Abstract
NG2 is a type I transmembrane glycoprotein known as chondroitin sulfate proteoglycan 4 (CSPG4). In the healthy central nervous system, NG2 is exclusively expressed by oligodendrocyte progenitor cells and by vasculature pericytes. A large body of immunohistochemical studies showed that under pathological conditions such as acute brain injuries and experimental autoimmune encephalomyelitis (EAE), a number of activated microglia were NG2 immuno-positive, suggesting NG2 expression in these cells. Alternative explanations for the microglial NG2 labeling consider the biochemical properties of NG2 or the phagocytic activity of activated microglia. Reportedly, the transmembrane NG2 proteoglycan can be cleaved by a variety of proteases to deposit the NG2 ectodomain into the extracellular matrix. The ectodomain, however, could also stick to the microglial surface. Since microglia are phagocytic cells engulfing debris of dying cells, it is difficult to identify a genuine expression of NG2. Recent studies showing (1) pericytes giving rise to microglial after stroke, and (2) immune cells of NG2-EYFP knock-in mice lacking NG2 expression in an EAE model generated doubts for the de novo expression of NG2 in microglia after acute brain injuries. In the current study, we took advantage of three knock-in mouse lines (NG2-CreERT2, CX3CR1-EGFP and NG2-EYFP) to study NG2 expression indicated by transgenic fluorescent proteins in microglia after tMCAO (transient middle cerebral artery occlusion) or cortical stab wound injury (SWI). We provide strong evidence that NG2-expressing cells, including OPCs and pericytes, did not differentiate into microglia after acute brain injuries, whereas activated microglia did express NG2 in a disease-dependent manner. A subset of microglia continuously activated the NG2 gene at least within the first week after tMCAO, whereas within 3 days after SWI a limited number of microglia at the lesion site transiently expressed NG2. Immunohistochemical studies demonstrated that these microglia with NG2 gene activity also synthesized the NG2 protein, suggesting activated microglia as an additional source of the NG2 proteoglycan after acute brain injuries.
Highlights
IntroductionNerve/glia antigen 2 (NG2), encoded by the cspg gene (for simplicity we will use NG2 gene instead of cspg gene), is a single membrane-spanning proteoglycan with a large extracellular domain, and a short cytoplasmic tail [43, 57]
Nerve/glia antigen 2 (NG2), encoded by the cspg4 gene, is a single membrane-spanning proteoglycan with a large extracellular domain, and a short cytoplasmic tail [43, 57]
NG2 was expressed in a subset of microglia after transient middle cerebral artery occlusion (tMCAO) Previous studies demonstrated the NG2 immunoreactivity in a significant number of microglia within the lesion area after tMCAO
Summary
Nerve/glia antigen 2 (NG2), encoded by the cspg gene (for simplicity we will use NG2 gene instead of cspg gene), is a single membrane-spanning proteoglycan with a large extracellular domain, and a short cytoplasmic tail [43, 57]. With a house-made antibody Hampton et al, detected only ~ 20% of microglia with NG2 immunoreactivity along the lesion site at 2 days post SWI, and no NG2 immuno-positive microglia at 7 days post injury [18]. These seemingly contradictory results raise doubts that NG2 immuno-positive microglia are NG2-expressing cells in acute brain injury. The situation was further complicated by recent studies suggesting that pericytes (with bona fide expression of NG2) could generate microglia after stroke [46] In this case, NG2 could be detected as a remnant of a previous cell differentiation event
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