P2Y12 expression on microglia in the human brain

Microglia are resident myeloid cells of the central nervous system that play a principal role in the maintenance of normal tissue homeostasis and plasticity. Microglia have a capacity to constantly migrate, and scavenge invading pathogens, apoptotic debris, unwanted synapses and pathologically accumulated protein aggregates by sensing them with a panel of pattern recognition receptors. In addition to pattern recognition receptors, microglia express purinergic receptors activated by extracellular nucleotides released from damaged neural tissues. They are composed of the metabotropic P2YR family, such as P2Y2, P2Y4, P2Y6, P2Y12 and P2Y14, and the ionotropic P2XR family, such as P2X4 and P2X7, with differential binding properties against adenosine triphosphate and its metabolites. Among them, the Gi/o-coupled P2Y12 receptor is expressed selectively on microglia, but not other cell types in the rodent brain. The P2Y12 receptor plays a central role in adenosine diphosphate-induced microglial chemotaxis and process extension at sites of injury and inflammation in the central nervous system by activating phospholipase C, phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinasemediated pathways, and integrin b1. Microglia derived from P2Y12-deficient mice are unable to polarize, migrate or extend processes toward nucleotides. P2Y12 expression is the highest in resting microglia, whereas the expression is greatly reduced in activated microglia. However, it remains unknown whether microglia with upregulated expression of P2Y12 plays a specialized role in neuroprotection or neurotoxicity with relevance to human neurological diseases. P2Y12 expression in the human brain remains controversial. One study showed that P2Y12 is expressed abundantly in myelinated fibers and interlaminar astrocytes, but not on microglia, and P2Y12 expression is reduced in demyelinated lesions of secondary progressive multiple sclerosis. In contrast, a different study showed that P2Y12 is expressed specifically on alternatively activated M2-polarized microglia in the human brain. To clarify these discrepancies, we studied P2Y12 expression by immunohistochemistry using rabbit anti-P2Y12 antibody (HPA014518, 1:1000; Sigma, St. Louis, MO, USA), along with Iba1 immunolabeling, in serial sections of the frontal cortex derived from four subjects who died of non-neurological causes (age 79 9 years), five with Nasu–Hakola disease (NHD; age 41 6 years), 10 with Alzheimer’s disease (age 70 8 years), four with amyotrophic lateral sclerosis (age 68 6 years) and three with Parkinson’s disease (age 79 4 years) cases. Written informed consent was obtained from all cases. In all brains, we identified intense P2Y12 immunoreactivity on Iba1-positive ramified microglia located in the grey matter, to a lesser extent, in the white matter (Fig. 1a,b). In contrast, perivascular monocytes/macrophages, intensely labeled by Iba1, did not express P2Y12 in any cases (Fig. 1c,d), whereas a subset of amoeboid microglia infiltrating white matter demyelinated lesions of NHD expressed P2Y12 (Fig. 1c inset). A small population of P2Y12immunopositive ameboid microglia expressed CD68 in NHD brains (Fig. S1a). We did not identify P2Y12 immunoreactivity on oligodendrocytes, astrocytes or neurons in any cases examined. We did not find P2Y12 expression on foamy macrophages. By quantitative analysis with the Image J software (National Institute of Health, Bethesda, MD, USA), the area of P2Y12 immunoreactivity was not significantly different among five groups described above because of great interindividual variations (Fig. S2a,b). Notably, two NHD cases showed extensive microgliosis, composed chiefly of ramified microglia in the cortex intensely labeled by P2Y12 and Iba1 (Fig. 1e,f). These observations are very important, because NHD is caused by loss-of-function mutations of microglial signaling genes, TREM2 or DAP12. However, P2Y12-immunoreactive ramified microglia were not labeled by CD80 (a M1 marker; Fig. 1g; Fig. S1b), CD163 (a M2 marker; Fig. 1h; Fig. S1c), or human leukocyte antigen-DR (HLA-DR) (Fig. 1i; Fig. S1d) in NHD brains. These results suggest that microglial proliferation did not attenuate P2Y12 expression, and P2Y12-positive ramified microglia are not polarized either to M1 or M2 in NHD brains. Importantly, the processes of P2Y12-immunoreactive ramified microglia often showed a close contact with amyloid-b (Ab) plaques in Alzheimer’s disease brains, suggesting that they migrate to Ab plaques by surveying aggregated Ab with their extended processes (Fig. 1j). However, the great majority of P2Y12immunoreactive ramified microglia in Alzheimer’s