Abstract

Neuroinflammation contributes to delayed (secondary) neurodegeneration following traumatic brain injury (TBI). Tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling may promote post-TBI neuroinflammation, thereby exacerbating secondary injury. This study investigated the pathogenic functions of TRAF6 signaling following TBI in vivo and in vitro. A rat TBI model was established by air pressure contusion while lipopolysaccharide (LPS) exposure was used to induce inflammatory-like responses in cultured astrocytes. Model rats were examined for cell-specific expression of TRAF6, NF-κB, phosphorylated (p)-NF-κB, MAPKs (ERK, JNK, and p38), p-MAPKs, chemokines (CCL2 and CXCL1), and chemokine receptors (CCR2 and CXCR2) by immunofluorescence, RT-qPCR, western blotting, and ELISA, for apoptosis by TUNEL staining, and spatial cognition by Morris water maze testing. These measurements were compared between TBI model rats receiving intracerebral injections of TRAF6-targeted RNAi vector (AAV9-TRAF6-RNAi), empty vector, MAPK/NF-κB inhibitors, or vehicle. Primary astrocytes were stimulated with LPS following TRAF6 siRNA or control transfection, and NF-κB, MAPKs, chemokine, and chemokine receptor expression levels evaluated by western blotting and ELISA. TRAF6 was expressed mainly in astrocytes and neurons of injured cortex, peaking 3 days post-TBI. Knockdown by AAV9-TRAF6-RNAi improved spatial learning and memory, decreased TUNEL-positive cell number in injured cortex, and downregulated expression levels of p-NF-κB, p-ERK, p-JNK, p-p38, CCL2, CCR2, CXCL1, and CXCR2 post-TBI. Inhibitors of NF-κB, ERK, JNK, and p38 significantly suppressed CCL2, CCR2, CXCL1, and CXCR2 expression following TBI. Furthermore, TRAF6-siRNA inhibited LPS-induced NF-κB, ERK, JNK, p38, CCL2, and CXCL1 upregulation in cultured astrocytes. Targeting TRAF6-MAPKs/NF-κB-chemokine signaling pathways may provide a novel therapeutic approach for reducing post-TBI neuroinflammation and concomitant secondary injury.

Highlights

  • Severe traumatic brain injury is a frequent cause of physical disability, cognitive dysfunction, and accidental death

  • Our preliminary study found that C-C motif ligand 2 (CCL2) was mainly co-localized with the astroglial marker glial fibrillary acidic protein (GFAP), while the CCL2 receptor chemokine C-C motif receptor 2 (CCR2) was mainly co-localized with the neuronal nuclear marker NeuN in traumatic brain injury (TBI) model rats, suggesting that CCL2–CCR2 signaling is a major driver of neuroinflammation, neurodegeneration, and concomitant motor and cognitive dysfunction following TBI (Liu et al, 2013a)

  • We further demonstrated that in vitro administration of pro-inflammatory lipopolysaccharide (LPS) significantly upregulated expression levels of CCL2 and C-X-C motif ligand 1 (CXCL1) as well as phosphorylation of the stress-associated transcription factor nuclear factor-kappa B (NF-κB) and the mitogen-activated protein kinases (MAPKs), c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 in cultured astrocytes prepared from cerebral cortices of neonatal rats

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Summary

Introduction

Severe traumatic brain injury (sTBI) is a frequent cause of physical disability, cognitive dysfunction, and accidental death. Neuroinflammation is a major pathogenic mechanism for secondary brain injury following TBI (Chiu et al, 2016; Simon et al, 2017; Liu et al, 2019), characterized by edema, microglial and astrocytic activation and migration, and the release of inflammatory cytokines and chemokines (Gyoneva and Ransohoff, 2015; Karve et al, 2016; Webster et al, 2017). Inhibition of these processes following sTBI may significantly reduce the progressive deficits associated with secondary brain injury. Neuroinflammation was modulated via regulation of LPS-induced MAPK/NF-κB-CCL2/CXCL1 signaling pathways (Liu et al, 2018)

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