*2.8. Patterns of Expression of P2RY12-Positive Microglia and Phosphorylated Tau-Positive Tangles*

Neurofibrillary tangles and dystrophic neurites are also a hallmark pathological feature of AD pathology. In this study, we identified them using antibody AT8 that recognizes phosphorylated forms of tau (serine 202/threonine 205)(pTAU) that accumulate in tangles and neurites. The cases studied had been staged based on plaque not tangle pathology. There was little difference in tangle scores between LPND and HPND cases, but a large increase in the AD cases (Table 1-set 1). AT8 staining was only prominent in the AD cases. A complete series of sections in this study were double-stained using enzyme histochemistry for P2YR12 (purple) and AT8 (brown), and a subset of these also examined by confocal microscopy. Figure 10A and 10B shows P2RY12-immunoreactive microglia interacting with sparse pTau-positive neurites (Figure 10A) and surrounding an early-stage intracellular tangle (arrow) (Figure 10B). Figure 10, panels C–E show features of P2RY12 immunoreactive microglia surrounding different AT8-immunoreactive structures. It can be seen that these microglia do not have the morphology of resting microglia. This is particular noticeable in Figure 10D with a microglia closely interacting with a tangle-containing neuron. A frequent observation in regions with heavy density of AT8 staining were the small numbers of microglia that were strongly immunoreactive for P2RY12 (Figure 10E). A rare feature observed in only one of our LPND case was the presence of AT8-positive glial cells (brown) (Figure 10F) with closely-associated P2RY12-positive microglia. Overall, the AT8-positive tangled structures did not appear to provide the inflammatory stimuli to cause downregulation of P2RY12 expression. Using confocal microscopy, Figure 10G shows an early intracellular tangle with an intact nucleus in a LPND case surrounded by P2RY12 microglia. More mature tangles and tangled

neurites (Figure 10H) in AD cases did not have directly interacting P2RY12-positive microglia. Based on the characteristic morphology, the AT8-positive structure in Figure 10I is considered to represent a neuritic plaque, an accumulation of phosphorylated tau-containing neurites associated with an Aβ plaque.

**Figure 10.** 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.
