CCAAT-enhancer binding protein-β expression and elevation in Alzheimer's disease and microglial cell cultures.

CCAAT-enhancer binding proteins are transcription factors that help to regulate a wide range of inflammatory mediators, as well as several key elements of energy metabolism. Because C/EBPs are expressed by rodent astrocytes and microglia, and because they are induced by pro-inflammatory cytokines that are chronically upregulated in the Alzheimer's disease (AD) cortex, we have investigated whether C/EBPs are expressed and upregulated in the AD cortex. Here, we demonstrate for the first time that C/EBPβ can be detected by Western blots in AD and nondemented elderly (ND) cortex, and that it is significantly increased in AD cortical samples. In situ, C/EBPβ localizes immunohistochemically to microglia. In microglia cultured from rapid autopsies of elderly patient's brains and in the BV-2 murine microglia cell line, we have shown that C/EBPβ can be upregulated by C/EBP-inducing cytokines or lipopolysaccharide and exhibits nuclear translocation possibly indicating functional activity. Given the known co-regulatory role of C/EBPs in pivotal inflammatory mechanisms, many of which are present in AD, we propose that upregulation of C/EBPs in the AD brain could be an important orchestrator of pathogenic changes

C/EBPβ confocal fluorescent immunocytochemistry in human microglia cultures.

<p>Parallel wells received (A) no cytokine treatment or 50 ng/ml (B) IL-1β, (C) IL-6, or (D) TNF-α for 4 hours. Microglia treated with cytokines show increased expression and nuclear localization of C/EBPβ. All wells were imaged concurrently with the same photomultiplier tube intensity, gain, and offset settings using an Olympus Fluoview Confocal microscope–200X (scale bar = 100 µm).</p

C/EBPβ bright field immunohistochemistry.

<p>Single and dual-label immunohistochemistry demonstrates increased immunoreactivity for C/EBPβ on activated AD microglia invested in amyloid plaques in AD tissue as compared to ND tissue. C/EBPβ immunoreactivity in (A) ND SFG tissue, (B and C) AD SFG tissue, and (D) AD entorhinal cortex tissue. (A–B–200X, scale bars = 800 µm. C–D–400X, scale bars = 400 µm). Percent area of C/EBPβ immunostaining is higher in AD (B: 2.23%) vs. ND (A: 0.44%). (E) C/EBPβ immunoreactive cells (black) and 4G8-immunoreactive Aβ plaques (brown) in AD SFG tissue. (F) C/EBPβ immunoreactive cells (black) and thioflavine S-labeled Aβ plaques (green fluorescence) in AD SFG tissue. (E–F–200X, inset 400X). Significantly higher percentages of C/EBPβ immunopositive cells were associated with Aβ plaques (E: 96.9%, F: 89.4%, both Student’s <i>t</i>-test, P<0.01) than not associated with Aβ plaques.</p

C/EBPβ bright field immunohistochemistry across multiple brain regions.

<p>Comparisons of C/EBPβ immuno-labeling across six different brain regions demonstrates increased immunoreactivity for C/EBPβ on activated AD microglia in AD tissue as compared to ND tissue. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086617#pone-0086617-g005" target="_blank">Figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086617#pone-0086617-g007" target="_blank">7</a>, clustered activated microglia in the AD brain are almost invariably associated with Aβ plaques. Percent area of C/EBPβ immunostaining by image analysis is shown in parentheses. (A) Superior parietal lobule (AD 0.54% vs. ND 0.3%, AD/ND ratio = 1.8). (B) Locus coeruleus (AD 2.2% vs. ND 0.29%, AD/ND ratio = 7.6). (C) Temporal lobe (AD 1.18% vs. ND 0.6%, AD/ND ratio = 2). (D) Visual cortex (AD 0.56% vs. ND 0.47%, AD/ND ratio = 1.2). (E) Superior frontal gyrus (AD 1.36% vs. ND 0.47%, AD/ND ratio = 2.9). (F) Mid-frontal gyrus (AD 0.63% vs. ND 0.55%, AD/ND ratio = 1.1). (All micrographs are at 200X, scale bars = 800 µm).</p

C/EBPβ in treated BV-2 cell Western blots.

<p>(A) BV-2 cells treated with 1 µg/ml LPS for differing times. Maximal expression of C/EBPβ (including the LIP isoform) is induced in nuclear fractions (NF) at 4 hours relative to 1, 24, or 48 hours, or 48 hour untreated controls. Cytoplasmic fractions (CF) show little to no expression of C/EBPβ. Lanes: (1) 1 hour NF (2) 1 hour CF (3) 4 hour NF (4) 4 hour CF (5) 24 hour NF (6) 24 hour CF (7) 48 hour NF (8) 48 hour CF (9) 48 hour Control NF (10) molecular weight marker–not visible (11) 48 hour Control CF (12) Blank. β-actin control shown in lower panel. (B) BV-2 cells treated with 1 µg/ml LPS or 10 ng/ml IL-6 and/or 1 or 5 µM aggregated Aβ(1–42). C/EBPβ expression was increased in cells treated with LPS and further increased by IL-6 treatment. Odd numbered lanes are corresponding cytoplasmic fractions for nuclear fractions (NF). Even numbered lanes are nuclear fractions: (2) Control untreated (4) LPS (6) IL-6 (8) Aβ 5 µM (10) Aβ 1 µM+IL-6 (12) Aβ 5 µM+IL-6.</p

C/EBPβ confocal fluorescent immunohistochemistry of AD superior frontal gyrus brain tissue.

<p>(A) C/EBPβ positive cells (green) and HLA-DR positive activated microglia (red) (MHC Class II–activated microglial marker) colocalize together (yellow), positively identifying C/EBPβ immunoreactive cells as activated microglia–200X (scale bar = 100 µm), with 55% of microglia being C/EBPβ immunopositive. (B–D) C/EBPβ positive activated microglia (green) colocalize with 4G8-immunoreactive Aβ plaques (red)–200X and 400X (scale bar = 50 µm). (D–F) C/EBPβ positive activated microglia (green) colocalize with 4G8-immunoreactive Aβ-laden microvasculature (red)–400X. Percentages of C/EBPβ immunopositive cells associated with Aβ: (B) 72.4%, P<0.01 (D) 55.4%, (E) 59.1%, (F) 48.1%. Upon averaging, the number of C/EBPβ immunopositive cells associated with Aβ was significantly higher than C/EBPβ immunopositive cells not associated with Aβ (Student’s <i>t</i>-test, P<0.01).</p

C/EBPβ Western blots in AD and ND frontal lobe gray matter.

<p>When normalized to β-actin, the data show a significant increase in C/EBPβ protein expression in AD compared to ND samples (t₁₆ = 2.9, P<0.01). Summary densitometry data for the blots (mean ratios of C/EBPβ/β-actin integrated optical density values, IDV) are provided in the bottom panel.</p

C/EBPβ immunocytochemistry of cultured murine BV-2 cell line.

<p>C/EBPβ is increased both in numbers of immunopositive cells and in intensity of fluorescence in the nucleus of cells treated with (B) 1 µg/ml LPS as compared with (A) controls (scale bar = 10 µm). Some control cells were positive for C/EBPβ but the immunofluorescence was notably less intense. The average mean gray value of pixels in LPS treated cells (51.14±3.15, SEM) was significantly higher than the control (38.45±1.86, SEM) (P<0.05). Integrated density in LPS treated cells (6.87±0.42, SEM) was also significantly higher than the control (4.23±0.31, SEM) (P<0.01).</p