Rapid tumor necrosis factor alpha-induced exocytosis of glutamate receptor 2-lacking AMPA receptors to extrasynaptic plasma membrane potentiates excitotoxicity

Abstract

The postinjury inflammatory response in the CNS leads to neuronal excitotoxicity. Our previous studies show that a major component of this response, the inflammatory cytokine tumor necrosis factor alpha (TNFalpha), causes a rapid increase in AMPA glutamate receptors (AMPARs) on the plasma membrane of cultured hippocampal neurons. This may potentiate neuron death through an increased vulnerability to AMPAR-dependent excitotoxic stress. Here, we test this hypothesis with an in vitro lactose dehydrogenase death assay and examine in detail the AMPAR surface delivery time course, receptor subtype, and synaptic and extrasynaptic distribution after TNFalpha exposure. These data demonstrate that surface levels of glutamate receptor 2 (GluR2)-lacking Ca2+-permeable AMPARs peak at 15 min after TNFalpha treatment, and the majority are directed to extrasynaptic sites. TNFalpha also induces an increase in GluR2-containing surface AMPARs but with a slower time course. We propose that this activity contributes to excitotoxic neuron death because TNFalpha potentiation of kainate excitotoxicity is blocked by a Ca2+-permeable AMPAR antagonist [NASPM (1-naphthyl acetyl spermine)] and a specific phosphoinositide 3 kinase (PI3 kinase) inhibitor (LY294,002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]) previously shown to block the TNFalpha-induced increase in AMPAR surface delivery. This information forms the basis for future in vivo studies examining AMPAR-dependent potentiation of excitotoxic neuron death and dysfunction caused by TNFalpha after acute injury and during neurodegenerative or neuropsychiatric disorders.

Figure 1.

TNFα induces rapid and transient delivery of endogenous GluR1 to the plasma membrane as shown by quantitative immunofluorescence microscopy. A, B, The 17–21 div hippocampal neurons were either treated with TNFα for 15, 30, 60 min or left untreated (control). Neurons were fixed without membrane permeabilization and processed for indirect immunofluorescence using extracellular epitope-directed anti-GluR1 (A) and anti-GluR2 (B) antibodies. Images were acquired using wide-field fluorescence microscopy and were thresholded and quantified using MetaMorph software (see Materials and Methods). Compiled data for GluR1 and GluR2 is expressed in bar graph (C). Error bars indicate SEM. *p < 0.01; **p < 0.05; n = 56–87 cells from eight different experiments.

Figure 2.

TNFα induces a rapid delivery of endogenous GluR1 and a more delayed delivery of GluR2 to the plasma membrane as shown by biochemical analysis. A, Control, untreated neurons or neurons treated with TNFα for 15 min or glycine [long-term potentiation chemical induction protocol (chemLTP); another method for inducing surface AMPAR delivery] and surface biotinylated. A total of 100 μg of soluble protein was processed for streptavidin-agarose pulldowns (see Materials and Methods). A total of 5 μg of total lysate from each condition was immunoblotted for total GluR1 and actin as loading controls (Loading Control). B, Immunoblots in A were quantified by densitometry and normalized to lysate controls, and the compiled data were graphed. *p < 0.01; n = 7. C, Control neurons or neurons were treated with TNFα for 15, 30, or 60 min and biotinylated as above. Streptavidin-agarose was used to precipitate and purify surface biotinylated proteins (left panels, surface receptor); supernatant from these precipitates was immunoprecipitated with either anti-GluR1 or anti-GluR2 antibodies (right panels, intracellular receptor). Precipitates were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the antibodies to the proteins indicated. A total of 5 μg of lysate (bottom panel) from each condition was immunoblotted as loading controls. D, Immunoblots of surface receptors in C were quantified by densitometry and normalized to actin lysate controls, and the compiled data were graphed. E, Immunoblots of intracellular receptors in C were quantified by densitometry and normalized to actin lysate controls, and the compiled data were graphed. Error bars indicate SEM. *p < 0.05; n = 4 for each condition.

Figure 3.

Live confocal imaging of SEP-GluR1-transfected hippocampal neurons treated with TNFα demonstrates exocytosis and rapid surface expression of intracellularly derived SEP-GluR1. The 14 div rat hippocampal neurons AMAXA transfected with SEP-GluR1 were imaged live at 37°C by scanning confocal microscopy (each image is a maximum projection of 11 images taken at 0.5 μm intervals). Fluorescent signal from SEP-GluR1 is visualized only at the neuron plasma membrane. To maximize detection of SEP-GluR1 recently delivered to the neuron surface, 1 min of fluorescent illumination was used to selectively bleach surface SEP-GluR1 already at the plasma membrane (see Materials and Methods). The left three panels show a process from a control live neuron before bleaching (Pre-Bleach), after 1 min of bleaching (Post-Bleach), and 15 min of recovery after bleaching (15 min Recov.). The right three panels depict a neuron imaged before bleaching (Pre-Bleach), after 1 min of bleaching (Post-Bleach), and allowed to recover in presence of TNFα (15 min Recov.). The arrows highlight example areas where AMPARs are either added to existing puncta of surface AMPARs (right arrow) or to areas where new AMPAR puncta appear (left arrow). Image quantitation of four experiments per condition demonstrates ∼50% increase in TNFα-treated neurons (percentage change in surface fluorescence, 148 ± 9%) compared with control, untreated neurons (percentage change in surface fluorescence, 94 ± 12%; n = 4 neurons; p < 0.05). Scale bar, 5 μm.

Figure 4.

Extrasynaptic surface GluR1 is significantly increased by TNFα treatment. A, Hippocampal neurons were costained with the synaptic marker anti-PSD-95 (green) and anti-GluR1 (red) and imaged by indirect immunofluorescence confocal microscopy. The image at the right displays overlaid images with overlapping fluorescence in yellow. The arrows in the top panels indicate colocalization of GluR1 with PSD-95. The arrowheads in the bottom panels indicate GluR1 not colocalizing with PSD-95. Compiled image quantitation from three experiments demonstrates an increase of 19 ± 7% compared with control (p < 0.001; n = 16). Scale bar, 5 μm. B, To biochemically isolate surface AMPARs, hippocampal cultures were treated with TNFα for 15 min or not treated (control), surface proteins were biotinylated, and neurons were subjected to biochemical synaptic fractionation based on detergent solubility (see Materials and Methods). Total cell lysates from these fractionations (5 μg) were immunoblotted for the synaptic marker NR1 (NMDA receptor subunit) and PSD95, which were enriched in the synaptic fraction, whereas GluR1 is mostly extrasynaptic. C, Surface biotinylated proteins from both extrasynaptic and synaptic fractions were purified by streptavidin-agarose and immunoblotted with anti-GluR1 and anti-GluR2 antibodies. D, Graph of actin-normalized compiled data from immunoblots of TNFα-treated neurons performed as in C. Surface GluR1 is significantly enriched at both extrasynaptic (black bars; *p < 0.001; n = 8) fractions and synaptic fractions (white bars; *p < 0.001; n = 5), whereas GluR2 levels do not change. Error bars indicate SEM.

Figure 5.

TNFα induces extrasynaptic dendritic accumulation of surface GluR2-lacking GluR1 AMPARs. A, Top, High-magnification, single-plane, confocal images of control, untreated hippocampal dendrites costained for surface GluR1 (red) and GluR2 (green) as in Figure 1. The arrows highlight examples of overlapping colocalization (yellow regions in overlaid image). Bottom, High-magnification images of TNFα-treated neurons stained as above. The arrowheads highlight regions where GluR1 does not overlap with GluR2. Image quantitation demonstrates a decrease of 28 ± 5% compared with control (p < 0.001; n = 15, 3 experiments). Scale bar, 5 μm. B, To biochemically isolate GluR2-lacking AMPARs, hippocampal synaptic lysates were prepared as in Figure 4 and immunoprecipitated with polyclonal anti-GluR2 antibodies to remove GluR2-containing AMPARs. The remaining biotinylated surface proteins in the supernatant were purified by streptavidin-agarose. Isolated proteins were immunoblotted for GluR1. C, Extrasynaptic lysates were immunoprecipitated with anti-GluR2 antibodies followed by streptavidin-agarose purification. Isolated proteins were immunoblotted for GluR1. D, Data from experiments shown in B and C were quantified and compiled. Surface biotinylated extrasynaptic extracts of TNFα-treated cultures showed a significant increase in surface GluR2-lacking GluR1 AMPARs (129 ± 9% of control; *p < 0.001; n = 8), whereas no significant change was detected in surface GluR2-lacking GluR1 AMPARs in synaptic extracts of TNFα-treated neuron cultures (86 ± 14% of control; n = 5). Error bars indicate SEM.

Figure 6.

Hippocampal neurons preexposed to TNFα display increased vulnerability to excitotoxicity. Neurons were left untreated (control) or pretreated with TNFα (TNF) alone or in combination with 50 μm LY294,002 (LY), 50 μm NASPM (NASPM) for 15 min. Kainate at 20 μm was then added to untreated (KA), TNFα-pretreated (TNF+KA), drug pretreated (LY+KA; NASPM+KA), or neurons pretreated with drug and TNFα (LY+KA+TNF; NASPM+KA+TNF) and incubated for 18 h. The graph is the compiled data of lactose dehydrogenase activity released from dead or dying cells into the incubation media normalized to untreated controls (see Materials and Methods). n = 4 experiments, 4 samples per experiment; *p < 0.05 compared with kainate alone. Error bars indicate SEM.