Insights into potential mechanisms underlying astrocytic resistance to GC-induced apoptosis.

<p>Astrocytes show less susceptibility to GC- (<b>A</b>) and staurosporine (STA)-induced (<b>B</b>) apoptosis, as compared to neuronal cells. Both neurons and astrocytes respond to GC treatment with a significant increase of ROS (measured by fluorescent DHE nuclear translocation) (<b>C</b>); note that, as compared to neurons, astrocytes generate markedly lower levels of ROS under basal conditions and after GC treatment. The ratios of expression of mRNAs for pro- vs. anti-apoptotic members of the Bcl2 family (<i>bax</i> vs. <i>bcl-X_L</i> and <i>bcl-2</i>) are different in neurons and astrocytes (<b>D and E</b>); mRNA levels were determined by qPCR. Neurons and astrocytes also respond differentially to GC treatment in terms of their activated caspase 3 responses (measured by immunoblotting) (<b>F</b>), with astrocytes showing smaller increases in levels of activated caspase 3. Numerical data are shown as mean ± SD. * p<0.05 vs. neuron CON; # p<0.05 vs. astrocyte CON; + p<0.05 GC-treated neurons vs. GC-treated astrocytes. Scale bars: 25 µm.</p

Astrocytes are spared from GC-triggered apoptosis in primary hippocampal cultures.

<p>Hippocampal cultures were genetically tagged with either Tα-tubulin-GFP or GFAP-GFP plasmids, to identify neurons and astrocytes, respectively. Approximately 50% of astrocytes in typical cultures displayed GR immunoreactivity (<b>A–C</b>, examples shown by arrowheads). After exposure to GC or vehicle, apoptosis in the different cell populations was visualized by TUNEL and Hoechst 33342 histochemistry (<b>D–F and G–I</b>). Solid arrowheads exemplify GFP⁺ cells that entered apoptosis after GC treatment; open arrowheads indicate non-apoptotic GFP-transfected cells. Numerical data (mean ± SD) from analysis of TUNEL staining in either all cells in culture, Tα-tubulin-GFP or GFAP-GFAP sub-populations are depicted in (<b>J</b>). * p<0.05 vs. CON, # p<0.05 vs. DEX. Scale bars: 50 µm in <b>A–C</b> and 20 µm in <b>D–I</b>.</p

Enriched astrocytic cultures also respond to GC with moderate activation of caspase 3, but fail to show signs of early- or late-stage apoptosis.

<p>After re-plating, enriched astrocytic cultures were treated with GC (48 h) in medium containing either charcoal-stripped serum (data not shown) or B27 supplement (representative images in <b>A–L</b>). Enriched astrocytes responded to GC treatment DEX with moderately increased immunostaining for activated caspase 3; these cells did not enter late-stage (stage II) apoptosis, as shown by TUNEL (<b>A–F</b>). In contrast, staurosporine (STA) induced a marked activation of caspase 3 and apoptosis (<b>G–I</b>). The immunocytochemical results shown for activated caspase 3 in <b>A–I</b> were confirmed by immunoblotting (J). Staurosporine, but not GC, treatment of enriched astrocytic cultures significantly increased levels of immunoreactive phospho-H2A.X, a marker of early apoptosis, as shown by immunoblotting studies (<b>K</b>). Similarly, astrocytes exposed to STA, but not GC, displayed high molecular weight (HMW) DNA fragments, when lysates where subjected to pulse-field gel electrophoresis (PFGE) (<b>L</b>); all lanes were loaded with DNA from the same number of astrocytes, and arrow indicates 50 kb HMW DNA fragments. Scale bars: 50 µm.</p

Temporal mRNA expression profiles of growth- and survival-regulating peptides in GC-treated astrocytes.

<p><b>A,</b> molecules implicated in the extrinsic death pathway; FasL mRNA was transiently increased, TRAIL and TWEAK mRNAs showed transient reductions and TNFα mRNA showed a sustained reduction after application of GC. <b>B</b>, expression patterns of the mRNAs encoding the neurotrophic factors BDNF (transient upregulation) and NGF (sustained downregulation) after GC treatment. <b>C</b>, levels of mRNA encoding for the mitogenic factors bFGF (increased) and VEGF (decreased) following exposure of astrocytes to GC. In all analyses, <i>gapdh</i> and <i>actin</i> served as housing-keeping gene controls. Values shown derive from 3 independent experiments (mean ± SD).</p

GC treatment drives neurons but not astrocytes into apoptosis in neonatal and adult rats.

<p><b>A</b>–<b>L</b>, Representative confocal images showing double staining for calbindin-D28K (<b>A</b>, <b>D</b>, <b>G</b>, <b>J</b>) and phosho-H2A.X (<b>B</b>, <b>E</b>, <b>H</b>, <b>K</b>) in the dentate gyrus of control and GC-treated neonatal (<b>A</b>–<b>C, G</b>–<b>I</b>) and adult (<b>D</b>–<b>F, J</b>–<b>L</b>) rats. Hoechst 33342 staining was used to identify cell nuclei and to help delineate the SGZ and GCL. Arrows indicate the representative positive phosphor-H2A.X staining in calbindin-D28K positive neurons. <b>M</b> and <b>O</b>, are representative images showing double-staining of GFAP and phospho-H2A.X in the <i>stratum radiatum</i> of the hippocampal CA3 and CA1 subfields (CA3-r, CA1-r) in GC-treated neonatal (<b>M</b>) and adult (<b>O</b>) rats. Arrowheads indicate GFAP-positive astrocytes that were negative for phospho-H2A.X, an early marker of apoptosis. Arrows indicate the representative phosphor-H2A.X staining in GFAP-negative cells. <b>N</b> and <b>P</b> illustrate the significant increase of apoptosis in calbindin-positive neurons, but not GFAP-labeled astrocytes, in neonatal (<b>N</b>) and adult (<b>P</b>) rats treated with GC (dexamethasone, 200 µg/kg/d on days 1–3, tapering to 100 µg/kg/d on days 4–7). The counts are from all hippocampal subregions displaying positive signal for calbindin (granule cell layer of DG) or GFAP (molecular and polymorphic cell layers of DG, and the <i>strata oriens</i> and <i>radiatum</i> of CA1-CA3). <b>Q</b>, Stacking figure showing that GC treatment does not induce apoptosis in astrocytes in any hippocampal subfield, as indicated by double-staining of GFAP and phopho-H2A.X. The relative numbers (%) of phospho-H2A.X⁺/GFAP⁺ cells relative to total GFAP⁺ cells in each subfield were calculated; each value was used to create the stacking figure in which each column represents the % of apoptotic events in astrocytes in each subfield <i>vs.</i> the total number of apoptotic events in astrocytes in the whole hippocampal formation (100%). o, <i>stratum oriens</i>; m, molecular layer; p, polymorphic cell layer. r, <i>stratum radiatum</i>. * p<0.05 compared to CON. Scale bars: 20 µm.</p

Conditioned medium from GC-treated astrocytes influences neurogenesis in hippocampal cultures.

<p><b>A</b>, Treatment of primary hippocampal cultures with DCM (conditioned medium harvested from GC-treated astrocytes) attenuated the mitotic effects of CM (conditioned medium from normal astrocytes), measured by BrdU incorporation and Ki67 immunostaining. Conditioned media were prepared by either serial concentration (to reduce dexamethasone concentrations to <3.10⁻¹¹ M; CM and DCM) or physical absorption of dexamethasone (CM-hydro and DCM-hydro). The effects of DCM could not be antagonized with GR antagonist, RU38486 (10⁻⁸ M). Neither dexamethasone (3.10⁻¹¹ M) nor RU38486 (10⁻⁸ M) exerted significant effects on neural proliferation, and DCM-hydro attenuated the mitotic effects of CM-hydro to similar extents. <b>B</b>, CM caused a dose-dependent increase in neural proliferation, whereas DCM reduced the stimulatory effects of CM. <b>C</b>, Apoptosis in primary hippocampal cells in culture were reduced by CM and DCM to similar extents. Numerical data represent mean ± SD. * p<0.05 vs. CON, + p<0.05 vs. corresponding CM group.</p

Caspase 3 is activated by GC-treated in astrocytes grown in mixed hippocampal cultures.

<p>As compared to vehicle-treated GFAP-GFP-labeled astrocytes (<b>A–C</b>), those treated with GC (<b>D–F</b>) displayed moderate levels of activated caspase 3. Open arrowheads indicate GFAP-GFP⁺/cleaved caspase 3⁻ staining; solid arrowheads indicate GFAP-GFP+/cleaved caspase 3⁺ cells. The GR antagonist RU38486 significantly attenuated GC-stimulated activation of caspase 3 (<b>G</b>). Extended exposure of cultures to GC (48–144 h) led to a progressive increase in activated caspase 3 immunoreactivity in GFAP-GFP tagged astrocytes (<b>H</b>), without causing significant apoptosis monitored by TUNEL and Hoechst staining (not shown). All numerical data represent mean ± SD. * p<0.05 vs. CON, # p<0.05 vs. GC. Scale bar: 50 µm.</p

Anti-proliferative actions of GC in astrocytes are GR-dependent.

<p><b>A,</b> Representative immunostaining of GFAP in the enriched astrocytic culture. <b>B–E</b>, are representative images showing BrdU incorporation in control (<b>B, C</b>) and GC-treated (dexamethasone, at 10⁻⁵ M for 48 h in medium with charcoal-stripped serum) astrocytes (<b>D, E</b>). Hoechst 33342 counterstaining demonstrates comparable cell densities. BrdU (20 µM) was added to cultures 12 h before fixation. <b>F,</b> shows that the anti-proliferative actions of GC are counteracted by addition of the GR antagonist, RU38486 (10⁻⁵ M). <b>G</b>, Representative Western blots showing GC (dexamethasone; 10⁻⁵ M in medium supplemented with charcoal-stripped serum; 48 h) regulation of various key regulators of the cell cycle in cultured astrocytes; the semi-quantitative (n = 4) data from these immunoblotting experiments are shown in <b>H.</b> Note that while GC treatment downregulates cyclin D1 protein expression, the treatment results in a concomitant increase in the levels of the cell cycle inhibitor, p27. Cyclin E and CDK6 expression levels are not changed after exposing astrocytes to GC. Numerical data represent mean ± SD. * p<0.05 vs. CON, # p<0.05 vs. GC. Scale bar: 50 µm.</p