A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells

Figure 1

Nuclear localization of an amino-terminal fragment of apoE4 is confirmed in microglia cells but not in astrocytes. (a–c) U87 cells, representing an astrocytic cell line, were plated on glass chamber slides in normal growth media and treated for 24 hours with the nApoE4_1-151 fragment. Following treatment, cells were fixed and immunocytochemistry was carried out using an anti-His rabbit, polyclonal antibody at 1 : 2,000, followed by the HRP-conjugated secondary antibody at 1 : 200 (see Materials and Methods for details). Under these experimental conditions, although there was evidence of the cytoplasmic uptake of the fragment (a), little nuclear localization of the nApoE4_1-151 fragment was observed following treatment (c, merge). Scale bars represent 10 μm. (d) A representative set of images showing nuclear localization in the microglial cell line, BV2, following exogenous treatment with the nApoE4_1–151 fragment. BV2 microglial cells were placed on glass chamber slides in normal growth media and treated for 24 hours with the nApoE4_1–151 fragment. Following treatment, cells were fixed and immunocytochemistry was carried out as described above. Double-label immunofluorescence confocal z-stacks were acquired to detect nApoE4_1–151 (green) together with DAPI (blue). The merged images indicated the strong nuclear and cytoplasmic presence of the amino-terminal fragment following extracellular incubation of BV2 cells.

(a)

(b)

(c)

(d)