Cell cycle proteins exhibit altered expression patterns in lentiviral-associated encephalitis - PubMed (original) (raw)

Cell cycle proteins exhibit altered expression patterns in lentiviral-associated encephalitis

Kelly L Jordan-Sciutto et al. J Neurosci. 2002.

Abstract

Cell cycle proteins regulate processes as diverse as cell division and cell death. Recently their role in neuronal death has been reported in several models of neurodegeneration. We have reported previously that two key regulators of the cell cycle, the retinoblastoma susceptibility gene product (pRb) and transcription factor E2F1, exhibit altered immunostaining patterns in simian immunodeficiency virus encephalitis (SIVE). Here we show that E2F1 and the inactivated, hyperphosphorylated form of pRb (ppRb) also exhibit altered immunostaining patterns in human immunodeficiency virus encephalitis (HIVE). Quantification of E2F1 and ppRb staining by immunofluorescent confocal microscopy confirms a significant increase in E2F1 and ppRb in both HIVE and the simian model. This increase in E2F1 and ppRb staining correlates with an increase in the presence of activated macrophages, suggesting a link between changes in cell cycle proteins and the presence of activated macrophages. Changes in ppRb and E2F1 staining in SIVE also correlate with alterations in E2F/DNA binding complexes present in the nuclear and cytoplasmic fractions from both midfrontal cortex and basal ganglia. These findings suggest that changes in cell cycle proteins occur in both HIVE and the simian model and that these changes have functional implications for gene expression in neural cells under encephalitic conditions mediated by macrophage activation or infiltration.

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Figures

Fig. 1.

Fig. 1.

In HIVE, increased E2F1 staining was observed predominantly in neuronal cytoplasm and not astrocytes.A_–_F, Triple-label immunofluorescent confocal microscopy for E2F1 (green), nuclear DNA (propidium idodide; red), and astrocytes labeled with GFAP (blue). In HIV autopsies without encephalitis (A–C), minimal E2F1 staining is observed. In HIV autopsies with encephalitis (D–F), abundant E2F1 staining is observed but does not localize to astrocytes.G_–_L, Triple-label immunofluorescent confocal microscopy for E2F1 (green), nuclear DNA (propidium iodide; red), and MAP2 (blue). In HIV autopsies without encephalitis (G–I), minimal E2F1 staining is observed. In HIV autopsies with encephalitis (J–L), increased E2F1 staining is observed in neuronal cytoplasm [colocalization of blue (MAP2) and_green_ (E2F1) staining appears aqua_]. The_columns contain images from the following brain regions:Basal Ganglia, Hippocampus, and midfrontal cortex (Frontal Cortex). DNA staining may not be seen in all nuclei because of the staining protocol used. In these cases, MAP2 and GFAP, as cytoskeletal components, are used to delineate the cytoplasm. All micrographs are the same magnification. Scale bar, 20 μm (for all images).

Fig. 2.

Fig. 2.

ppRb localized predominantly to nuclei of neurons in HIVE. A_–_F, Triple-label immunofluorescent confocal microscopy for ppRb (green), nuclear DNA (propidium idodide;red), and astrocytes labeled with GFAP (blue). In HIV autopsies without encephalitis (A–C), minimal ppRb staining was observed. In HIV autopsies with encephalitis (D–F), abundant ppRb staining was observed but only occasionally localized to GFAP-positive astrocytes. G_–_L, Triple-label immunofluorescent confocal microscopy for ppRb (green), nuclear DNA (propidium iodide;red), and MAP2 (blue). In HIV autopsies without encephalitis (G–I), minimal ppRb staining was observed. In HIV autopsies with encephalitis (J–L), increased ppRb staining was observed in neuronal cytoplasm and nuclei [colocalization of blue(MAP2) and green (E2F1) staining appears_aqua_, and colocalization between green(E2F1) and red (DNA) appears _yellow_]. Columns contain images from the following brain regions: Basal Ganglia, Hippocampus, and midfrontal cortex (Frontal Cortex). DNA staining may not be seen in all nuclei because of the staining protocol used. In these cases, MAP2 and GFAP, as cytoskeletal components, are used to delineate the cytoplasm. All micrographs are the same magnification. Scale bar, 20 μm (for all images).

Fig. 3.

Fig. 3.

E2F1, pRb, and ppRb staining was increased in SIVE. Three SIV and three SIVE tissue sections containing the basal ganglia and midfrontal cortex were stained for E2F1, pRb, or ppRb. Anatomic features were identified and marked on the slide as cortical gray matter (CTX), caudate (CAD), and putamen (PUT). Five random 67,600 μm2 fields were chosen from each region and imaged using confocal microscopy as described in Results. The number of pixels present in the captured image was quantified as described in Materials and Methods. Averages of the pixels per field for each brain region are indicated for the three SIV (stippled) and three SIVE (diagonal striped) cases for E2F1, pRb, and_ppRb_. SDs are indicated by error bars. (∗ indicates significant difference between staining in SIV and SIVE cases for the indicated brain region; p < 0.001).

Fig. 4.

Fig. 4.

E2F1 and ppRb staining was increased in HIVE. Five HIVE and five HIV tissue sections containing the midfrontal cortex, basal ganglia, and hippocampus were stained for E2F1 or ppRb. Neuro-anatomic features were identified and marked on the slides corresponding to midfrontal cortical gray matter (GM), midfrontal cortical white matter (WM), putamen (PUT), and hippocampal CA1 region (CA1). In each case, five random 67,600 μm2 fields were chosen and imaged for each brain region. The amount of staining for a given field was quantified by counting the number of pixels present. Shown are the average number of pixels per field for each brain region within the five HIV (stippled) and five HIVE (diagonal striped) cases. SDs are indicated by error bars; ∗ indicates a significant difference between the average pixels per field in HIVE as compared with HIV (p < 0.001).

Fig. 5.

Fig. 5.

E2F1, pRb, and ppRb staining was present near SIV-infected and activated macrophages. Tissue sections from SIV and SIVE necropsies were stained for E2F1 (left column,green), pRb (middle column,green), or ppRb (right column, green) and gp110 (A_–_F, red) or HAM56 (G_–_L, red). Shown is staining from the midfrontal cortex, but similar results were seen when the caudate or putamen was used (data not shown). Little E2F1, pRb, or ppRb staining was observed in the SIV cases (A_–_C,G_–_I). Abundant staining for E2F1, pRb, and ppRb was observed near SIV-infected cells [gp110(red); _D_–_F_]. Abundant staining for E2F1, pRb, and ppRb was also observed near activated macrophages [HAM56(red);_J_–_L_]. Scale bar, 50 μm (for all images).

Fig. 6.

Fig. 6.

E2F1 and ppRb staining was present near HIV-infected and activated macrophages. Tissue sections from the basal ganglia of HIV and HIVE autopsies were stained for ppRb (A_–_C, green) or E2F1 (D_–_F, green) and HLA-DR (left and middle columns,red) or p24 (right column,red) (and data not shown). Little if any ppRb, E2F1, HLA-DR, or p24 staining was observed in HIV autopsies without encephalitis (A, D). In HIVE, abundant E2F1 and ppRb staining was noted in regions with activated macrophages (B, E) or HIVE-infected macrophages (C, F). Scale bar, 50 μm (for all images).

Fig. 7.

Fig. 7.

E2F/DNA complexes were altered in SIVE. E2F DNA binding activity was assessed in protein extracts from midfrontal cortex and basal ganglia of control, SIV, and SIVE cases by electrophoretic mobility shift assay. A, When nuclear extracts from the midfrontal cortex were assayed, E2F complexes were observed in all samples: control (C 1–C 3), SIV (SIV 1, SIV 2), and SIVE (SIVE 1_–_SIVE 3). The complexes observed in the encephalitic cases exhibited distinct properties as indicated by their further migration in the gel. Similar results were seen in extracts from basal ganglia (data not shown). B, E2F complexes were also observed in all cytoplasmic samples from the midfrontal cortex. In addition to the E2F complex present in both control and SIVE lanes, the extracts from encephalitic cases produced a slower migrating complex. Similar results were also seen in the basal ganglia (data not shown). C, To show that the complexes that we observed in the nuclear and cytoplasmic extracts were specific for the E2F DNA binding assay, we added unlabeled competitor oligonucleotides that contained the E2F site (+E2F comp) and an unrelated sequence (+unrelated comp) in increasing amounts of 50 and 150 ng. Shown are the results using the SIVE samples. The bands specific for E2F are indicated by the bracket to the left and are competed by E2F oligonucleotides but not the unrelated sequence. Experiments on control and SIV samples yielded similar results (data not shown). The first lane in each panel contains only the radiolabeled, double-stranded oligonucleotides used as probe in all other lanes.

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References

    1. Achim CL, Wiley CA. Expression of major histocompatibility complex antigens in the brains of patients with progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol. 1992;51:257–263. - PubMed
    1. Achim CL, Wiley CA. Inflammation in AIDS and the role of the macrophage in brain pathology. Curr Opin Neurol. 1996;9:221–225. - PubMed
    1. Achim CL, Wang R, Miners DK, Wiley CA. Brain viral burden in HIV-infection. J Neuropathol Exp Neurol. 1994;53:284–294. - PubMed
    1. Adams P, Kaelin WJ. Transcriptional control by E2F. Semin Cancer Biol. 1995;6:99–108. - PubMed
    1. Arendt T, Rodel L, Gartner U, Holzer M. Expression of the cyclin-dependent kinase inhibitor p16 in Alzheimer's disease. NeuroReport. 1996;7:3047–3049. - PubMed

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