Astrocyte elevated gene-1 regulates astrocyte responses to neural injury: implications for reactive astrogliosis and neurodegeneration

Abstract

Background: Reactive astrogliosis is a ubiquitous but poorly understood hallmark of central nervous system pathologies such as trauma and neurodegenerative diseases. In vitro and in vivo studies have identified proinflammatory cytokines and chemokines as mediators of astrogliosis during injury and disease; however, the molecular mechanism remains unclear. In this study, we identify astrocyte elevated gene-1 (AEG-1), a human immunodeficiency virus 1 or tumor necrosis factor α-inducible oncogene, as a novel modulator of reactive astrogliosis. AEG-1 has engendered tremendous interest in the field of cancer research as a therapeutic target for aggressive tumors. However, little is known of its role in astrocytes and astrocyte-mediated diseases. Based on its oncogenic role in several cancers, here we investigate the AEG-1-mediated regulation of astrocyte migration and proliferation during reactive astrogliosis.

Methods: An in vivo brain injury mouse model was utilized to show AEG-1 induction following reactive astrogliosis. In vitro wound healing and cell migration assays following AEG-1 knockdown were performed to analyze the role of AEG-1 in astrocyte migration. AEG-1-mediated regulation of astrocyte proliferation was assayed by quantifying the levels of cell proliferation markers, Ki67 and proliferation cell nuclear antigen, using immunocytochemistry. Confocal microscopy was used to evaluate nucleolar localization of AEG-1 in cultured astrocytes following injury.

Results: The in vivo mouse model for brain injury showed reactive astrocytes with increased glial fibrillary acidic protein and AEG-1 colocalization at the wound site. AEG-1 knockdown in cultured human astrocytes significantly reduced astrocyte migration into the wound site and cell proliferation. Confocal analysis showed colocalization of AEG-1 to the nucleolus of injured cultured human astrocytes.

Conclusions: The present findings report for the first time the novel role of AEG-1 in mediating reactive astrogliosis and in regulating astrocyte responses to injury. We also report the nucleolar localization of AEG-1 in human astrocytes in response to injury. Future studies may be directed towards elucidating the molecular mechanism of AEG-1 action in astrocytes during reactive astrogliosis.

Figure 1

Astrocyte elevated gene-1 (AEG-1) colocalizes with reactive astrocytes during astrogliosis. AEG-1 expression in activated astrocytes of mouse brain, 4 days post stereotactic phosphate-buffered saline (PBS) injection, was analyzed by immunofluorescent microscopy. The non-injected contralateral hemisphere was used as a control. Immunostaining for glial fibrillary acidic protein (GFAP) (red, astrocyte marker) and 4', 6-diamidino-2-phenylindole (DAPI) (blue, nuclear marker) identified the needle tract of activated astrocytes (A,B). Non-injected (inset image, (C) and PBS-injected contralateral hemispheres costained for AEG-1 and GFAP, were assessed for AEG-1 expression and colocalization with GFAP (yellow, inset image, (D).

Figure 2

Astrocyte elevated gene-1 (AEG-1) mediates astrocyte migration during wound healing. Human astrocytes were transfected with AEG-1 specific siRNA (siAEG-1) or non-targeting, scrambled siRNA (siCon) by nucleofection and plated for 48 h. Messenger RNA was isolated at 24 h and 120 h post recovery and AEG-1 levels were measured by real-time polymerase chain reaction (PCR) (***P <0.001, (A)). In parallel experiments, immunoblotting was performed for AEG-1 at 48 h; β-actin was used as normalizing loading control (**P <0.001, (B)). Transfected astrocytes were plated to confluence for 48 h into Oris^TM migration assay plates and then injured by removal of the cell-seeding stopper. Migrating astrocytes were visualized with Hoechst nuclear stain at various time points. Micrographs are shown for siCon-transfected (C1,C3,C5,C7) and siAEG-1-transfected astrocytes (C2,C4,C6,C8). The number in the lower right corner of the white box (C1-C8) show number of cells in the area of the injury. Separate images from four replicate wells were analyzed to quantify the number of cells present (***P <0.001, (D)). Representative data from three individual donors assayed in triplicate is shown.

Figure 3

Astrocyte proliferation requires astrocyte elevated gene-1 (AEG-1). Astrocytes transfected with AEG-1 specific siRNA (siAEG-1) or non-targeting, scrambled siRNA (siCon) were immunostained with Ki67 or proliferating cell nuclear antigen (PCNA) (green, proliferation marker) and glial fibrillary acidic protein (GFAP) (red, astrocyte marker) antibodies. Untransfected and mock-transfected astrocytes were stained in parallel. Micrographs at 20 × original magnification of Ki67 or PCNA with GFAP costaining for non-transfected control (A1,A2), mock-transfected (B1,B2), siCon-transfected (C1,C2) and siAEG-1-transfected astrocytes (D1,D2) are shown. (E) This panel shows the number of Ki67 positive cells in ten micrographs from replicate wells for each condition (***P <0.001). (F) This panel shows the number of PCNA positive cells in ten micrographs from replicate wells for each condition (***P <0.001). All micrographs are 20 × original magnification, unless otherwise noted. Immunostaining data is representative of three individual donors assayed in triplicate.

Figure 4

Astrocyte elevated gene-1 (AEG-1) localizes to nuclear pockets during wound healing in cultured human astrocytes. Confluent astrocyte cultures were scratched to mimic injury and subsequent wound healing by migrating astrocytes was monitored for up to 48 h. Phase-contrast images of the wound tract are shown from 0 to 48 h (A1-F1). In parallel, starting from 8 h post injury, cells were periodically fixed and immunostained with glial fibrillary acidic protein (GFAP) (red, astrocyte marker) and AEG-1 (green) antibodies (A2-E2). Higher resolution micrograph of AEG-1 and GFAP costaining in migrated astrocytes is shown (40 ×, (G)). All images are 20 × original magnification, unless otherwise noted. Cytoplasmic and nuclear protein extracts of astrocytes following wound healing were immunoblotted for AEG-1 (H); cytoplasmic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and nuclear lamin A/C were used as subcellular fraction normalizing controls (*P <0.01, (J)). Representative data from three individual donors assayed in triplicate. Immunoblotting data is representative of three individual donors.

Figure 5

Astrocyte elevated gene-1 (AEG-1) localizes to the astrocyte nucleolus during wound healing. At 48 h post injury astrocytes were immunostained for fibrillarin (red, nucleolar marker, (A)), AEG-1 (green, (B)) and 4', 6-diamidino-2-phenylindole (DAPI) (blue, nuclear marker, (C)) and micrographed by confocal microscopy. Z-stack micrographs of fibrillarin fluorescence were overlaid upon AEG-1 and DAPI (D). Higher magnification micrograph of AEG-1 and fibrillarin costaining is shown (E). A scatter plot of overlapping AEG-1 and fibrillarin pixels was generated (P = 1, (F)). Representative confocal images are 200 × original magnification from 2 individual donors assayed in triplicate.

Figure 6

Role of astrocyte elevated gene-1 (AEG-1) in reactive astrogliosis. Central nervous system (CNS) insults ranging from mild cellular disturbances to severe tissue damage and cell death lead to release of molecular mediators of reactive astrogliosis such as inflammatory cytokines interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) and molecules of oxidative stress such as reactive oxygen species (ROS). These mediators in turn activate the local healthy astrocyte population by inducing a spectrum of changes in the microenvironment and intracellular signaling pathways resulting into reactive astrogliosis. Injury triggers increased AEG-1 localization to the cytoplasmic regions and the dense fibrillar nuclear regions of the astrocyte. This change in intracellular localization of AEG-1 in astrocytes undergoing reactive astrogliosis is a plausible mechanism of AEG-1-mediated regulation of astrocyte proliferation and migration during reactive astrogliosis.