Co-localization of TRAF6 with neuron and astrocyte markers in cerebral cortex of traumatic brain injury (TBI) model rats
To determine the specific cell types expressing TRAF6, we performed double immunofluorescence staining using anti-TRAF6 and a second cell-specific marker (GFAP, NeuN, or IBA-1). TRAF6 mainly co-localized with the astrocyte marker GFAP (Fig. 1a-c) and the neuronal marker NeuN (Fig. 1d-f) but demonstrated almost no co-localization with the microglial marker IBA-1 (Fig. 1g-i). According to manual cell counting, 55% ± 3% of astrocytes, 39% ± 2% of neurons, and 6% ± 1% of microglia expressed detectable TRAF6 (Fig.1j).
Fig. 1 Upregulation of TRAF6 in neurons and astrocytes at the injury site following experimental TBI in rats. (a-i) TRAF6 was co-localized mainly with the astrocytic marker GFAP (a-c), less frequently with the neuronal marker NeuN (d-f), and rarely with the microglial marker IBA-1 (g-i). (j) Quantification of co-expression showing that TRAF6 was upregulated mainly in cells positive for the astrocytic marker GFAP and the neuronal marker NeuN.
TBI induced region-specific upregulation of TRAF6 expression in cerebral cortex
Western blot was used to detect changes in cortical TRAF6 expression at 1, 3, 7, and 10 days post-TBI (Fig. 2). On day 1 post-TBI, TRAF6 expression was significantly greater in the injured area of the TBI group compared to the sham group. Expression reached a peak at 3 days post-TBI and then gradually declined.
Fig. 2 Transiently increased cortical expression of TRAF6 following TBI. The values are presented as the mean ± SEM. ***P < 0.001 vs. sham group.
AAV9-TRAF6-RNAi injection reduced TBI-induced TRAF6 overexpression and rescued spatial cognition
To evaluate whether TRAF6 signaling pathways contribute to cognitive dysfunction after TBI, rat subgroups receiving Sham, TBI, AAV9-NC+TBI, or AAV9-TRAF6-RNAi+TBI were compared for spatial learning and memory in the Morris water maze. The average latency to the hidden platform during learning trials was significantly lower in the AAV9-TRAF6-RNAi+TBI group compared to TBI and AAV9-NC+TBI groups (Fig. 3a), while in the probe trial, the mean number of platform location crossing was significantly greater in the AAV9-TRAF6-RNAi group (Fig. 3b). Thus, suppression of the post-TBI increase in TRAF6 partially rescued spatial learning and memory.
Fig. 3 Knockdown of TRAF6 after TBI rescued spatial learning and memory. Sham and TBI model rats receiving intracerebral injection of knockdown vector (AAV9-TRAF6-RNAi) or negative control vector (AAV9-NC) were compared for spatial learning and memory in the Morris water maze at 3 days post-TBI (or sham surgery). a Escape latency is presented as mean ± SEM (s). Administration of AAV9-TRAF6-RNAi reduced average latency to the escape platform, indicating improved spatial learning following TBI. **P < 0.01 vs. AAV9-NC+TBI group. b The number of platform location crossings is presented as mean ± SEM. Administration of AAV9-TRAF6-RNAi increased the number of platform crossings following TBI, indicating improved spatial memory. **P < 0.01 vs. AAV9-NC+TBI group.
AAV9-TRAF6-RNAi injection reduced neuronal apoptosis after TBI
We used TUNEL staining of cortical sections to examine the effect of TBI-induced TRAF6 signaling on apoptosis, a major cell death pathway contributing to secondary injury. Cell counting revealed a significant decrease in TUNEL-positive cells 3 days post-TBI in TBI model rats receiving AAV9-TRAF6-RNAi compared to the AAV9-NC-treated TBI group (Fig. 4).
Fig. 4 Knockdown of TRAF6 reduced apoptotic death rate following TBI. TUNEL (red), DAPI (blue), and TUNEL+DAPI (merged) images showing apoptotic cortical neurons. a-c Sham-treated rats. d-f TBI model rats. g-i TBI model rats injected with AAV9-NC. j-l TBI model rats injected with AAV9-TRAF6-RNAi. Scale bars are equal to 50 μm. m AAV9-TRAF6-RNAi treatment decreased neuronal apoptosis. Values are presented as mean ± SEM. ***P< 0.001 vs. AAV9-NC group.
AAV9-TRAF6-RNAi downregulated the expression levels of p-NF-κB, p-JNK, p-ERK, and p-p38 in injured cortex
Based on the above results and our previous research, we speculated that p-NF-κB, p-JNK, p-ERK, and p-p38 may be TRAF6-activated downstream signaling factors contributing to secondary degeneration following TBI. To test this hypothesis, we compared the expression changes in p-NF-κB, p-JNK, p-ERK, and p-p38 among TBI model rats receiving AAV9-TRAF6-RNAi or AAV9-NC pretreatment. Consistent with activation of these signaling pathways by TRAF6, the expression levels of p-NF-κB, p-JNK, p-ERK, and p-p38 at the site of injury were significantly lower in the AAV9-TRAF6-RNAi+TBI group compared to the AAV9-NC+TBI group on day 3 post-TBI (Fig. 5).
Fig. 5 TRAF6 knockdown suppressed p-NF-κB, p-JNK, p-ERK, and p-p38 expression in the injured cortex after TBI. *P < 0.05, **P < 0.01, ***P< 0.001 vs. AAV9-NC group.
AAV9-TRAF6-RNAi downregulated mRNA and protein expression levels of CCL2, CCR2, CXCL1, and CXCR2 in injured cortex
We also speculated that CCL2 and CXCL1 may be downstream effectors of TRAF6‒MAPK‒NF-kB signaling. To test this hypothesis, we compared mRNA and protein expression levels of CCL2, CXCL1, CCR2, and CXCR2 between TBI model rats receiving AAV9-TRAF6-RNAi or AAV9-NC pretreatment (Figure 6). Indeed, consistent with regulation by TRAF6, AAV9-TRAF6-RNAi suppressed the mRNA expression levels (Fig. 6a and 6b) and protein expression levels (Fig. 6c and 6d) of CCL2, CCR2, CXCL1, and CXCR2 on day 3 post-TBI.
Fig. 6 TRAF6 knockdown suppressed CCL2, CCR2, CXCL1, and CXCR2 expression at both mRNA and protein levels in injured cortex after TBI. a-d AAV9-TRAF6-RNAi downregulated (a) CCL2 and CCR2 mRNA, (b) CXCL1 and CXCR2 mRNA, (c) CCL2 and CCR2 protein, and (d) CXCL1 and CXCR2 protein expression in injured cortex on day 3 post-TBI. *P< 0.05, **P< 0.01, *** P< 0.001 vs. AAV9-NC group.
NF-κB, ERK, JNK, and p38 inhibitors also reducedprotein expression levels of CCL2, CCR2, CXCL1 and CXCR2in injured cortex
In our previous study, we demonstrated that NF-κB, ERK, and JNK inhibitors suppressed CCL2 and CXCL1 expression in activated astrocytes. To verify these effects in vivo, we measured changes in CCL2, CXCL1, CCR2, and CXCR2 protein expression levels among model rats treated with low (2.5 mg/10 mL) or high (25 mg/10 mL) doses of NF-κB, ERK, JNK, and p38 inhibitors. Indeed, a high-dose of the NF-κB inhibitor BAY117082, ERK inhibitor PD98059, JNK inhibitor SP600125, or p38 inhibitor SB203580 suppressed expression of CCL2, CCR2, CXCL1, and CXCR2 at the protein level (Figure 7a-d) on day 3 post-TBI. Thus, TRAF6-MAPK/NF-κB signaling pathways appear to mediate upregulation of CCL2, CCR2, CXCL1, and CXCR2 following TBI.
Fig. 7 Inhibitors of p-NF-κB and MAPKs suppressed upregulation of CCL2, CCR2, CXCL1, and CXCR2 following TBI. (a-d) A 25 mg/10 mL dose of the p-NF-κB inhibitor BAY117082 (a), p-JNK inhibitor PD98059 (b), p-ERK inhibitor SP600125 (c), or p-p38 inhibitor SB203580 (d) suppressed CCL2, CCR2, CXCL1, and CXCR2 protein expression in the injured cortex as measured by ELISA, while lower doses (2.5 mg/10 mL) had no significant effect. *P< 0.05, **P < 0.01, ***P < 0.001 vs. TBI group. ###P < 0.001 vs. sham group.
LPS induced TRAF6 upregulation in cultured astrocytes
To verify the link between neuroinflammation and TRAF6 upregulation in astrocytes (the predominant cell type showing TRAF6 upregulation after TBI, Fig. 1), we measured TRAF6 protein expression changes in primary astrocytes at 1, 3, and 6 h following stimulation (activation) by the inflammatory inducer LPS (Figure 8). Expression levels of TRAF6 in primary astrocytes were higher at 1 h post-LPS compared to the sham-treated control group and peaked at 3 h before gradually decreasing.
Fig. 8 Upregulation of TRAF6 in primary cortical astrocytes in response to LPS. LPS exposure (1 μg/mL) rapidly upregulated TRAF6 protein expression in cultured primary rat astrocytes. Values are presented as the mean ± SEM. *P< 0.05, **P< 0.01 vs. control group.
TRAF6 knockdown suppressed p-NF-κB, p-JNK, p-ERK, and p-p38 expression in LPS-treated cultured astrocytes
To examine if TRAF6 upregulation in astrocytes activates NF-κB, JNK, ERK, and p38 as downstream effectors, we compared LPS-induced changes in the expression levels of the phosphorylated forms between control astrocytes and astrocytes transfected with TRAF6 siRNA (Fig. 9). Indeed, TRAF6 siRNA significantly suppressed LPS-induced upregulation of p-NF-κB, p-JNK, p-ERK, and p-p38 at 3 h post-stimulation. Collectively, these findings (Figs. 8 and 9) suggest that MAPK/NF-κB signaling pathways are induced in activated astrocytes at the site of TBI via upregulation of TRAF6.
Fig. 9 TRAF6 knockdown suppressed LSP-induced upregulation of p-NF-κB, p-JNK, p-ERK, and p-p38 in cultured astrocytes. **P< 0.01, ***P< 0.001 vs. NC siRNA group.
TRAF6 knockdown suppressed CCL2 and CXCL1 expression in LPS-treated astrocytes
Finally, to examine if CCL2 and CXCL1 are downstream effectors of TRAF6, expression levels were compared between control astrocyte cultures and cultures pretreated with TRAF6 siRNA. As shown in Figure 10a and b, TRAF6 siRNA significantly suppressed CCL2 and CXCL1 expression levels in astrocytes treated for 3 or 6 h with LPS treatment.
Fig. 10 TRAF6 knockdown suppressed CCL2 and CXCL1 expression in LPS-activated astrocytes. a TRAF6 siRNA downregulated CCL2 expression. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NC siRNA group. b TRAF6 siRNA downregulated CXCL1 expression. ***P < 0.001 vs. NC siRNA group.