Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha

Abstract

The proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) causes a rapid exocytosis of AMPA receptors in hippocampal pyramidal cells and is constitutively required for the maintenance of normal surface expression of AMPA receptors. Here we demonstrate that TNFalpha acts on neuronal TNFR1 receptors to preferentially exocytose glutamate receptor 2-lacking AMPA receptors through a phosphatidylinositol 3 kinase-dependent process. This increases excitatory synaptic strength while changing the molecular stoichiometry of synaptic AMPA receptors. Conversely, TNFalpha causes an endocytosis of GABA(A) receptors, resulting in fewer surface GABA(A) receptors and a decrease in inhibitory synaptic strength. These results suggest that TNFalpha can regulate neuronal circuit homeostasis in a manner that may exacerbate excitotoxic damage resulting from neuronal insults.

Figures

Figure 1.

TNFα, unlike other cytokines, acts on neurons to increase surface expression of AMPARs. A, Representative micrographs from sister cultures of nonpermeabilized isolated neurons immunostained for surface expression of the AMPA subunit GluR1. The cells on the right were treated with 60 nm TNFα for 15 min. B, Group data from untreated or TNFα-treated cultures (n = 70 for both conditions), showing a substantial increase in surface GluR1 staining. C, Composite data of surface GluR1 from cultures treated with TNFα (60 nm; n = 214), IL-1β (50 ng/ml; n = 140), IL-6 (32 ng/ml; n = 159), or IL-10 (40 ng/ml; n = 130). D, Group data of surface GluR1 from cultures treated for 24 h with sTNFR (10 μg/ml; n = 59) or an antibody against IL-1β (40 μg/ml; n = 72). For this and all subsequent figures, *p < 0.05; **p < 0.001; ***p < 0.0001 when compared with untreated cultures.

Figure 2.

TNFα acts via TNFR1 to increase surface AMPARs. A, Representative micrographs and composite data from cultures treated for 24 h with sTNFR (10 μg/ml; n = 54), which decreased the surface expression of GluR1 relative to untreated control cells. Treatment for 24 h with a neutralizing antibody for TNFR1 (TNFR1 N; 5 μg/ml; n = 66) also causes a similar decrease in GluR1 surface expression. Neutralizing antibodies for TNFR2 (TNFR2 N; 15 μg/ml; n = 84) were ineffective at decreasing GluR1 surface expression. B, Sample micrographs and composite data demonstrating that an activating antibody for TNFR1 (TNFR1 A; 2 μg/ml; n = 81) increases the surface expression of GluR1 to a similar degree as sister cultures treated with TNFα (n = 69), whereas an activating antibody for TNFR2 (TNFR2 A; 2 μg/ml; n = 88) was ineffective at increasing surface GluR1.

Figure 3.

PI3 kinase is required for the increase in surface AMPARs induced by TNFα. A, Pretreatment (2 h) with inhibitors for PKA (PKI, 1 μm), CaMKII (KN-93, 20 μm), COX-2 (aspirin, 10 μm), p38 MAP kinase [SB 20 3580 (SB), 50 μm], or p42-44 MAP kinase [PD 98059 (PD), 50 μm] all failed to prevent the TNFα-mediated increase in surface GluR1 (black bars; n = 40-85 per condition) relative to untreated sister cultures (gray bars). B, Group data indicating that pretreatment with wortmannin (100 nm; n = 172) or LY 294,002 (50 μm; n = 60) prevented the TNFα-induced increase in surface expression of GluR1 (black bars) compared with untreated sister cultures (gray bars). C, Sample micrographs showing the surface expression of GluR1 from sister cultures: untreated, treated with 60 nm TNFα, or pretreated with wortmannin and then treated with TNFα.

Figure 4.

TNFα does not increase the surface expression of GluR2. A, Representative micrographs from cultured neurons double labeled for surface GluR1 and GluR2, after treatment with TNFα. Despite a clear increase in GluR1 surface expression, GluR2 surface levels are not increased relative to untreated cultures. B, Quantification of all doubled-labeled cells, showing a significant increase in GluR1 surface expression after TNFα treatment (black bars; n = 177) compared with untreated cells (gray bars; n = 156), whereas the same cells had no significant increase in GluR2 surface expression relative to untreated controls. C, Representative blots from surface biotinylation experiments from sister cultures, probed for GluR1 or GluR2, after treatment with TNFα. D, Quantification of all experiments for the levels of biotinylated receptors (n = 7 cultures for GluR1, 6 for GluR2).

Figure 5.

TNFα treatment induces surface expression of GluR2-lacking AMPARs at synapses. A, Voltage-clamp recording of mEPSCs from untreated and TNFα-treated cultured hippocampal neurons, before and during application of HPP-spermine (10 μm). Traces at right show the average mEPSC before and after HPP-spermine application. Ctrl, Control. B, Group data showing the normalized mEPSC amplitude after HPP-spermine for control (n = 10) and TNFα-treated cells (n = 12). C, Cumulative probability graphs from control cells (left) and TNFα-treated cells (right) of normalized mEPSC amplitudes before (black) and after (gray) HPP-spermine application. D, Group data of average mEPSC amplitude of untreated (n = 13) and TNFα-treated (n = 15) cells, showing a significant increase in average mEPSC amplitude. E, The cumulative distribution of mEPSC amplitudes (left graph) demonstrates a significant rightward shift of TNFα-treated mEPSC amplitudes (black) compared with untreated cells (gray). The right graph shows the cumulative distribution of mEPSC amplitudes after the application of HPP-spermine, in which there is no significant difference between the untreated (gray) and TNFα-treated (black; p > 0.5) cells.

Figure 6.

TNFα decreases the surface expression of GABAA receptors. A, Representative micrographs and group data from cells double labeled for surface GluR1 and the β2/3 subunit of the GABAA receptors (GABAR). In cells showing a robust increase in GluR1 after TNFα treatment, there was a small but significant decrease in surface expression of GABAR compared with cells from untreated sister cultures. B, Sample images and composite data from cells labeled for endocytosed GluR1 or GABAR. TNFα treatment (black bars) increased the endocytosis of GABAR but not GluR1 relative to cells from untreated cultures (gray bars).

Figure 7.

TNFα increases the ratio of excitatory to inhibitory synaptic transmission in acute hippocampal slices. A, TNFα increases mEPSC amplitude. Left, Representative average mEPSCs from cells treated with TNFα and untreated slices. Middle, Composite data of average mEPSC frequency and amplitude of cells from untreated (gray; n = 10) and TNFα-treated (black; n = 12) slices. Right, Cumulative probability of mEPSC amplitude from the same cells, showing a significant rightward shift in TNFα-treated cells. B, Representative mIPSCs (left), composite average mIPSC frequency and amplitude (middle), and cumulative probability of mIPSC amplitudes (right) of cells from TNFα-treated (black; n = 12) and untreated (gray; n = 13) hippocampal slices. C, Representative traces of compound postsynaptic potentials of cells from TNFα-treated slices and untreated slices from the same animal. TNFα-treated cells (n = 11) had a significantly higher ratio of the EPSP to IPSP amplitude than untreated cells (n = 10).