY12-positive microglia interacting with phosphorylated tau-containing structures. (A–F). Dual-color DAB enzyme histochemistry illustrating different features of P2RY12-positive microglia (purple) with phosphorylated tau-positive structures (brown). Abbreviations: LPND; low plaque non-demented. HPND; high plaque non-demented. AD; Alzheimer’s disease. MTG; middle temporal gyrus. Scale bars represent 50 μm. (G–I). Dual-color laser confocal histochemistry showing interaction of P2RY12 positive microglia with early intracellular tangle in LPND case (G). Features of P2RY12-positive microglia interacting with mature tangle (H) and neuritic plaque (I) in AD cases. Scale bars represent 50 μm.

Fig 3: Confocal microscopy of P2RY12 and progranulin-positive microglia in pathologically staged cases. (A–C). Low magnification merged images of P2RY12 (green), progranulin (PGRN) (red) and DAPI (blue) in MTG of LPND (A), HPND (B) and AD cases (C) to show the distribution of P2RY12 and PGRN immunoreactivity through cortical layers. Scale bar represents 50 μm. (D–F). Higher magnification images of PGRN (red) in MTG of LPND (D), HPND (E) and AD cases (F) to show the distribution of immunoreactivity. Similar amounts of PGRN immunoreactivity was present in each disease group. Scale bar represents 50 μm. (G–I). Merged images of P2RY12with PGRNimages shown in (D–F) with DAPI (blue) showing expression in same cells (yellow arrows). Scale bar represents 25 μm.

Fig 4: Features of P2RY12-immunoreactive microglia. (A,B). Morphology of P2RY12-immunoreactive microglia (purple) in a low plaque non-demented (LPND) case (A) and AD case (B). Sections of middle temporal gyrus (MTG) were single-stained with antibody to P2RY12. Red arrows in panel B illustrate the lack of P2RY12 immunoreactive cells in an area occupied by plaque. (C,D). P2RY12-immunoreactive microglia (purple) are a feature in hippocampus sections from non-demented (ND) case (H) and Alzheimer’s disease (AD) case. Section shows staining in CA2 region of hippocampus. Continued presence of P2RY12-positive microglia in AD hippocampus (D) was noticeable. (E,F). Double-staining of section of ND and AD case with P2RY12 (purple) and HLA-DR (brown) showed limited overlap. HLA-DR-positive microglial clusters over plaques were P2RY12-negative except for single cells observed within the cluster (arrows). (G,H). Interaction of P2RY12-immunoreactive microglia (purple) and Aβ plaques (brown). The panels show two types of interactions of P2RY12-positive microglia with plaques. Positive microglia are not present in close association with mature cored plaque (G), while they are present in close association with diffuse type of plaques (H). Specificity controls for P2RY12 staining of microglia. (I,J). Staining of representative sections with P2RY12 (Novus) antibody preabsorbed with immunizing peptide (I, +Pep) compared to staining of matched section with P2RY12 antibody non-absorbed (J, -Pep). (K,L). Staining of matched sections with alternative P2RY12 antibody. Same staining pattern of microglia revealed with P2RY12 (Novus) antibody (C) as with P2RY12 (Alomone Labs) antibody (D). Sections reacted with Alomone Lab P2RY12 required antigen retrieval to obtain positive staining pattern. All sections shown had been counterstained with neutral red to identify nuclei (red color). Abbreviations: ND: non-demented. AD: Alzheimer’s disease. MTG: middle temporal gyrus.—Pep: antibody without immunizing peptide. + Pep: antibody with immunizing peptide. Scale bars represent 50 μm.

Fig 5: Proposed scheme of arrangement of different P2RY12-expressing microglia around Aβ plaques. Suggested scheme to describe localized areas of microglial inflammation around plaques. Zone 1: microglia interacting with mature plaques (HLA-DR high, P2RY12 negative) producing proinflammatory cytokines. Zone 2: Area adjacent to plaque with low or negative P2RY12 positive microglia. Zone 3. P2RY12 high expression in surrounding area defining the boundary between proinflammatory area (Zone 1 and 2) and non-affected area (Zone 3 and beyond). As described in this report, exceptions to this scheme were observed.