Pathogenic Functions of Tumor Necrosis Factor Receptor-Associated Factor 6 Signaling Following Traumatic Brain Injury

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.

Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1

“…Briefly, rats were anesthetized under 4% isoflurane and placed into a stereotactic frame. As in our previous study [17], three holes were created in the right parietal bone with a dental drill (1.5 mm posterior to bregma and 1.5 mm lateral to the midline; 1.5 mm posterior to bregma and 3 mm lateral to the midline; 3 mm posterior to bregma and 1.5 mm lateral to the midline) for injection of vehicle or virus (2 μL/hole) at 0.2 μL/ min and a depth of 1.3 mm from the skull surface using a Hamilton syringe. After injection, the syringe needle was left in place for 10 min to ensure distribution of the virus.…”

“…Traumatic brain injury was induced by controlled cortical impact (CCI) as described in our previous study [17]. Briefly, male SD rats were anesthetized under 4% isoflurane and placed into a stereotactic frame.…”

“…Linear RNAs were digested using exonuclease (Epicentre Technologies, Madison, WI, USA) and circRNA sequencing was then performed using the Novaseq 6000 PE150 platform (Illumina). CircRNAs were mapped using STAR Chimeric Post (STARChip) alignment software [17] and then identified and quantified using the DCC program [18]. Differentially expressed genes were identified using edgeR.…”

“…Primary rat astrocytes were obtained from the cerebral cortices of neonatal rats as described [17]. Briefly, cortices were quickly isolated and placed in cold D-Hank's buffer, carefully cleared of membranes and large blood vessels, then triturated into a cell suspension by passing through a 70 mesh filter.…”

Structural Characterization of TRAF6 N-terminal for Therapeutic Uses

“…Besides, the connection between upregulation and/or accumulation of TRAF6 and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Amyotrophic lateral sclerosis (ALS) have been reported since TRAF6 triggers neuronal apoptosis and central nervous system (CNS) disruption. In CNS, TRAF6 also contributes to inflammatory responses in stroke and neuropathic pain [27][28][29][30].…”