Genetic deletion of TNF receptor suppresses excitatory synaptic transmission via reducing AMPA receptor synaptic localization in cortical neurons

Abstract

The distribution of postsynaptic glutamate receptors has been shown to be regulated by proimmunocytokine tumor necrosis factor α (TNF-α) signaling. The role of TNF-α receptor subtypes in mediating glutamate receptor expression, trafficking, and function still remains unclear. Here, we report that TNF receptor subtypes (TNFR1 and TNFR2) differentially modulate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) clustering and function in cultured cortical neurons. We find that genetic deletion of TNFR1 decreases surface expression and synaptic localization of the AMPAR GluA1 subunit, reduces the frequency of miniature excitatory postsynaptic current (mEPSC), and reduces AMPA-induced maximal whole-cell current. In addition, these results are not observed in TNFR2-deleted neurons. The decreased AMPAR expression and function in TNFR1-deleted cells are not significantly restored by short (2 h) or long (24 h) term exposure to TNF-α. In TNFR2-deleted cells, TNF-α promotes AMPAR trafficking to the synapse and increases mEPSC frequency. In the present study, we find no significant change in the GluN1 subunit of NMDAR clusters, location, and mEPSC. This includes applying or withholding the TNF-α treatment in both TNFR1- and TNFR2-deleted neurons. Our results indicate that TNF receptor subtype 1 but not 2 plays a critical role in modulating AMPAR clustering, suggesting that targeting TNFR1 gene might be a novel approach to preventing neuronal AMPAR-mediated excitotoxicity.

Figure 1.

TNFR1 deletion decreases the number of AMPAR clusters. A) Cortical neurons were labeled at 7 DIV by immunofluorescence with antibodies against TNFR1 and TNFR2. TNFR1 (green) and TNFR2 (red) expression is diffuse, little clustering on the dendrite and somatic membrane. B) Immunostaining directed GluA1 subunit of AMPAR is visualized in WT, TNFR1^−/−, and TNFR2^−/− cortical neurons with and without TNF-α treatment. C) Statistical analysis shows that, in comparison with WT cells, the number of GluA1 clusters significantly decreases in TNFR1^−/− neurons, but not in TNFR2^−/− cells (P>0.05). With TNF-α treatment for 2 and 24 h, the number of GluA1 clusters significantly increases in WT and TNFR2^−/− neurons. However, no significant change in GluA1 clusters is observed in the cells with TNFR1 deletion when exposed to TNF-α. *P < 0.05; **P < 0.01. D) Western blot shows no obvious changes of GluA1 expression levels among WT, TNFR1^−/−, and TNFR2^−/− cortical neurons without (control) or with TNF-α treatment for 24 h. Scale bars = 5 μm.

Figure 2.

TNFR1 deletion reduces the synaptic location of AMPA receptors. A) Representative photograph shows the colocalization (yellow) of PSD-95 (red), a postsynaptic protein, and GluA1 clusters (green) with and without TNF-α application. B) A significant decrease in the percentage of the colocalization of GluA1 clusters to PSD-95 is observed in TNFR1^−/− neurons. With the treatment of TNF-α for 2 h or 24 h, the percentage of synaptic GluA1 clustering significantly is increased in WT and TNFR2^−/− neurons, whereas no significant changes appeared in TNFR1^−/− neurons (P>0.05). C) Representative photograph shows the colocalization of SYP (red), a presynaptic protein, and GluA1 (green) with and without TNF-α application. D) A significant increase in the percentage of the colocalization of GluA1 clusters and SYP staining is observed in WT and TNFR2^−/− neurons, but no significant changes appeared in TNFR1^−/− neurons with the treatment of TNF-α for 2 or 24 h. *P < 0.05.

Figure 3.

TNFR1 deletion decreases the synaptic localization of AMPAR in focal cerebral ischemic mice. A) Gallyas silver stain shows the left demyelinating infarct region (red arrow) after the ligation of the left internal carotid artery. B) Hematoxylin histology shows an injury tissue structure following the left internal carotid artery ligation. C) Double immunoreactive staining shows an apparently increased synaptic localization of GluA1 (yellow dots indicate the overlaps of the GluA1 and SYP) in the ipsilateral ischemic tissues in comparison with the contralateral normal control of WT mice with ischemic injury. Little localization of GluA1 to synapses is seen in the contralateral brain tissues of ischemia, and there are no obvious increases in the double-staining of GluA1 and SYP in the ipsilateral tissues of the ischemic TNFR1^−/− mice. Scale bars = 100 μm (B); 10 μm (C).

Figure 4.

TNFR1 deletion decreases synaptic excitatory transmission and prevents TNF-α-induced increase of synaptic excitatory transmission. A) Representative imagography shows mEPSC recording from primary cortex neurons of WT, TNFR1^−/−, and TNFR2^−/− mice in the absence and presence of TNF-α application. B) Average mEPSC peak amplitude before and after TNF-α treatment. C) Cumulative probability of the peak amplitude and frequency of mEPSC before and after TNF-α treatment. *P < 0.01.

Figure 5.

TNFR1 deletion decreases AMPAR function in whole-cell recordings. A) Representative whole-cell traces of AMPA (100 μM)-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons. B) Calculated peak (I_p), steady state (I_s), and kinetics (ratio of I_s vs. I_p) values of AMPA-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons. C) Representative whole-cell traces of AMPA (100 μM)-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons untreated and treated with TNF-α. D) Comparison of calculated I_p values of AMPA-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons untreated and treated with TNF-α. **P < 0.01.

Figure 6.

TNF-α treatment does not alter NMDAR whole-cell function. A) Group of representative photographs shows GluN1 subunit clusters of NMDARs in WT, TNFR1^−/−, and TNFR2^−/− cortical neurons with and without TNF-α treatment. Scale bar = 5 μm. B) Quantitative analysis of GluN1 clusters per 20 μm proximal dendrites shows no significant changes (P>0.05) in all three types of WT, TNFR1^−/−, and TNFR2^−/− neurons with and without TNF-α treatment for 2 or 24 h. C) Representative whole-cell traces of NMDA (100 μM)-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons. D) I_p, I_s, and the ratio of I_s vs. I_p values of NMDA-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons. E) Representative whole-cell traces of NMDA (100 μM)-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons untreated and treated with TNF-α. F) Comparison of calculated I_p values of NMDA-induced inward currents from WT, TNFR1^−/−, and TNFR2^−/− neurons untreated and treated with TNF-α.