Microglia express GPNMB in the brains of Alzheimer's disease and Nasu-Hakola disease

Abstract

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein first identified in low-metastatic human melanoma cell lines as a regulator of tumor growth. GPNMB is widely expressed in various tissues, where it is involved in cell differentiation, migration, inflammation/anti-inflammation, tissue regeneration, and neuroprotection. GPNMB is identified in microglia of adult rat brains, neurons and astrocytes of GPNMB transgenic (Tg) mouse brains, and motor neurons of amyotrophic lateral sclerosis (ALS) patients. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either TYROBP (DAP12) or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Pathologically, the brains of NHD patients exhibit leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and remarkable activation of microglia predominantly in the white matter of frontal and temporal lobes and the basal ganglia. At present, molecular mechanisms responsible for development of leukoencephaolpathy in NHD brains remain totally unknown. Recent evidence indicates that disease-associated microglia (DAM) that cluster around amyloid plaques express high levels of GPNMB in Alzheimer's disease (AD) brains. Because microglia act as a key regulator of leukoencephalopathy in NHD brains, it is proposed that GPNMB expressed on microglia might play a protective role in progression of leukoencephalopathy possibly via active phagocytosis of myelin debris. In the present study using immunohistochemistry, we have attempted to clarify the expression of GPNMB in NHD brains, compared with AD brains. We found that microglia accumulating in the white matter express an intense GPNMB immunoreactivity in both NHD and AD brains, suggesting that the accumulation of GPNMB-immunoreactive microglia is a general phenomenon in neurodegenerative brains.

Keywords: Alzheimer's disease; GPNMB; Nasu-Hakola disease; leukoencephalopathy; microglia; osteoactivin.

Figure 1.

Validation of the specificity of anti-GPNMB antibody. Western blot analysis of a V5-tagged recombinant GPNMB protein with (A) anti-GPNMB antibody AF-2550, (B) anti-V5 antibody, and (C) anti-G3PDH antibody, as a loading control. (lane 1) non-transfectant and (lane 2) transfectant.

Figure 2.

Immunohistochemistry of frontal white matter with anti-GPNMB antibody. (a) frontal white matter, NC, (b) hippocampus, AD, (c) frontal white matter, AD and (d) frontal white matter, NHD. Scale bars indicate (a, c, d) 50 μm and (b) 100 μm.

Figure 3.

Immunohistochemistry of NHD brains with anti-GPNMB antibody. (a-d) NHD brains. (a, b) frontal white matter, (c) frontal cortex and (d) hippocampus. Scale bars indicate (a-d) 50 μm.

Figure 4.

GPNMB-immunolabeled areas of frontal white matter and frontal cortex. The differences in the GPNMB-positive areas among AD, NHD, and NC subjects were evaluated statistically by one-way analysis of variance (ANOVA) followed by post-hoc Tukey's test. (a) frontal white matter and (b) frontal cortex.

Figure 5.

Double immunolabeling with anti-GPNMB antibody and cell type-specific antibodies. (a-f) AD, (a-c, f) hippocampus and (d, e) frontal cortex. Double immunolabeling of GPNMB (brown) with (a) Iba1 (red), (b) GFAP (red), (c) NeuN (red), (d) amyloid-β (red), (e) ApoE (red), or (f) AT8-tau (red). Scale bars indicate (a-c, e, f) 20 μm and (d) 50 μm.

Figure 6.

Immunohistochemistry of plaque-forming microglia with anti-GPNMB antibody. (a-d) AD, (a, c, d) hippocampus and (b) frontal cortex. Scale bars indicate (a) 50 μm and (b-d) 20 μm.