A novel protein complex in membrane rafts linking the NR2B glutamate receptor and autophagy is disrupted following traumatic brain injury

Abstract

Hyperactivation of N-methyl-D-aspartate receptors (NRs) is associated with neuronal cell death induced by traumatic brain injury (TBI) and many neurodegenerative conditions. NR signaling efficiency is dependent on receptor localization in membrane raft microdomains. Recently, excitotoxicity has been linked to autophagy, but mechanisms governing signal transduction remain unclear. Here we have identified protein interactions between NR2B signaling intermediates and the autophagic protein Beclin-1 in membrane rafts of the normal rat cerebral cortex. Moderate TBI induced rapid recruitment and association of NR2B and pCaMKII to membrane rafts, and translocation of Beclin-1 out of membrane microdomains. Furthermore, TBI caused significant increases in expression of key autophagic proteins and morphological hallmarks of autophagy that were significantly attenuated by treatment with the NR2B antagonist Ro 25-6981. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin-1 in membrane rafts after TBI.

FIG. 1.

TBI induces changes in NR2B signaling intermediates and autophagy proteins in the injured cortex. (A) Immunoblot analysis of NR2B, pCaMKII, and PSD95. Rats were subjected to moderate TBI, and cerebral cortices were harvested at the indicated times after injury. Levels of NR2B and pCaMKII were increased at 1 and 4 h after injury. (B) Immunoblot analysis of Beclin-1, LC3, ATG5, ATG7, pMTOR, p-p70 S6K, MTOR, and p70 S6K. Levels of Beclin-1, LC3, ATG5, and ATG7 significantly increase after injury but with different time courses. In contrast, levels of the negative regulator of autophagy pMTOR and p-p70 S6K show significant decreases after trauma. *p ≤ 0.05 versus sham at 15 min. MTOR and p70 S6K were used as internal standards. β-Tubulin was used as a loading control. n = 8 for each group.

FIG. 2.

Beclin-1 and LC3 autophagy proteins are present in cortical neurons, and TBI induces alterations in their expression patterns. Confocal images show cortical neurons near the injury epicenter of sham and injured brains at 24 h after TBI. Sections were stained for Beclin-1 and LC3 (red) and the neuronal markers NeuN and MAP2 (green). Beclin-1 immunoreactivity (red and merged, rows 1 and 3) was observed in a diffuse pattern in the perinuclear region of NeuN-positive cells and processes of MAP2-positive cells. At 24 h after TBI, there was increased immunostaining of Beclin-1 in cortical neurons that was distributed in a punctate pattern in the cell soma and in processes (rows 2 and 4). LC3 immunoreactivity was present as punctate inclusions in NeuN-positive cells in sham-operated animals (red and merged, row 5) and showed increased staining 24 h after injury (red and merged, row 6). Scale bar = 10 μm.

FIG. 3.

Ultrastructural evidence for autophagy in normal cortical neurons and 3 days after TBI in adult male rats. Transmission electron micrographs of cortical neurons from injured animals near the injury epicenter at 3 days after trauma (A,B). Arrowhead points to double membrane vacuoles (A). Arrows point to initial autophagic vacuoles (AVi); degradative autophagic vacuoles (AVd). (C) Quantification of number of AVi and AVd per cortical neuron in sham and injured brains at 3 days after trauma. *p ≤ 0.05 versus sham at 15 min. Scale bar = 500 nm. n = 6 for each group.

FIG. 4.

Partitioning of NR2B, signaling intermediates, and Beclin-1 in membrane rafts in the normal and injured cortex. (A) Density gradient profile of detergent-resistant membranes from cortices of sham-operated animals. Cortical membranes were extracted with 1.0% Triton X-100 at 4°C and separated on density gradients formed from 40%, 30%, and 5% sucrose. Eight fractions (from top to bottom of the gradient) were immunoblotted for the indicated proteins. Fraction 2 of the gradient was enriched for caveolin 1, flotillin 1, Thy 1, and GM1, indicating the membrane raft containing fraction (R), while fraction 8 contained the Na⁺/K⁺-pump, indicating the non-membrane raft or soluble fraction (TS). (B) TBI induces recruitment of NR2B and pCaMKII into membrane rafts and translocation of Beclin-1 out of membrane rafts. Equal aliquots of the fractions were subjected to SDS-PAGE, and the protein distribution was assessed by immunoblotting using specific antibodies against NR2B, pCaMKII, PSD 95, Beclin-1, and caveolin 1. *p ≤ 0.05 versus sham at 15 min. n = 8 for each group.

FIG. 4.

Partitioning of NR2B, signaling intermediates, and Beclin-1 in membrane rafts in the normal and injured cortex. (A) Density gradient profile of detergent-resistant membranes from cortices of sham-operated animals. Cortical membranes were extracted with 1.0% Triton X-100 at 4°C and separated on density gradients formed from 40%, 30%, and 5% sucrose. Eight fractions (from top to bottom of the gradient) were immunoblotted for the indicated proteins. Fraction 2 of the gradient was enriched for caveolin 1, flotillin 1, Thy 1, and GM1, indicating the membrane raft containing fraction (R), while fraction 8 contained the Na⁺/K⁺-pump, indicating the non-membrane raft or soluble fraction (TS). (B) TBI induces recruitment of NR2B and pCaMKII into membrane rafts and translocation of Beclin-1 out of membrane rafts. Equal aliquots of the fractions were subjected to SDS-PAGE, and the protein distribution was assessed by immunoblotting using specific antibodies against NR2B, pCaMKII, PSD 95, Beclin-1, and caveolin 1. *p ≤ 0.05 versus sham at 15 min. n = 8 for each group.

FIG. 5.

Membrane raft associated NR2B signaling complex is altered following TBI. (A) Co-immunoprecipitation with anti-NR2B of membrane raft fractions of cortical lysates obtained from sham animals and at 15 min to 48 h after TBI. NR2B immunoprecipitates were blotted for NR2B, PSD 95, Shank, Homer, Beclin-1, pCaMKII, and Flotillin 1. In sham animals, anti-NR2B immunoprecipitated NR2B, PSD 95, Shank, Homer, and Beclin-1, thus indicating association of these proteins in a multiprotein complex. TBI-induced disassociation of this multiprotein signaling complex within 15 min. (B) Co-immunoprecipitation using anti-NR2B demonstrated similar protein associations of NR2B signaling intermediates in cortical membrane rafts in the contralateral hemisphere of sham animals. However, the NR2B multiprotein signaling complex was not present in Triton X-100 insoluble fractions (TS) isolated from cortical lysates. (C) Reciprocal co-immunoprecipitation with Beclin-1 of membrane rafts from sham and injured cortices, and rafts obtained from injured animals. Anti-Beclin-1 immunoprecipitated Beclin-1 and NR2B in sham animals only, further indicating association of these proteins in a multiprotein complex in membrane rafts.

FIG. 6.

NR2B antagonist but not NR2A antagonist inhibits TBI–induced increases in expression of NR2B signaling intermediates in the injured cortex. Representative immunoblots of cortical lysates from sham animals and injured animals injected intravenously with 1 mg/ml/kg of Ro 25-6981 or NVP0AAM077 at 30 min prior to TBI. (A) Ro 25-6981 (NR2B antagonist) blocked TBI induced increases in NR2B signaling intermediates. (B) NVP-AAM077 (NR2A antagonist) had no effect on expression of NR2B signaling intermediates, where protein profiles were similar to those in untreated animals subjected to TBI (Fig. 1). *p ≤ 0.05 versus sham at 15 min. n = 6 for each group.

FIG. 7.

NR2B antagonist but not NR2A antagonist inhibits TBI–induced increases in expression of autophagic proteins in the injured cortex. Representative immunoblots of cortical lysates from sham animals and injured animals injected intravenously with 1 mg/ml/kg of Ro 25-6981 or NVP-AAM077 at 30 min prior to TBI. (A) Pretreatment with the Ro 25-6981 (NR2B antagonist) blocked the TBI-induced increases in protein expression of autophagic proteins after TBI. (B) NVP-AAM077 (NR2A antagonist) did not alter expression of autophagic proteins after TBI, where protein profiles in both signaling pathways were similar to untreated animals subjected to TBI (Fig. 1). *p ≤ 0.05 versus sham at 15 min. n = 6 for each group.

FIG. 8.

NR2B but not NR2A antagonist inhibits TBI–induced alterations of protein associations of NR2B signaling proteins in membrane rafts. Co-immunoprecipitation with anti-NR2B of membrane raft fractions of cortical lysates obtained from sham animals and animals pretreated with injected intravenously with 1 mg/ml/kg of Ro 25-6981 (A) or 1 mg/ml/kg of NVP-AAM077 (B) at 30 min prior to injury. Membrane rafts were isolated from animals at 15 min to 48 h after TBI and immunoprecipitated with anti-NR2B. NR2B immunoprecipitates were blotted for the indicated proteins. In sham animals, anti-NR2B immunoprecipitated NR2B, PSD 95, Shank, Homer, and Beclin-1, thus indicating association of these proteins in a multiprotein complex. Animals pretreated with Ro 25-6981 (NR2B antagonist) did not show TBI-induced disassociation of this multiprotein signaling complex at 15 min after trauma as observed in untreated animals subject to trauma (Fig. 5A). However, animals pretreated with the NVPAAM077 (NR2A antagonist) demonstrated similar pattern of association of NR2B signaling proteins as those observed in untreated sham and injured animals (Fig. 5).

FIG. 9.

Glutamate treatment of primary neuronal cultures results in activation of autophagy and cell death that is blocked by the NR2B antagonist Ro 25-6981. (A) Primary cortical neurons were treated with 100 μM glutamate for 8 h resulted in cell death as assayed by propridium iodide. Ro 25-6981 (10 ng/ml to 25 μg/ml) administered 30 min prior to glutamate treatment significantly decreased glutamate induced cell death. (B) Immunoblots of primary cortical neurons treated with 100 μM glutamate for 8 h resulted in significant increases in Beclin-1 and LC3II at 2–6 h after glutamate treatment. (C) Ro 25-6981 (100 ng/ml) administered 30 min prior to glutamate treatment prevented increases in Beclin-1 and LC3II. *p ≤ 0.05 versus control. n = 6 wells per each group.