Extended Data Fig. 5: Brain immune cells generate ROS in AD.
a scRNA sequencing in mouse cortex and human brain: the major cerebral ROS-generating NADPH oxidase, NOX2, is only expressed in microglia/PVMs. Upper graph abbreviations: mRNA level (counts), pericytes (PCs), venous SMCs (vSMCs), arteriolar SMCs (aSMCs), arterial SMCs (aaSMCs), microglia (MG), fibroblast classes (FB1, FB2), oligodendrocytes (OL), broadly-distributed endothelial cell classes (EC1-EC3), venous ECs (vEC), capillary ECs (capilEC), arterial ECs (aEC), astrocytes (AC), from43. Lower graph: 45 clusters (c-0 to c-44) of neuronal and glial cells including MG, ACs, oligodendrocyte progenitor cells (OPCs) and subtypes of excitatory neurons, inhibitory neuons and OLs, from ref. 94 (v23.proteinatlas.org). b Top: confocal images of live AD acute cortical slice with DHE-labeling of ROS in microglia (white arrow) but not pericytes (yellow arrow) or extravascular cells (blue asterisks). Bottom: DHE labeling quantification in WT and AD cortical slices in pericyte somata, capillary segments without (w/o) pericyte somata and (non-immune) extra-vascular cells. c Mitochondrial ROS measured using MitoSOX in microglia or pericyte somata does not differ in WT and AD slices. P values: b-c from Mann-Whitney tests,d from paired t-test. d Laser speckle imaging in barrel cortex of AD mice in vivo shows intravenous glutathione (GSH) does not alter capillary perfusion. e NG2-expressing microglia are in cortex but not cerebellum of mature AD mice nor in cortex and cerebellum of WT mice. f Top: NG2-expressing microglia (arrow heads) but not pericytes (PCs) express Iba1 and P2Y12Rs. Bottom: NG2-expressing microglia cluster around Aβ plaques (labeled with 82E1 antibody). g Percentage of Iba1 cells co-labeling for NG2. h Most Iba1-NG2-labeled cells co-express P2Y12Rs. i Most NG2-expressing microglia are at Aβ plaques. j Number of NG2-expressing microglia increases with ageing in AD cortex. P-value for AD cortex (cx) graph assesses whether the slope of linear regression fit deviates from 0 (cb=cerebellum). k ROS generation measured using CellROX is increased in immune cells at and away from plaques in AD cortex. The antioxidant NAC reduces ROS production. P values are from Mann-Whitney tests for Iba1+ cells and unpaired t-tests for NG2-expressing microglia. P-values are 2-tailed. Error bars are s.e.m.
