Fig 2: 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 3: Expression of P2RY12 mRNA and protein by in vitro cultures of human microglia. (A). Interleukin-4 stimulates P2RY12 mRNA expression. Bar chart showing results real time PCR analysis for P2RY12 mRNA of human microglia stimulated with indicated agents. Results of analysis of single human microglia case (each in triplicate) and representative of other analyses. Abbreviations: Con, control unstimulated: IL-4, interleukin-4 (40 ng/mL): Aβ2 and Aββ5 (Aβ (1–42) 2 μM and 5 μM): IFNγ, interferon-γ (100 ng/mL): LPS, lipopolysaccharide (100 ng/mL): LPS/IFNγ (doses combined): IL-6, interleukin-6 (40 ng/mL). **** p < 0.0001. * p < 0.05. (B). Western blot of human microglia protein samples probed with antibody to P2RY12. Increased amounts of P2RY12 (58 kDa) in IL-4 treated samples.

Fig 4: 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.

Fig 5: Distribution of P2RY12 microglia within cortical layers in relation to amyloid beta plaques in pathologically staged samples (A–C). Lower magnification photomicrographs showing the changes in P2RY12 microglia distribution compared to Aβ plaques within cortical layers of MTG. Sections from low plaque, high plaque and AD cases stained for P2RY12 (purple) and Aβ (brown). Sections were counterstained with neutral red to identity cellular morphology. Scale bars represent 200 μm. (D–F). Higher magnification photomicrographs of the areas in panels (A–C) indicated by frames. Scale bars represent 50 μm.