Beta-amyloid stimulates murine postnatal and adult microglia cultures in a unique manner

Abstract

Reactive microglia are commonly observed in association with the beta-amyloid (Abeta) plaques of Alzheimer's disease brains. This localization supports the hypothesis that Abeta is a specific activating stimulus for microglia. A variety of in vitro studies have used postnatal derived rodent microglia cultures to characterize the ability of Abeta to stimulate these cells. However, it is unclear whether this paradigm accurately models conditions in aged animals. To determine whether Abeta stimulatory phenotypes differ between young and adult microglia, we established cultures of acutely isolated adult murine cortical microglia to compare with postnatal derived microglial cultures. Although cells from both ages expressed robust immunoreactivity for CD68 and CD11b, their responses to activating stimuli differed. Fibrillar Abeta was rapidly phagocytosed by postnatal microglia and both oligomeric and fibrillar peptide stimulated increased tumor necrosis factor alpha (TNFalpha) secretion. However, Abeta oligomers but not fibrils stimulated TNFalpha secretion from adult microglia. More importantly, adult microglia had diminished ability to phagocytose Abeta fibrils. These findings demonstrate that adult microglia respond to Abeta fibril stimulation uniquely from postnatal cells and suggest that adult rather than postnatal microglia cultures are more appropriate for modeling proinflammatory changes in the aged CNS.

Figure 1.

Adult and postnatal brain-derived mouse microglia cultures display similar cell surface marker immunoreactivity. Cultures of postnatal-derived microglia were collected from mixed glial cultures at 14 d in vitro and plated onto glass chamberslides for 24 h. Adult microglia were isolated from brains and plated for 24 h in vitro. Cells were fixed with 4% paraformaldehyde and immunostained using anti-CD68 (A, B) or -CD11b (C, D) antibodies. Antibody binding was visualized using Texas Red-conjugated secondary antibody, and cellular nuclei were counterstained with DAPI for imaging. Scale bar, 20 μm.

Figure 2.

Postnatal brain-derived microglia secrete TNFα after stimulation with fibrillar and oligomeric Aβ1–42 peptide. Postnatal microglia isolated from 14 d in vitro mixed glial cultures were stimulated 24 h in the absence or presence of 5, 10, and 20 μm oligomeric Aβ1–42 (Aβ_o) or fibrillar Aβ1–42 (Aβ_f). A, Conditioned media was collected from stimulated cells, and TNFα concentrations were determined via commercial ELISA. B, Cell viability after 24 h stimulation was determined by quantitating LDH release into the media using a commercial assay. Graphs are representative of three independent experiments ± SD (∗p < 0.001 from respective control).

Figure 3.

Adult brain-derived microglia secrete TNFα after stimulation with oligomeric Aβ1–42 peptide. Adult microglia were isolated from brains and stimulated for 24 h in vitro in the absence or presence of 5, 10, and 20 μm oligomeric Aβ1–42 (Aβ_o) or fibrillar Aβ1–42 (Aβ_f). A, TNFα concentrations were quantitated from media aliquots from stimulated cells via commercial ELISA. B, Cell viability after the stimulation was assessed by quantitating LDH release into the media using a commercial assay. Graphs are representative of three independent experiments ± SD (∗p < 0.001 from respective control).

Figure 4.

Postnatal brain-derived microglia have an increased capacity to phagocytose fibrillar Aβ1–42 compared with adult microglia. A concentration of 500 nm FITC-Aβ1–42 was incubated in the absence or presence of 1 μg/ml anti-Aβ antibodies, BAM-10, or 6E10. A, B, Postnatal (A) and acutely isolated adult microglia (A, B) were incubated in the absence or presence of FITC-Aβ fibrils (500 nm), antibody only, Aβ plus antibody, or FITC-bioparticle (0.25 mg/ml; positive control) for 6 h. After incubation, the medium was removed and the signal from unphagocytosed peptide was quenched with trypan blue. Fluorescence intensity from phagocytosed peptide in each condition was quantitated using a fluorescent plate reader (480 nm excitation and 520 nm emission) and averaged (±SD). Graphs are representative of three independent experiments (∗p < 0.05; ^#p < 0.001 from respective control).